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Development and characterization of a liposomal subunit vaccine against Neisseria Gonorrhoeae Parmar, Manjeet M. 1999

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DEVELOPMENT AND CHARACTERIZATION OF A LIPOSOMAL SUBUNIT VACCINE AGAINST NEISSERIA  GONORRHOEAE  By MANJEET M PARMAR B.Sc, The University of British Columbia, 1994 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Pharmacology & Therapeutics, Faculty of Medicine) We accept this as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA September 1999 © Manjeet M. Parmar, 1999  In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l l m e n t o f t h e requirements f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d b y t h e h e a d o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n .  D e p a r t m e n t o f P^,4AM/J^C^C>6^ The U n i v e r s i t y o f B r i t i s h Vancouver, Canada  Date  Serr^ZeZ  }  dM>) TtftifflfcajICS  Columbia  11  A B S T R A C T  This thesis is concerned with the development and characterization of a subunit vaccine against Neisseria gonorrhoeae. The major gonococcal outer membrane protein, Protein I (Por), was selected as the subunit component because it is antigenically conserved between gonococcal strains.  Isolated proteins, however, often do not elicit a protective  immune response, either because they are not efficiently taken up by antigen presenting cells (APC) or because antibodies generated against the denatured protein do not recognize the native conformation. In the present research, therefore, Protein I was reconstituted into liposomes. These lipid bilayer structures should be capable of maintaining Por in its native conformation and may be efficiently accumulated by APC.  The protein reconstitution  process was characterized with regard to the efficiency of protein insertion and the ease of detergent removal. Subsequently, the Por proteoliposomes were characterized as to their size, lamellarity, Por orientation in the bilayer and antibody binding efficiency. The detergents octyl glucopyranoside (OGP) and sodium cholate were compared with respect to efficiency of detergent removal and protein incorporation. The rate of OGP removal was greater than for cholate during dialysis. During OGP-mediated reconstitution, essentially complete protein incorporation was achieved at a protein-lipid ratio of 0.01:1. It was observed, however, that the degree of protein incorporation was dependent on the initial protein-lipid ratios. Increasing the concentration of Por protein relative to phospholipid in the reconstitution mixture resulted in inefficient protein incorporation at ratios of 0.02:1 or higher. Reconstitution studies using cholate indicated that protein insertion into liposomes was less efficient than during OGP-mediated reconstitution at the same initial protein-lipid ratios. Subsequent experiments examined Por reconstitution into liposomes consisting of a  Ill  solely bilayer-forming lipid, 1-palmitoyl, 2-oleoyl phosphatidylcholine (POPC) or mixtures of POPC with a non-bilayer-forming lipid, 1-palmitoyl, 2-oleoyl phosphatidylethanolamine (POPE). These studies showed no significant differences in incorporation as a function of POPC POPE ratio. Examination of Por orientation in these proteoliposomes suggested that over 80% of the protein was oriented facing outwards in the same "hairpin loop" fashion found in the native bacterial membrane. The conclusion from these studies is that OGP is a preferred detergent for protein reconstitution, providing efficient insertion into the liposomal bilayer in an orientation comparable to the native conformation. Por protein was also reconstituted into liposomes containing positively or negatively charged lipids. Protein reconstitution into systems composed of negatively charged lipids, 1palmitoyl, 2-oleoyl phosphatidylserine (POPS) or 1-palmitoyl, 2-oleoyl phosphatidylglycerol (POPG), exhibited similar protein incorporation efficiencies as neutral POPC systems when the acidic lipid was present at 5% (by wt). However, increasing the amount of anionic lipid up to 25% resulted in a decrease in protein incorporation efficiency. Essentially complete protein incorporation was achieved when Por was reconstituted into positively charged, dioleyl dimethylammonium chloride- or DODAC-containing liposomes at 5, 10 or 25% D O D A C . Interestingly, increasing the concentration of cationic lipid resulted in a shift of the protein/lipid peak towards the top of the isopycnic density gradient, indicating a decrease in density of the reconstituted  systems.  These results indicate that efficient  protein  incorporation can be achieved for liposomal systems of differing lipid composition. Reconstituted Por proteoliposomes were characterized by quasi-elastic light scattering size analysis (QELS) and cryo-electron microscopy (CTEM) to determine proteoliposome size and morphology. Such systems exhibited a mean vesicle diameter of greater than 0.3  iv microns and were observed as heterogeneous structures with regard to size and lamellarity using CTEM. A potential subunit vaccine would have to be sterilized before it could be safe for use in humans. Conventional techniques such as heat or steam sterilization would be inappropriate due to potential denaturation of the protein subunit. Terminal filtration through 0.2 micronfilterswould be adequate for vaccine sterilization; however, reconstituted systems with a mean diameter of 0.3 microns would be too large and unsuitable for direct sterile filtration. Therefore, these proteoliposomes would have to be size-reduced prior to the filtration/sterilization step. In this thesis, it is shown that reconstituted systems can be sizereduced to 100 nm unilamellar vesicles by extrusion, without significant loss of protein or lipid. These extruded systems were then suitable for sterilization by terminal filtration. In a comparative study, the reconstitution of meningococcal outer membrane protein (MOMP) was characterized with regard to degree of protein insertion and detergent removal. The rate of octyl glucoside removal during MOMP reconstitution followed the same kinetics as seen for gonococcal Por reconstitutions. However, the efficiency of protein incorporation was lower than that of Por incorporation at the same initial protein and lipid concentrations. In addition, MOMP reconstitution was examined in the presence of a zwitterionic detergent, Empigen BB, which has been shown to restore the antigenicity of purified meningococcal proteins. Isopycnic density gradient centrifugation studies showed that liposomes were not formed, and hence no protein incorporation occurred during dialysis from an Empigen BBcontaining reconstitution mixture. The results of this comparative study would suggest that protein reconstitution occurs more efficiently in the case of a single polypeptide, such as Por, than for mixtures of different membrane proteins, such as MOMP.  ELISA assays were performed to determine the antibody binding activities of various Por liposome formulations using both anti-Por monoclonal antibodies and immunized rabbit sera.  Consistently higher levels of antibody binding were obtained for Por liposomes  prepared as described herein compared to reconstituted systems prepared as described in earlier publications from a different research group. Moreover, neutral proteoliposomes had a higher antibody binding activity compared to negatively or positively charged liposomes. Following the in vitro antigenicity analysis, the in vivo immunogenic properties of Por proteoliposomes and free Por were characterized and compared in a murine model. Mice were immunized by intraperitoneal or intradermal injections of either free Por protein or proteoliposomes containing neutral, cationic or anionic lipids.  In addition to the overall  antibody titers, the immune sera were characterized with regard to the ratio of the particular serotypes to determine whether the immune response elicited was humoral, indicated by elevated IgGl, or cell-mediated, marked by a predominant IgG2a response.  Analysis of  mouse immune sera showed that neutral and positively charged proteoliposomes, as well as free Por, induced similar antibody titers and these titers were greater than titers elicited by anionic proteoliposomes.  These differences in immune response were seen following  administration by either the intraperitoneal or intradermal routes of immunization. Cationic proteoliposomes, however, induced irritation and inflammation at the site of intradermal injection.  Examination of the antibody serotypes in the immune sera indicated that  immunization via the intraperitoneal route induced predominant IgGl responses whereas intradermal inoculation elicited greater IgG2a antibody titers.  These results suggest that  intradermal immunization might be more effective than the intraperitoneal route for  vi  generating a cell-mediated i m m u n e response, w h i c h is required f o r f i g h t i n g intracellular infections.  vii  T A B L E  O F  C O N T E N T S  Page ABSTRACT  ii  T A B L E OF CONTENTS  vii  LIST OF FIGURES  xi  LIST OF T A B L E S  xiv  ABBREVIATIONS  xv  ACKNOWLEDGEMENTS  xviii  DEDICATION  xix  C H A P T E R 1: INTRODUCTION  1  1.1 Vaccines and Immunology 1.1.1 Vaccine strategies 1.1.2 Humoral immunity 1.1.3 Cell-mediated immunity  1 2 4 10  1.2 Neisseria gonorrhoeae 1.2.1 Characteristics of the gonococcal organism 1.2.2 Characteristics of gonococcal infection 1.2.3 Treatment of gonococcal infection 1.2.4 Potential vaccine target antigens  16 16 19 20 23  1.3 Gonococcal Protein I 1.3.1 Characteristics of gonococcal protein I 1.3.2 Structure of protein I 1.3.3 Function of protein I 1.3.4 Antigenic properties of gonococcal protein I  26 26 30 33 34  1.4 Immunological Adjuvants 1.4.1 Liposomes: Model biomembranes 1.4.2 Liposome preparation and characterization 1.4.3 Liposomes as drug delivery systems 1.4.4 Liposomes as immunological adjuvants  36 37 41 47 48  viii  1.5 Research Hypotheses  52  1.6 Specific Research Objectives  52  C H A P T E R 2: M A T E R I A L S A N D M E T H O D S  54  2.1 Lipids, chemicals, and reagents  54  2.2 Reconstitution of gonococcal protein I (Por) into liposomes  55  2.2.1 Reconstitution of soluble Por 2.2.2 Reconstitution of lyophilized Por 2.2.3 Reconstitution of Por into anionic and cationic proteoliposomes  55 56 57  2.3 Reconstitution of meningococcal proteins (MOMP)  58  2.4 Analytical Procedures 2.4.1 Isopycnic density gradient centrifugation 2.4.2 Protein and phospholipid quantitation  59 59 59  2.5 Protease digestion  60  2.6 SDS-Polyacrylamide gel electrophoresis  60  2.7 Gel scanning densitometry  61  2.8 Size reduction of reconstituted proteoliposomes by extrusion  61  2.9 Quasi-elastic light scattering (QELS)  62  2.10 Cryo-transmission electron microscopy  62  2.11 Antibody binding evaluation  62  2.11.1 Sample preparation for antigenicity tests 2.11.2 Porin monoclonal antibodies 2.11.3 ELISA and inhibition E L I S A assays  62 63 63  2.12 Antibody binding activity of anionic and cationic Por proteoliposomes 2.12.1 Biotinylation of goat anti-mouse IgG 2.12.2 ELISA antibody binding assays  65 65 65  2.13 In vivo antigenicity of Por proteoliposomes 2.13.1 Immunization 2.13.2 ELISA antibody assays 2.13.3 ELISA antibody isotyping assays  67 67 67 68  IX  2.14 Tissue histology  69  2.15 Statistical methods  69  C H A P T E R 3: FACTORS I N F L U E N C I N G PROTEIN INCORPORATION INTO LIPOSOMES  70  3.1 INTRODUCTION  70  3.2 RESULTS 3.2.1 Detergent removal during reconstitution of Por protein 3.2.2 Influence of Por protein/lipid ratio on reconstitution efficiency 3.2.3 Proteoliposome size 3.2.4 Reconstitution of meningococcal outer membrane protein (MOMP): Residual detergent levels 3.2.5 Characterization of M O M P incorporation during reconstitution 3.2.6 Empigen BB-mediated M O M P reconstitution into liposomes  73 73 76 83 85  3.3 DISCUSSION  92  C H A P T E R 4: I N F L U E N C E OF LIPID COMPOSITION O N POR RECONSTITUTION A N D C H A R A C T E R I Z A T I O N OF T H E R E S U L T I N G PROTEOLIPOSOMES  87 90  98  4.1 INTRODUCTION  98  4.2 RESULTS 4.2.1 Por Protein reconstitution determined by isopycnic density gradient centrifugation 4.2.2 Por orientation in reconstituted proteoliposomes 4.2.3 Size reduction of reconstituted Por proteoliposomes 4.2.4 Reconstitution of Por into liposomes composed of charged lipids 4.2.5 Vesicle morphologies  101 101 105 109 113 120  4.3 DISCUSSION  127  C H A P T E R 5: THE ANTIGENIC C H A R A C T E R I Z A T I O N OF POR PROTEOLIPOSOMES  130  5.1 INTRODUCTION  13 0  5.2 RESULTS 5.2.1 Antibody binding to Por proteoliposomes determined using an ELISA assay 5.2.2 In vitro antibody binding activity of charged Por proteoliposomes 5.2.3 In vivo immune responses to proteoliposomes  132 132 137 137  5.2.4 Antibody isotyping of immune sera  142  5.2.5 Histology  145  5.3 DISCUSSION  147  CHAPTER 6: SUMMARY  152  REFERENCES  157  XI  L I S T  O F  F I G U R E S  Page Figure 1.1 The humoral immune response  6  Figure 1.2 The pathways of complement activation  9  Figure 1.3 The cell-mediated immune response  12  Figure 1.4 The surface components of the gonococcal outer membrane  18  Figure 1.5 Models of proteins IA and IB orientations in the gonococcal outer membrane  28  Figure 1.6 D N A sequence of Protein IA of gonococcal strain FA19  31  Figure 1.7 D N A sequence of Protein IB of gonococcal strain RIO  32  Figure 1.8 The eukaryotic plasma membrane  38  Figure 1.9 General phospholipid structure  40  Figure 1.10 Structure of lipid model membranes  42  Figure 111 Protein reconstitution into liposomes  45  Figure 3.1 N-Octyl-(3-D-glucopyranoside levels during dialysis  74  Figure 3.2 Sodium cholate levels during dialysis  75  Figure 3.3 OGP-mediated Por reconstitution into POPC liposomes (P/L=0.01)  78  Xll  Figure 3.4 OGP-mediated Por reconstitution into POPC liposomes (P/L=0.02)  80  Figure 3.5 Cholate-mediated Por reconstitution into POPC liposomes (P/L=0.01)  82  Figure 3.6 OGP levels during M O M P reconstitution  86  Figure 3.7 OGP-mediated M O M P reconstitution into POPC liposomes  89  Figure 3.8 Empigen BB/cholate-mediated M O M P reconstitution  91  Figure 4.1 Isopycnic density gradient centrifugation profile for Por reconstituted into POPC liposomes  102  Figure 4.2 Isopycnic density gradient centrifugation profile for Por reconstituted in POPC POPE (1:1) liposomes  103  Figure 4.3 Isopycnic density gradient centrifugation profile for insoluble Por reconstituted in POPC POPE (1:1) liposomes  104  Figure 4.4 Trypsin and a-chymotrypsin cleavage of detergent-solubilized and reconstituted Por protein.  106  Figure 4.5 Recovery of protein and phospholipid following extrusion of Por proteoliposomes.  112  Figure 4.6 Por protein incorporation into POPC/POPG liposomes  117  Figure 4.7 Por protein incorporation into POPC/POPS liposomes  119  Figure 4.8 Por protein incorporation into POPC/DODAC liposomes  122  Figure 4.9 Cryo-electron micrographs of reconstituted Por proteoliposome formulations viewed under different magnifications Figure 5.1 In vitro Por antibody binding activity Figure 5.2 In vitro Por antibody binding activity with rabbit anti-sera Figure 5.3 Effect of charged liposomes on antibody binding activity of proteoliposome formulations Figure 5.4 Anti-Por IgG titers of mice immunized with Por protein preparations Figure 5.5 Effect of immunization route on IgG isotypes of anti-Por antibodies Figure 5.6 Photograph of skin cross-section of the intradermal injection site  xiv  L I S T  O F  T A B L E S  Page Table 1.1 Outline of the regimen and mechanism of action of antibiotics against Neisseria gonorrhoeae  21  Table 1.2 Amino acid compositions of gonococcal proteins IA and EB isolated from bacterial strains FA19 and RIO, respectively  29  Table 3.1 QELS Size analysis of Por proteoliposomes reconstituted from OGP and cholate  84  Table 4.1 Trypsin and a-chymotrypsin cleavage of reconstituted Porproteoliposomes: Densitometric analysis of SDS-PAGE gel  108  Table 4.2 Size reduction of reconstituted Por proteoliposomes by extrusion  110  Table 4.3: QELS size analysis and protein incorporation efficiency of Por proteoliposomes reconstituted with varying lipid composition  114  A B B R E V I A T I O N S  Ab  antibody  ADCC  antibody-dependent cytotoxic cells  Ag  antigen  APCs  antigen presenting cells  BCA  bicinchoninic acid  BCR  B cell receptor  CD  cluster designation  CFA  complete Freund's adjuvant  CMC  critical micelle concentration  CMI  cell-mediated immunity  CRs  complement receptors  CS  circumsporozoite  CTL  cytotoxic T lymphocyte  Da  dalton  DGI  disseminated gonococcal infection  ELISA  enzyme-linked immunosorbent assay  FA  Freund's adjuvant  FATMLV  freeze and thawed multilamellar vesicles  Fc  crystallizable fragment  FCR  crystallizable fragment receptor  g  force of gravity  xvi GMP  general manufacturing procedure  HBS  HEPES-buffered saline  HBV  hepatitis B virus  HEPES  N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]  HI  humoral immunity  HIV  human immunodeficiency virus  id.  intradermal  i.p.  intraperitoneal  IFNy  interferon-gamma  IgG  immunoglobulin G  IL  interleukin  kDa  kilodalton  LOS  lipooligosaccharide  LPS  lipopolysaccharide  LUV  large unilamellar vesicle  MAb  monoclonal antibody  MAC  membrane attack complex  mBc  memory B cell  MHC  major histocompatibility complex  MLV  multilamellar vesicle  M0  macrophage  MOMP  meningococcal outer membrane protein  mTc  memory cytotoxic T cell  xvii MW  molecular weight  OGP  octyl-P-D-glucopyranoside  PG  peptidoglycan  PI  gonococcal protein I  PID  pelvic inflammatory disease  PII  gonococcal protein II  PHI  gonococcal protein III  POPC  l-Palmitoyl-2-oleoyl-sn-glycero- 3-phosphocholine  POPE  1 -Palmitoyl, 2-oleoyl-sn-glycero-3-phosphoethanolamine  POPG  1 -Palmitoyl, 2-oleoyl-sn-glycero-3 -phosphoglycerol  POPS  1 -Palmitoyl, 2-oleoyl-sn-glycero-3-phosphoserine  QELS  quasi-elastic light scattering  RER  rough endoplasmic reticulum  RES  reticuloendothelial system  SDS  sodium dodecyl sulfate  SUV  small unilamellar vesicle  TCR  T cell receptor  T  T helper cell  H  TNF  tumor necrosis factor  xvm ACKNOWLEDGEMENTS  Several colleagues and friends were instrumental in the preparation, development and eventual completion of this doctoral thesis.  I would like to acknowledge my committee  members Drs. Tom Madden, David Godin, Morley Sutter, and Lawrence Mayer for their contributions and assistance to my research project. The research supervisor is essential for providing guidance and support in a graduate student's research and education. I would like to thank Tom Madden for giving me the opportunity to work with him and providing invaluable supervision and input into my thesis and other written work. Laboratory work is a crucial part of science that often becomes stressful. As a supervisor, Tom has provided much needed reprieves from the long days in the lab as we have been rock climbing, skiing, and, on a number of occasions, dinner outings. I would also like to acknowledge my lab colleagues present and past: Miranda, Cliff, Jeff, Gitanjali, Ed, Cindy, Xue Min, and John for their contributions in and out of the laboratory. M y thanks go to Dana Masin for her assistance in the animal immunology studies. I would also like to acknowledge Drs. Milan Blake and Katarina Edwards for their assistance in this research. This research was supported by grants from National Institute of Health and scholarships from the Science Council of British Columbia.  D E D I C A T I O N  TO MY WIFE AND FAMILY  FOR THEIR CONSTANT SUPPORT AND MOTIVATION  1 CHAPTER 1 INTRODUCTION  The following chapter provides an introduction to vaccines and the host immune response. In addition, the characteristics of the gonococcus, mechanisms by which it avoids host defenses to cause infection and disease, current therapy of infection, as well as the rationale for using liposomes as a carrier for a gonococcal subunit vaccine are discussed.  1.1 Vaccines and Immunology The primary aim of vaccination is to generate an adaptive immune response that is highly specific for a particular pathogen and provides a memory of the pathogen such that subsequent challenge by the organism will be met with an enhanced immune response leading to inactivation and elimination of the pathogen. Vaccines have proven to be effective weapons for disease prevention. Vaccination has led to substantially lower incidences of disease, particularly diseases such as measles, mumps, rubella, pertussis, tetanus and poliomyelitis. The benefits of immunization can be illustrated by the virtual eradication of smallpox.  However, there are a number of diseases for which vaccines are not readily  available.  Diseases such as malaria, cholera, human immunodeficiency virus (HIV) and  toxin-producing E. coli kill millions of people annually. Obstacles that have become evident and have slowed the development of vaccines against these diseases are antigenic variation, hypersensitivity to the antigen, adverse reactions due to contamination with endotoxins and reversion of the attenuated organism to the wild type or pathogenic form.  2 1.1.1 Vaccine strategies Immunization has involved the administration of live, attenuated organisms (polio, measles and mumps) or killed whole organism vaccines (pertussis, influenza and typhoid). Microorganisms can be attenuated by mutation and selection of an avirulent strain which is then grown in culture. antigen retention.  Mutants are constantly monitored for absence of virulence and  The problems with attenuated vaccines are antigen instability and the  danger of reversion to virulent forms, which can then cause disease (Ross, 1998). Researchers  have indicated that genetic  mutations  contribute  to the  reversion to  neurovirulence of attenuated vaccines, as observed in cases of vaccine-associated paralytic poliomyelitis (Nkowane et al, 1987 and L i et al, 1996). In contrast, whole, killed-organism vaccines are prepared by chemical inactivation or irradiation with y-rays.  Advantages of using inactivated vaccines are that they are more  stable and will not revert to virulent forms as with attenuated preparations. However, killed vaccine formulations have been associated with safety problems. For instance, endotoxin contamination in pertussis vaccination has been associated with severe febrile convulsions and encephalitis in infants leading to, in severe cases, brain damage and even death (Miller et al, 1981 and Cherry, 1992). Alternative vaccine strategies using specific antigens from pathogens have been examined. Use of purified antigens can decrease the risk of adverse reactions associated with attenuated or killed preparations.  Capsular polysaccharides, exotoxins, recombinant  antigens, D N A and synthetic peptides have been investigated as potentiafantigen targets for vaccines (Kuby, 1997). Polysaccharide vaccines against Neisseria meningitidis groups A and C, but not B, have proven effective for eliciting humoral immunity through thymus-  3 independent B cell activation (Buchanan et al. 1998). However, polysaccharides do not bind to major histocompatibility complex (MHC) molecules and thus are unable to activate T helper cells or trigger the development of memory cells. Conjugation of polysaccharide to a protein carrier permits binding to the MHC molecules leading to T cell activation and H  induction of memory B cells in response to the polysaccharide antigen. However, this type of vaccine lacks the ability to induce memory T cells against the pathogen. Other vaccine preparations, such as diphtheria or tetanus toxoid vaccines, are effective in inducing antitoxoid antibodies that bind and neutralize bacterial toxin (Kuby, 1997). One of the concerns of using exotoxins in a vaccine relates to the need to ensure that there is complete detoxification without excessive modification of the epitope. Recombinant protein or synthetic peptide vaccines have been developed, in particular, against the hepatitis B virus.  Although recombinant vaccines are capable of inducing  humoral immunity (HI), they are taken up by endocytosis and processed as exogenous antigens and are therefore unable to activate major histocompatibility complex (MHC) class I-restricted T cells that are required for induction of cell-mediated immunity (CMI). Synthetic peptides are often poorly immunogenic or they tend to preferentially elicit HI over CMI. In order to improve the immune response to weak antigens, peptides and proteins are often combined with adjuvants, as described in a later section. A host's ability tofightan infection is influenced by the type of immune response, HI or CMI. The humoral response plays an important role in combating extracellular pathogens, whereas the cell-mediated immunity is involved in fighting intracellular infections.  The  development and characterization of a vaccine should take into account the type of immune response that is being generated and whether or not this response will be able to confer  4 protection against the specific pathogen. The components and mechanisms of humoral and cell-mediated immunity are described below.  1.1.2 Humoral Immunity Humoral immunity is that arm of the immune system that is mediated by immunoglobulins which are a group of glycoproteins present in serum and tissue fluids. Immunoglobulins are also commonly known as antibodies and can either be free in blood and lymph or can be surface-attached on B cells acting as receptors for antigens.  Antigen  recognition by B cells stimulates their maturation, proliferation and differentiation into antibody-forming cells (AFCs) or plasma cells which secrete large amounts of antibody against the particular antigen (Figure 1.1, adapted from Roitt et al, 1996). In addition, a certain fraction of the B cell population develops into longer-lived memory B cells that retain the antigen-binding specificity. Antibody molecules consist of four polypeptide chains, two identical light chains and two heavy chains linked together by disulphide bonds. The amino terminal ends of the heavy chains constitute the ends of the Fab portion of the antibody molecule and contain the site for antigen binding. The C-terminal ends or Fc portion of the heavy chains function as the ligand for binding to surface receptors (FCR) on phagocytic cells, B cells, and antibody-dependent cytotoxic (K) cells (ADCC). Antibody Fab portion recognition and binding of surface-exposed antigen on bacteria leads to opsonization in which Fc receptors on the surface of macrophages ( M 0 ) or ADDCs bind to Fc portion of the antibody attached to bacterial surface antigen resulting in bacterial cell phagocytosis and lysis. Macrophages can act as antigen-presenting cells (APCs) by ingesting a pathogen or foreign particles and processing and presenting antigen in association with cell surface  5 Figure 1.1: The humoral immune response. Antigen binds to B cells that have the appropriate surface B cell receptor (BCR). Cells are stimulated leading to clonal selection, cell proliferation and maturation into antibody-forming cells (AFCs) and memory B cells (mB). Antibody secreted by the AFCs and the memory cells have the same antigen-binding specificity. Secreted antibodies then bind to antigen resulting in opsonization of antigen or microbes expressing antigen on the cell surface. Antibody-antigen complex binds to Fc receptor (FCR) on the surface of the macrophage (Mphg) and triggers cell-mediated immunity, as well as phagocytosis of the antigen. Antigen-microbe complexes bind to antibody-dependent cytotoxic cells (ADCC) via the FCR, resulting in microbe lysis.  6  V  •  •  Antigen  f^BCR f Bcellj  Clonal Selection  Proliferation +Maturation  AFc)  ( A F C ) (AFf?)  Y y y +  V  •Antigen • Opsonization V  Y  Mfcr  (Mphg)  Cell-mediated Immunity  Phagocytosis  7 molecules called major histocompatibility complexes (MHCs).  Antigen presentation on  M H C molecules can trigger recognition by B cells leading to maturation, proliferation and differentiation into AFCs and memory B cells.  In addition to phagocytosis and lysis,  antibody-antigen complexes can trigger complement activation and serum killing. The complement system is a network of proteins or zymogens that require proteolytic cleavage in order to become active. The role of complement is to provide defense against bacteria by mediating lymphocyte activation, as well as triggering opsonization and lysis of target cells.  Complexes formed by antibody binding to bacterial surface antigens trigger  activation of this system through the classical or alternative pathways in which serum C4 is cleaved to C4b which becomes surface bound by binding to protein or carbohydrates on the bacterium. Surface-bound C4b then binds C2 which activates a cascade of reactions leading to cleavage of C3 to C3b which deposits on the bacterial cell surface (Figure 1.2, adapted from Roitt et al, 1996). Covalently attached complement proteins C3b and C4b act as opsonins enhancing phagocytosis by functioning as ligands for complement receptors on phagocytic cells.  Furthermore, fixed C3b can bind serum C5 which is converted to a  convertase to generate C5b which subsequently binds C6, C7, C8, C9 to form C5b-9, the membrane  attack complex.  Complement-mediated killing occurs in several ways.  Complement C3b-coated target cell can bind to complement receptors (CRs) on phagocytic cells triggering endocytosis or phagocytosis and cell death. Alternatively, binding of effector cells to complement fragments on the target can trigger activation and chemotaxis of leukocytes capable of producing bacterial cell death.  In addition, the membrane attack  complex (MAC), C5b-9, forms a hydrophobic "plug" or pore-forming molecule. This plug inserts into the target cell membrane causing osmotic disruption and cell lysis.  8 Figure 1.2: The pathways of complement activation. Activation of the complement system, which is a part of the innate immune system functioning in opsonization and removal of foreign pathogens from the body, is illustrated. Classical pathway (Panel A) links the adaptive immune system, antibody, to the innate immune system, complement. Antibody-microbe complexes bind Clq and trigger a series of reactions to generate the enzyme C4b2a that cleaves C3 to C3b and deposits on the cell surface and acts as an opsonin. The alternative pathway (Panel B) involves the surfaceattached C3b, which binds factor B. Factor D then cleaves factor B to generate the enzyme complex C3bBb that combines with properdin (P) to enhance cleavage C3 to C3b The enzymes C4b2a and C3bBb cleave C5 to C5b leading to the deposition and binding of C6, C7, C8 and C9 to form the membrane attack complex (MAC), which is a pore-forming molecule that causes osmotic disruptions in the cell membrane resulting in cell death.  10 1.1.3 Cell-mediated immunity The second component of the immune system is cell-mediated immunity (CMI), which is mediated primarily by lymphocytes and phagocytes and where antibodies play a secondary role acting as links in some cell-mediated reactions.  T cell-independent  interactions of an organism and phagocytic cells can lead to uptake and killing of the organism. As mentioned above, deposition of complement on the organism enables binding of macrophages via cell-surface receptors and subsequent pathogen lysis. Alternatively, macrophages and other cells can release cytokines upon recognition of the organism, which leads to stimulation and activation of other leukocytes. T cell-dependent cell-mediated immune responses represent a major host defense mechanism. The uptake, processing and presentation of antigen in association with M H C molecules on the APC surface triggers T helper (T ) cell activation, in particular C D 4 - T H +  H  cells (Figure 1.3, adapted from Roitt et al, 1996). T helper cells modulate the various types of cellular cooperation by releasing specific combinations of cytokines. There are two major subsets of T H cells, TH1 and TH2, each having a specific cytokine profile (Mosmann and Coffman, 1989). The differentiation and selection of the effector mechanism is determined by the nature of the antigen presentation on the surface of the APC. TH1 type cells are generally stimulated by M H C class I-restricted antigen presentation.  These CD4 -T cells +  produce cytokines IL-2 and IFNy, which are effective stimulators of B cell activation and production of IgG2a, but not much IgGl.  In addition, EL-2 stimulates cytotoxic C D 8  +  lymphocytes via the IL-2 receptor to recognize and kill infected host cells. In contrast, antigen recognition in association with M H C class II molecules stimulates CD4 -T cells to +  produce cytokines IL-4 and IL-6 which are efficient helper cells for B cell activation and  11 Figure 1.3: The cell-mediated immune response. Antigen is recognized, taken up and processed by antigen-presenting cells (APCs), such as macrophages and dendritic cells. The antigen is then presented on the surface of the cell in association with major histocompatibility complex class I (MHCl) or class II (MHCII). C D - T helper (TH) recognize antigen via the T cell receptor (TCR) and stimulate the activation of T H cells. When the antigen is presented on M H C l molecules, TH1 cells are activated releasing cytokines, such as tumor necrosis factor (TNF), interferon gamma (fFN) and interleukins 2 (IL-2) and 12 (EL-12). These cytokines stimulate macrophages and natural killer (NK) cells, which mediate phagocytosis and lysis of the pathogen. Cytokines also stimulate C D cytotoxic T (Tc) cells, which recognize and kill infected host cells. Activation of Tc also generates memory cytotoxic T (mTc) cells that retain long-lived memory of the specific antigen. Antigen presentation on MHCII molecules triggers the activation of TH2 cells and release of cytokines, such as DL-2 and EL-4. These cytokines trigger B cell activation and the release of EL-4, EL-6 and IL-10, which stimulate antibodyforming cells (AFC) to secrete antibodies. 4+  8 +  12  •  •  Antigen  Antigen uptake & processing  \y*  • l Y APC + ffrH) CD4+ MHciV_yMHcn ^ v _ y T  Antigen Recognition & Activation of T H cells  MHCl Recogniton  MHCII Recognition  IL-2,  TNF.IFNY  IL-4  IL-2, IL-12  Tc  (Mphg)  (NK^)  IL-2R  CD8+ I L - 1 , I L - 6 . I L 10  V mTc IL-2R  CD8+  Recognition of Infected Host Cells  Phagocytosis of Pathogen  (AFC)  (  A  F  C  13  proliferation for antibody p r o d u c t i o n , e s p e c i a l l y I g G l a n d I g E . C h o w and c o w o r k e r s (1998) demonstrated that c o - i m m u n i z a t i o n o f hepatitis B v i r u s a n d T H 1 type c y t o k i n e s , E L - 1 2 o r I F N y , stimulated T H 1 cell d e v e l o p m e n t w i t h a c o n c o m i t a n t increase i n I g G 2 a p r o d u c t i o n . I n contrast, antigen a n d E L - 4 c o - i n j e c t i o n i n d u c e d elevated I g G l levels w i t h enhanced T H 2 c e l l development, but suppressed T H 1 differentiation a n d I g G 2 a p r o d u c t i o n . cells a n d c y t o k i n e s have been s h o w n t o increase the C D 8 M. tuberculosis-infected  +  Elevation o f T H 1  T cell c y t o l y t i c a c t i v i t y t o w a r d s  macrophage target cells ( S k i n n e r et al., 1997).  E x o g e n o u s antigen is taken u p into endosomes o r l y s o s o m e s and degraded b y a c i d h y d r o l y s i s t o peptides. glycoprotein  chains  M H C class II m o l e c u l e s are heterodimers o f h e a v y (a) a n d light (P) that  are synthesized  i n the r o u g h  endoplasmic  reticulum ( R E R )  c o m p l e x e d t o a p o l y p e p t i d e c h a i n c a l l e d the invariant c h a i n (Ii) and then transported t h r o u g h the G o l g i c o m p l e x to the a c i d i c e n d o s o m a l o r l y s o s o m a l c o m p a r t m e n t (Jensen, 1997). T h e invariant c h a i n is b o u n d to the M H C class II b i n d i n g g r o o v e w h i c h is c o m p r i s e d o f a P sheet supporting t w o a helical d o m a i n s . m o l e c u l e b i n d s peptide.  H e r e , the I i b e c o m e s d i s s o c i a t e d a n d the M H C class II  C r y s t a l l o g r a p h y studies have s h o w n that the ends o f the b i n d i n g  g r o o v e are open and o p t i m a l l y b i n d peptides o f 12-20 a m i n o a c i d residues w i t h at least three v a r i a b l e a n c h o r i n g residues (Stern and W i l e y , 1994 and M a l c h e r e k et al,  1995).  Peptides  w i t h fewer than 13 a m i n o a c i d residues g e n e r a l l y have less than m a x i m a l b i n d i n g activities. T h e endosome  is then transported to the cell surface w h e r e the M H C class I I - peptide  c o m p l e x is e x p o s e d to the external e n v i r o n m e n t .  T helper cells r e c o g n i z e and b i n d t o the  p e p t i d e - M H C class II c o m p l e x v i a the T c e l l receptor response.  T H 2 cells release  cytokines  (EL-4,  ( T C R ) a n d trigger a T H 2 - t y p e  I L - 6 , EL-10)  which  proliferation, maturation and differentiation into A F C s a n d m e m o r y cells.  stimulate  B  cell  14  E n d o g e n o u s or c y t o p l a s m i c antigen, i n A P C , is processed b y proteases and then is transported b y a transmembrane transporter to the r o u g h e n d o p l a s m i c r e t i c u l u m ( R E R ) .  In  the R E R , proteosome c o m p l e x e s are processed and b o u n d to M H C class I m o l e c u l e s .  In  contrast to the class II b i n d i n g g r o o v e , the ends o f the M H C class I b i n d i n g cleft are c l o s e d ( R a m m e n s e e et al, 1993 and Stern and W i l e y ,  1994).  M o r e o v e r , endogenously  bound  peptides are restricted i n length to eight or nine a m i n o acids and are b u r i e d deep i n the g r o o v e b y a n c h o r i n g residues at the a m i n o and c a r b o x y l t e r m i n i o f the peptide  through  h y d r o g e n b o n d i n g and V a n der W a a l s forces ( M a t s u m a r a et al, 1992, Y o u n g et al, 1995 and F r e m o n t et al., 1995).  T h e ternary c o m p l e x is transported to the cell surface v i a the G o l g i  c o m p l e x and exposed for T c e l l r e c o g n i t i o n . M H C m o l e c u l e s that do not have peptide b o u n d to their clefts are unstable and p r o m p t l y dissociate and are degraded i n t r a c e l l u l a r l y . A n t i g e n presentation o n M H C class I m o l e c u l e s triggers T H 1 type c e l l activation. T helper cells b i n d v i a the T C R and release c y t o k i n e s that stimulate T cells, B cells and monocytes.  In  particular, T H 1 cytokines, I L - 2 and I F N - y activate c y t o t o x i c T cells ( T c ) , as w e l l as natural k i l l e r ( N K ) cells and macrophages.  I n d u c t i o n o f T c cells, N K c e l l s and macrophages directs  c e l l - m e d i a t e d c y t o t o x i c i t y and hence the c e l l - m e d i a t e d i m m u n e response. C e l l - m e d i a t e d c y t o t o x i c i t y produces target c e l l l y s i s b y several different m e c h a n i s m s . C y t o t o x i c c e l l s c a n degranulate perforin that binds to the  and release  pathogen  cell  a m o n o m e r i c p o r e - f o r m i n g protein c a l l e d  membrane  i n the  presence  o f c a l c i u m ions.  M o n o m e r s p o l y m e r i z e to f o r m transmembrane channels that cause the target c e l l to b e c o m e leaky.  Subsequently,  the T c releases degradative  p o l y p e r f o r i n channels to produce cell death.  e n z y m e s that penetrate t h r o u g h  the  In a d d i t i o n , the release o f t u m o r necrosis factor  ( T N F a or P) and I F N y can b i n d to surface receptors o n the target cell and cause c e l l death.  15  T h e exact m e c h a n i s m is not k n o w n , but the l i g a t i o n o f the agents at the c e l l surface m a y induce alterations i n the internal m e t a b o l i c p a t h w a y s w i t h i n the o r g a n i s m t o cause death o f the cell.  16  1.2 NEISSERIA  GONORRHOEAE  G o n o r r h e a is a s e x u a l l y transmitted gonorrhoeae.  disease  caused  b y a bacterium,  Neisseria  I n 1995, nearly 4 0 0 , 0 0 0 cases o f g o n o r r h e a i n the U n i t e d States w e r e reported  to the Centers for D i s e a s e C o n t r o l ( N I A T D , 1998), whereas a p p r o x i m a t e l y 5,500 cases w e r e reported i n C a n a d a ( L C D C , 1998). H o w e v e r , it is estimated that 8 0 0 , 0 0 0 cases o f g o n o r r h e a o c c u r annually i n the U n i t e d States, w i t h a n annual cost estimated at close to $1.1 b i l l i o n for the treatment o f gonorrhea and its c o m p l i c a t i o n s . D u r i n g 1995, it is estimated that there w e r e over 60 m i l l i o n n e w cases o f g o n o r r h e a a m o n g adults w o r l d w i d e ( W H O , 1995).  The  prevalence o f this disease, m e d i c a l costs and the dramatic rise o f antibiotic-resistant strains o f g o n o c o c c u s underscore the need f o r a means o f p r e v e n t i n g a n d c o n t r o l l i n g gonorrhea. Therefore, the d e v e l o p m e n t o f a n effective g o n o c o c c a l v a c c i n e is a n important objective for m a n y research g r o u p s around the w o r l d .  research  T h e characteristics o f the g o n o c o c c a l  o r g a n i s m , current methods o f treatment and potential v a c c i n e targets are described b e l o w .  1.2.1 Characteristics o f the g o n o c o c c a l o r g a n i s m Neisseria gonorrhoeae b e l o n g s to the f a m i l y o f organisms c a l l e d N e i s s e r i a c e a e w h i c h are non-flagellated, spherical o r rod-shaped o r g a n i s m s c o m m o n l y o c c u r r i n g i n pairs o r as short-chained groups.  G o n o c o c c i are g r a m negative bacteria that c o l o n i z e m u c o s a l sites.  T h e structural characteristics o f the g o n o c o c c u s have been d e s c r i b e d b y several researchers ( M a e l a n d , 1977, M o r s e and B r o o k s , 1985 B r o o k s , 1985a). g o n o c o c c a l c e l l e n v e l o p e consists o f three major layers.  A s s h o w n i n F i g u r e 1.4, the  T h e c y t o p l a s m i c m e m b r a n e is the  innermost layer o f p r i m a r i l y l i p i d s and proteins s u r r o u n d i n g the c y t o p l a s m a n d the c e l l u l a r machinery.  T h e m i d d l e layer consists o f the p e p t i d o g l y c a n , w h i c h i s constructed b y a  17  Figure 1.4: The surface components of the gonococcal outer membrane. T h e outer m e m b r a n e structures that are k e y factors i n g o n o c o c c a l pathogenesis are s h o w n . T h e abbreviations are. P I , protein I; P I I , protein II; P H I , protein III; L P S , l i p o p o l y s a c c h a r i d e ; l i p i d , f o r m i n g the p h o s p h o l i p i d b i l a y e r o f the outer membrane; proteins  forming  peptidoglycan,  hair-like  extensions  that  a structure o f sugars and  are  amino  considerably acids.  pilus, polymer o f pilin longer  than  A r r o w s indicate the  illustrated; proposed  connections between inner and outer m e m b r a n e proteins to the p e p t i d o g l y c a n ( A d a p t e d f r o m B r i t i g a n e f a/., 1985 and B r o o k s , 1985a).  18  19  l i n k a g e o f a series o f sugars and a m i n o acids. T h e p e p t i d o g l y c a n ( P G ) forms a r i g i d structure and maintains the structural integrity o f the c e l l . T h e outermost layer o f the c e l l e n v e l o p e is a c o m p l e x outer membrane.  T h e structures w i t h i n this outer m e m b r a n e are considered to be  the k e y v i r u l e n c e factors i n the pathogenesis o f disease.  T h e interactions o f these c o m p l e x e s  w i t h host m u c o s a l surfaces and i m m u n e system mediate the d e v e l o p m e n t o f disease.  The  internal side o f the outer membrane is thought to be connected to the p e p t i d o g l y c a n v i a membrane  proteins.  T h e m i d d l e layer is c o m p o s e d o f p h o s p h o l i p i d s and h y d r o p h o b i c  components such as the h y d r o p h o b i c m e m b r a n e - b o u n d d o m a i n s o f proteins that assist i n the attachment to the p e p t i d o g l y c a n layer.  T h e external surface o f the g o n o c o c c u s presents the  h y d r o p h i l i c regions o f the m e m b r a n e - b o u n d protein and other structures such as p i l i and lipopolysaccharide.  These surface-exposed structures interact w i t h the external e n v i r o n m e n t  and function i n facilitating and m e d i a t i n g g o n o c o c c a l infection.  1.2.2 Characteristics o f g o n o c o c c a l i n f e c t i o n H u m a n s are the natural hosts for Neisseria gonorrhoeae  where gonococcal infection  results i n a w i d e spectrum o f sequelae ( B r o o k s and D o n e g a n , 1985 and B r i t i g a n et al., 1985). A n i m a l m o d e l s for g o n o c o c c a l i n f e c t i o n have been f o u n d to be difficult to maintain, as a n i m a l hosts tend to have natural resistance to i n f e c t i o n ( B r o o k s , 1985b). i n f e c t i o n i n a n i m a l s does not m i m i c the i n f e c t i o n i n humans.  I n addition,  U r e t h r i t i s is a c o m m o n  s y m p t o m o f g o n o c o c c a l i n f e c t i o n i n m e n and is characterized b y m i l d d y s u r i a and urethral discharge after a t w o to seven day i n c u b a t i o n p e r i o d . I n some m e n , the disease can d e v e l o p to e p i d i d y m i t i s , painful i n f l a m m a t i o n o f the e p i d i d y m i s . Infections i n females often lead to more  serious  conditions.  Initially,  gonococcal  infection  is  primarily,  although  not  20  e x c l u s i v e l y , manifested i n the e n d o c e r v i x and is associated w i t h u n u s u a l o r increased v a g i n a l discharge.  S o m e i n d i v i d u a l s experience d y s u r i a , increased frequency o f u r i n a t i o n , p e l v i c  p a i n a n d menstrual  abnormalities.  I n some instances,  w o m e n c a n develop  salpingitis,  i n f l a m m a t i o n o f the f a l l o p i a n tubes, w h i c h is characterized b y r a p i d onset o f p e l v i c , l o w e r a b d o m i n a l and, i n some cases, b a c k and leg p a i n ( B r o o k s , 1985c). can lead t o ectopic pregnancy  a n d infertility.  f a l l o p i a n tubes o r b y l y m p h a t i c drainage perihepatitis.  C o m p l i c a t e d salpingitis  I n addition, spread o f infection f r o m the  t o the l i v e r c a n cause l i v e r d y s f u n c t i o n o r  Other m u c o s a l infections that often o c c u r are rectal and p h a r y n g e a l infection,  p r i m a r i l y due t o rectal-genital and oral-genital contact, respectively. Complicated  gonococcal  infections  are referred  to as disseminated  gonococcal  infections ( D G I ) and are characterized b y fever and s k i n lesions i n the early stages ( B r o o k s , 1985d). L e s i o n s u s u a l l y o c c u r o n distal parts o f the arms and legs such as j o i n t s o f toes and fingers, i n c l u d i n g the general area o f the hands and feet. A s the c o n d i t i o n progresses, septic arthritis develops, m a r k i n g the onset o f the secondary  stage o f D G I .  I n severe disease  situations, D G I is manifested as endocarditis o r m e n i n g i t i s .  1.2.3 Treatment o f g o n o c o c c a l i n f e c t i o n A n t i b i o t i c s are the current therapy f o r c o m b a t i n g g o n o c o c c a l infections.  Penicillin  was w i d e l y prescribed f o r the treatment o f g o n o r r h e a a n d remains the standard d r u g f o r treatment i n m a n y d e v e l o p i n g nations.  H o w e v e r , d u e to the emergence o f p e n i c i l l i n a s e -  p r o d u c i n g strains o f Neisseria gonorrhoeae a n d the development antibiotics, change i n the therapy r e g i m e n has b e c o m e inevitable.  o f newer m o r e potent  T h e current first c h o i c e o f  a n t i m i c r o b i a l therapy is a c o m b i n a t i o n o f a m o x i c i l l i n and c l a v u l a n i c a c i d ( T a b l e 1.1, adapted  21  Regimen  Antibiotic Class  Antimicrobial Agents  First choice  P e n i c i l l i n s + (3 lactamase inhibitor  Amoxicillin + Clavulanic  Inhibition o f  Acid  p e p t i d o g l y c a n synthesis  Cephalosporins  Ceftriaxone, cefuroxime,  Inhibition o f p e p t i d o g l y c a n synthesis  cefotaxime Second choice  Fluoroquinolones  Ciprofloxacin, enoxacin, perfloxacin, norfloxacin  Mechanism of Action  Inhibition o f D N A synthesis  Table 1.1: O u t l i n e o f the r e g i m e n a n d m e c h a n i s m o f a c t i o n o f antibiotics against Neisseria gonorrhoeae.  22 f r o m R a n g et al,  1995).  A m o x i c i l l i n , a P-lactam antibiotic, s i m i l a r to p e n i c i l l i n ,  w i t h the formation o f the p e p t i d o g l y c a n layer o f the g o n o c o c c a l c e l l envelope.  interferes It acts b y  i n h i b i t i n g the transpeptidation e n z y m e responsible for c r o s s - l i n k i n g o f peptide chains d u r i n g p e p t i d o g l y c a n synthesis ( A r m s t r o n g et al,  1987). C l a v u l a n i c a c i d is a P-lactamase i n h i b i t o r  that protects the antibiotic from e n z y m a t i c degradation.  F o r the treatment o f P-lactamase-  p r o d u c i n g bacteria, s p e c t i n o m y c i n w a s a c o m m o n replacement for p e n i c i l l i n ; however, there is a propensity to develop resistance to s p e c t i n o m y c i n ( B r i t i g a n et al,  1985). A n o t h e r group  o f first-line a n t i m i c r o b i a l agents are the cephalosporins, ceftriaxone b e i n g the p r o t o t y p i c a l agent.  A s w i t h the  Increased  resistance  p e n i c i l l i n s , cephalosporins to  these  agents  has  interfere  developed  with peptidoglycan due  to  synthesis.  plasmid-mediated  and  c h r o m o s o m a l p-lactamases that have greater h y d r o l y t i c activity t o w a r d s cephalosporin.  Due  to m u l t i - d r u g resistance, newer agents have b e e n introduced for the treatment o f g o n o c o c c a l infection.  C i p r o f l o x a c i n is a broad-spectrum  compounds  c a l l e d the fluoroquinolones.  antibiotic b e l o n g i n g to a group o f synthetic  These a n t i m i c r o b i a l s e x h i b i t excellent activity  against many organisms resistant to p e n i c i l l i n , c e p h a l o s p o r i n and a m i n o g l y c o s i d e s . therapeutic a c t i o n o f these agents o c c u r s d u r i n g D N A r e p l i c a t i o n . synthesis to proceed, D N A gyrase (topoisomerase supercoil o f D N A and generate a negative  The  In order for D N A  II) must i n i t i a l l y u n w i n d the p o s i t i v e  supercoil.  supercoil i n D N A permits transcription or replication.  The introduction o f a  negative  D r u g s such as c i p r o f l o x a c i n i n h i b i t  D N A synthesis b y b i n d i n g and b l o c k i n g D N A gyrase function. A l t h o u g h a n t i m i c r o b i a l therapy is an effective m e t h o d o f eradicating g o n o c o c c a l infections, the emergence o f greater numbers o f antibiotic-resistant gonorrhoeae  requires  the  continued  development  o f newer,  more  strains o f expensive  Neisseria agents  to  23  c i r c u m v e n t the resistance mechanisms. is r e c e i v i n g considerable  attention  as  Therefore, the concept o f p r e v e n t i o n v i a v a c c i n a t i o n an a d d i t i o n a l m e t h o d  o f combating  gonococcal  infection, e s p e c i a l l y i n d e v e l o p i n g countries w h e r e antibiotic-resistant bacteria are prevalent.  1.2.4 Potential v a c c i n e target antigens F o r g o n o c o c c a l i n f e c t i o n to occur, attachment to host m u c o s a l cells is essential.  The  role o f v a r i o u s g o n o c o c c a l surface structures i n the attachment process has been r e v i e w e d b y several researchers ( B r i t i g a n et al,  1985 and B l a k e and G o t s c h l i c h , 1987). P i l i are h a i r - l i k e  extensions radiating f r o m the surface o f the g o n o c o c c a l c e l l .  E a c h p i l u s is c o m p o s e d o f a  series o f protein subunits, p i l i n proteins, that range i n m o l e c u l a r w e i g h t s o f 18,000-20,000 daltons.  T h e size and antigenic characteristics v a r y d e p e n d i n g o n the particular strain.  A d h e r e n c e o f the g o n o c o c c u s is mediated p r i m a r i l y b y the p i l i , as p i l i a t e d organisms e x h i b i t greater adherence to h u m a n e u k a r y o t i c c e l l s than n o n - p i l i a t e d g o n o c o c c i . T h e p i l u s c a n also mediate the spread o f i n f e c t i o n f r o m the e n d o c e r v i x to the f a l l o p i a n tubes b y f a c i l i t a t i n g attachment o f bacterial c e l l s to sperm and e n a b l i n g the penetration o f the c e r v i c a l m u c o u s barrier and m i g r a t i o n to the  f a l l o p i a n tissue.  In a d d i t i o n , p i l i c a n i n h i b i t neutrophil  phagocytosis o f bacterial cells.  T h e i m p o r t a n c e o f the p i l u s i n the pathogenesis o f i n f e c t i o n  has made p i l i n peptides targets for potential v a c c i n e candidates.  However, gonococcal pili  have been observed to have a h i g h degree o f antigenic heterogeneity ( B u c h a n a n , 1975). T h i s has been demonstrated b y m a r k e d p i l i n v a r i a b i l i t y amongst strains, as w e l l as w i t h i n a n i n d i v i d u a l g o n o c o c c a l strain ( S w a n s o n et al., 1987). P i l i n v a r i a b i l i t y , therefore, has l e a d to the targeting o f other surface structures as potential v a c c i n e antigens.  24  P r o t e i n II is an outer membrane p r o t e i n w i t h a m o l e c u l a r w e i g h t ranging b e t w e e n 2 4 , 0 0 0 - 3 0 , 0 0 0 daltons, depending o n the c o n d i t i o n s used i n p u r i f i c a t i o n and s o l u b i l i z a t i o n . S i m i l a r to p i l i , protein II is thought to p l a y an important role i n the adherence to certain m u c o s a l , as w e l l as p h a g o c y t i c cells s u c h as neutrophils.  I n particular, protein II is thought  to be i n v o l v e d i n the c l u m p i n g o f o r g a n i s m s s u c h that the adherence o f a single g o n o c o c c u s to the m u c o s a l surface m a y a l l o w the attachment o f m a n y m o r e g o n o c o c c i .  Antigenic  v a r i a t i o n i n protein II is quite prevalent and enables g o n o c o c c i to s u r v i v e at v a r i o u s different sites o f i n f e c t i o n i n m a l e and female hosts.  Studies have s h o w n that a single g o n o c o c c a l  strain can express either no, one or several types o f p r o t e i n II, each h a v i n g a m o l e c u l a r w e i g h t and b e i n g a n t i g e n i c a l l y distinct ( B l a c k et al,  1984).  The  different antigenic  diversity o f protein II species has been demonstrated b y the reaction o f different protein II species f r o m the same strain w i t h a specific protein II serum ( D i a z and H e c k e l s , 1982).  The  results were i n d i c a t i v e o f v e r y little cross-reactivity between different protein II m o l e c u l e s and a h i g h degree o f antigenic v a r i a b i l i t y i n the surface-exposed protein structure ( H e c k e l s , 1981). A s indicated, phase variations enable the o r g a n i s m to escape host i m m u n e responses and adapt efficiently, e n a b l i n g t h e m to c o l o n i z e particular m i c r o e n v i r o n m e n t s .  Therefore,  protein II has p r o v e n to be less than i d e a l as an antigen for a g o n o c o c c a l v a c c i n e . A n o t h e r outer membrane constituent that has b e e n considered as a v a c c i n e antigen is protein III. T h i s protein ranges i n m o l e c u l a r w e i g h t f r o m 3 0 , 0 0 0 - 3 1 , 0 0 0 daltons, d e p e n d i n g o n the r e d u c i n g c o n d i t i o n s and it is a h i g h l y a n t i g e n i c a l l y c o n s e r v e d protein c o m m o n to all strains o f g o n o c o c c i ( B l a k e and G o t s c h l i c h , 1983 and B l a k e et al,  1988).  It is located i n  close p r o x i m i t y to the major g o n o c o c c a l outer m e m b r a n e protein, protein I ( M c D a d e and Johnston, 1980). P r o t e i n III is i n v o l v e d i n m e d i a t i n g serum resistance b y p r o v i d i n g a b i n d i n g  25  site for b l o c k i n g antibodies.  Studies have s h o w n that antibodies to protein III can b l o c k the  bactericidal a c t i v i t y o f h u m a n sera ( R i c e et al, V i r j i and H e c k e l s , 1988).  1994, R i c e et al,  1985, R i c e et al,  T h e effect m a y be exerted b y c o m p e t i t i o n between  1986 and blocking  antibody and b a c t e r i c i d a l a n t i b o d y for c o m p l e m e n t a c t i v a t i o n and d e p o s i t i o n o n the c e l l surface (Joiner et al,  1985a).  Joiner and c o w o r k e r s (1985b) s h o w e d that c o m p l e m e n t C 3  d e p o s i t i o n w a s enhanced s i x - to n i n e - f o l d b y b l o c k i n g a n t i b o d y and that b l o c k i n g a n t i b o d y i n h i b i t e d k i l l i n g i n a dose-dependent manner.  These observations suggest that c o m p l e m e n t  depletion m a y be a m e c h a n i s m o f serum resistance.  F u r t h e r studies s h o w e d that enhanced  c o m p l e m e n t activation and d e p o s i t i o n b y the b l o c k i n g a n t i b o d y lead to the f o r m a t i o n o f a n o n - b a c t e r i c i d a l C 5 b - 9 c o m p l e x that w a s i n a different m o l e c u l a r c o n f i g u r a t i o n than  the  bactericidal C 5 b - 9 required for serum l y s i s and k i l l i n g o f bacteria (Joiner, 1983, Joiner, 1985c). T h e depletion o f c o m p l e m e n t factors m a y reduce the n u m b e r o f effective m e m b r a n e attack c o m p l e x e s and hence i m p a i r the h o s t ' s a b i l i t y to fight infection. D u e to the antigenic v a r i a t i o n o f p i l i , protein II and serum resistance  mediated b y b l o c k i n g antibodies,  the  g o n o c o c c a l o r g a n i s m has d e v e l o p e d several m e c h a n i s m s to evade and thwart the i m m u n e system and ensure its s u r v i v a l and facilitate the pathogenesis o f infection.  R e s e a r c h has  therefore focussed o n protein I, a major g o n o c o c c a l m e m b r a n e c o m p o n e n t as a potential antigen target.  26  1.3 G O N O C O C C A L P R O T E I N I  1.3.1 Characteristics o f g o n o c o c c a l p r o t e i n I T h e characteristics o f g o n o c o c c a l protein I have been r e v i e w e d b y G o t s c h l i c h and c o w o r k e r s (1988).  P r o t e i n I is the major g o n o c o c c a l m e m b r a n e protein ( 6 0 % ) , h a v i n g a  m o l e c u l a r w e i g h t i n the range o f 3 2 - 4 0 k D a (Johnston and G o t s c h l i c h , 1974).  It is an  antigenically conserved m e m b r a n e constituent that serves as the basis for c l a s s i f i c a t i o n o f a large n u m b e r o f g o n o c o c c a l strains (Johnston et al, and B l a k e and G o t s c h l i c h , 1983).  1976, B u c h a n a n and H i l d e b r a n d t , 1981  Studies have s h o w n that protein I is a channel f o r m i n g  outer membrane protein termed a p o r i n ( D o u g l a s et al,  1981).  Isolation, p u r i f i c a t i o n and  protease digestion studies have i n d i c a t e d the existence o f t w o types o f protein I that adopt different  conformations  w i t h i n the g o n o c o c c a l outer  membrane.  P r o t e i n I A has  been  observed to be inserted c o m p l e t e l y into the m e m b r a n e w i t h o n l y a s m a l l fragment exposed o n the surface ( B a r r e r a and S w a n s o n , 1984). P r o t e i n I B is oriented w i t h b o t h ends inserted into the b i l a y e r w i t h the l o o p p o r t i o n e x t e n d i n g out from the p e r i p l a s m i c surface ( B l a k e et 1981, B l a k e and G o t s c h l i c h ,  1982 and B l a k e and G o t s c h l i c h ,  1983).  A m o d e l o f the  p r o p o s e d protein I structures is illustrated i n F i g u r e 1.5 (adapted from B l a k e et al, B a r r e r a and S w a n s o n , 1984).  al,  1981 and  T h e general a m i n o a c i d c o m p o s i t i o n determined for proteins  I A and I B f r o m t w o g o n o c o c c a l strains is s h o w n i n T a b l e 1.2 (adapted from B l a k e and G o t s c h l i c h , 1982).  27  Figure 1.5: Models of proteins IA and IB orientations in the gonococcal outer membrane. P r o t e i n I A is almost c o m p l e t e l y b u r i e d w i t h i n the m e m b r a n e w i t h o n l y a s m a l l t e r m i n a l , exposed portion. Treatment o f m e m b r a n e - b o u n d P I A w i t h c h y m o t r y p s i n or t r y p s i n does not p r o d u c e any cleavage fragments. H o w e v e r , treatment w i t h proteinase K ( P K ) y i e l d s t w o fragments, a s m a l l fragment (1.5 k D a ) and a membrane-associated fragment (33.5 k D a ) . I n contrast, protein I B is situated w i t h its t e r m i n a l portions b u r i e d i n the outer m e m b r a n e w i t h an external l o o p region e x p o s e d o n the g o n o c o c c a l surface. C h y m o t r y p s i n treatment generates t w o membrane-associated fragments o f 2 2 ( C l ) and 14 k D a ( C 2 ) , whereas t r y p s i n , i n i t i a l l y , cleaves P I B into 28 ( T 2 ) and 10 k D a ( T l ) fragments. P r o l o n g e d t r y p s i n treatment cleaves the 28 k D a fragment to a m e m b r a n e - b o u n d 21 k D a fragment and a smaller, soluble fragment. In contrast to P I A , P I B is i n i t i a l l y c l e a v e d b y proteinase K into fragments r e s e m b l i n g those generated b y c h y m o t r y p s i n . P r o l o n g e d d i g e s t i o n cleaves P K 2 fragment into a smaller soluble fragment P K 3 . ( A d a p t e d f r o m B l a k e et al., 1981 and B a r r e r a and S w a n s o n , 1984).  29  A m i n o A c i d C o n t e n t ( m o i o ' r e s i d u e / m o l o f protein) Amino Acid Neutral Aliphatic Glycine  Strain F A 1 9 - P r o t e i n I A  Strain R I O - P r o t e i n I B  198 (0.64)*  223 (0.68)  155 (0.50) 37  179 (0.54) 43  Alanine  27  31  Valine  28  29  Leucine Isoleucine  16 6  19 8  Serine  23 18  31 18  38 (0.12)  37(0.11)  Phenylalanine  15  15  Tyrosine Tryptophan  18 5  18 4  0 0  2 (0.01) 2  0  0  Aspartic/asparagine  5 (0.02) 61 (0.20) 33  67 (0.20) 34  Glutamic/glutamine B a s i c a m i n o acids  28 48 (0.16)  33 39(0.12)  Threonine Aromatic  Sulphur-containing Methionine Cysteine I m i n o acids/proline D i c a r b o x y l i c a m i n o acids  5 (0.02)  12  8  Arginine  16  11  Lysine  20  20  307  329  Histidine  Total  Table 1.2: A m i n o a c i d c o m p o s i t i o n s o f g o n o c o c c a l proteins I A and EB isolated f r o m bacterial strains F A 1 9 and R 1 0 , respectively. T h e a m i n o a c i d content w a s determined from the specific D N A sequence o f each p r o t e i n I. * N u m b e r s i n parentheses represent the fraction o f the total n u m b e r o f residues represented i n each class.  30  1.3.2 Structure o f protein I A s mentioned, g o n o c o c c a l p r o t e i n I is d i v i d e d into t w o i m m u n o c h e m i c a l classes, P I A and P I B , based o n the reactions w i t h specific m o n o c l o n a l antibodies ( K n a p p et al,  1984).  P r o t e i n I A is u s u a l l y associated w i t h strains capable o f i n v a d i n g the h u m a n b l o o d stream and c a u s i n g systemic g o n o c o c c a l infections o r disseminated disease.  Strains bearing protein I B  are t y p i c a l l y responsible for u r o g e n i t a l disease or p e l v i c i n f l a m m a t o r y disease ( P I D ) . P r o t e i n I B has a m o l e c u l a r w e i g h t o f 34-38 k D a and w h e n m e m b r a n e - b o u n d is susceptible to t r y p s i n and c h y m o t r y p s i n cleavage, whereas p r o t e i n I A has  a lower molecular weight  generally resistant to cleavage b y these s p e c i f i c proteases ( B l a k e et al.,  1981).  and  is  Carbonetti  and S p a r l i n g (1987) c l o n e d the P I A gene and determined the D N A sequence e n c o d i n g protein I o f one g o n o c o c c a l strain. T h e predicted N - t e r m i n a l a m i n o a c i d sequence o f protein I A o f strain F A 1 9 ( F i g u r e 1.6) matched that o f a k n o w n protein I A (strain 120176-2) and w a s v e r y s i m i l a r to that o f the protein I B o f strain RIO ( F i g u r e 1.7) ( B l a k e and G o t s c h l i c h , 1982, B l a k e , 1985 and G o t s c h l i c h et al,  1987).  T h e predicted a m i n o a c i d sequence ( 3 0 7 A A ) o f  protein I A F A 1 9 predicts a m o l e c u l a r w e i g h t o f 3 3 , 7 8 6 daltons, w h i c h c l o s e l y approximates the apparent m o l e c u l a r w e i g h t o f 3 4 , 0 0 0 ( C a r b o n e t t i and S p a r l i n g , 1987).  S i m i l a r l y , the  calculated m o l e c u l a r w e i g h t based o n the a m i n o a c i d sequence ( 3 2 9 A A ) o f protein I B o f strain RIO agrees w i t h that o f the k n o w n v a l u e o f 36 k D a as determined b y ( G o t s c h l i c h et al,  1987).  SDS-PAGE  C o m p a r i s o n s o f the g o n o c o c c a l proteins I A and I B p r i m a r y  structures to that o f the major porins o f Escherichia  coli ( O m p F / O m p C ) s h o w e d a h i g h  degree o f sequence h o m o l o g y , i n d i c a t i n g the p o s s i b i l i t y o f a c o m m o n ancestral (Carbonetti and S p a r l i n g , 1987 and G o t s c h l i c h et al,  origin  1987). M o r e o v e r , the D N A sequences  o f b o t h P I A and PUB genes predicted the same t y p i c a l 19 a m i n o a c i d s i g n a l peptide as s h o w n  31  o ^  o VO  0 0  0 cs  0 00  >  ON  <L> >  3  - E-  < o  <  _ o  o o 3 CJ u H J o  ^y < o  l a  o S3 P  «>8 •5 o  -H  -  o  -a < < CJ  o <  H  < < < <  < o o  p CJ ESH < <  I8  o o _ CJ  £• o o o  °  CJ « cj  < o  5,2  OH  o  ^5  E—  2 <  o o  CJ  _  H  o  -  o  <  £>8 < CJ  o E— t— O  p 'CJ  o o o o <  < H CJ  O < < <  CJ  o  o  £.2  a. Q  O  I a* "Q  E—  o o CJ o o  CJ ECJ CJ CJ CJ  o  <  -J CJ CJ  •3  y  .2 o  < o  3U  (J  CJ E-T< Et  H  i-  H  b  «  <<  CJ u O  < o < O  ^ H  =5  <o  £ CJ  3  o  JS CJ  <  O o u.  CJ  fe o  <M < cfl CJ  X CJ  - E—  <  Cfl  _ ra > ^  H H O O  Cfl  o  55  c_  0» M  H  CJ H _ CJ  < < X  0  u. O  < <  > o  3 O  . CJ  CJ O  >.<  H .E-  < CJ  . 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CJ  cn  T3 e  U  So 3  7  cd  1  ^ -a u  s 3  cu  z Q  g ^ C  'S E  c« • °  CD 0  c CD §-  vo 3  Ml  ^ 1 H  Q.  32  o  t  cs  m  O  -  CS ON  fi  > 3  rt  o  H H EO < O E~ O  u y o 3  53 3 <  < O < E~  y  K  O  - o u. o 1  s» <  -3 IS < <  O O <  U  <  <  o o  < < < < < < o  <  _  > o  -h 03 f-, °" E- EE- <  o  EH O E— E—  < < H E-i O O EO O  o o u  y V  < o o  < u  s<  o = <  ^ o o o  •J <  ^8  Jj o  _ o $Z  E- < E-  3 <  rt E> O O O O  o  «8 a?  -J  o o 2 H  1^ <: < :>y  38 3  -J o &y < o 3  58 GO < .  a) O  -8  O O  y  <  <  y  o  oo -J <:  <o  <<  58  > O  > o  fe 8  s=  . o  cu ^ — < u O  Q  -< 5S  -1  <  .  O  ^ o o o i-  o  o o  <  B-O E- H -  H  < i - E-  <  y  O  5§  ^ o  ^8 E- <  o  o t* 3  U  E-  < o u •a y  58 ^8  » u  ^  >.o  s  <  _ o  ^ a  S3 O  _ < > o O  2<  . ^  EO O  O O  |8  cu H  < o  c  °  << to  *^  °  °  O  ^8  <  > a  9  3  3  E8 o  Q  O  E— H  |8  "  5 EJ O u.  o  o  c—.  o o  3  O  Q E~  •s U  r,  S  O 0  B-O  t- E-  o  »o  < o —  <  oS ^ o « o  ^ o  „ <  <  O O  fe o 3  y  u  S  O o u-  o O J= Ecu p  u O  < E—  o o  £ <  O O  58  ° E—  =y 2  -it  o §  2 u<  o o ^8  55 =y << < 2 CO  <,  53  wi E—  3  2  J o u, E~  3  2  n  <  S o  St  °H  < a  <  O O  fe O ai <  °-fe  < o — Ert f-<  > o i.  7=  D  u,  o  <  < o  u. O  O o  o  [-. H E^ O  <  0  _o  > o ES <  s o E-  1  — H  rt — t > 5 E— E-  •—  g  cci  o O E-  p  oo E-  ^8  00  CT\ 1  u,  "3  O E~  so  es  u u o u o e o  E — < — H rt E-< >3 O  2 J3t 3"  <o  o pa _c " S +J o u  Eu O u,  HH  ?8 o J  uu  < <  y  o  u &  H OJ O  <U  &  . y  H  H  CU  3  O  «  cr -g  S Q CU  o C^  cu  . ^ ^ H o  w  CTS CU  c 13 * .2 O -T3  X!  oo  C  oo  <D  CU  «»  CU S o 3 I  M  y < o  o o ;3 CU  ©  « y  < < —3 < < . y uC o rt o O O cu  ctf  O  <! o  > o  o  . y  E— ._ E—  oo _ rt >  00  2  o o  P^  O  3  O  s tj « o  a,  o3  &8  J= H C U UH _ O  S  OJ  o o  H O  a o  < O  o,  j < ^ y  O O  Cu  < O  3  ** - —<  ii<  < < o oo y u ES o ^3 < o o 3 ^° y 5 5  C  1  O O <  H  (Si <£,  0>  X o  58  E— H  "rt H > O O o E-  y  o o  *«  <3  18  o <  s8  oo u_  < O  .  c O  =<  ^y  fe o *s  > a  O  5- <  ^5 oo  e  < o  <<  J2  << •- <  Sj:  -2 O o  u,  &o E—  <8  E— i- H  < <<  o a o J= o •§  <  <D  «y 4 y  <  °-y  E- Et. E—  3  > o « u  3  o  ^§  2  > O e <  H H O  <  3  U  3  =  3  o o  b  E-  5  o o _ < > o  o  H  >> < J <  sq  9-  2  %o  > — ] P  o O  « <; < O  s8 a  3  o o s o  cu (J  M  .o  O O o O  <  „ o < 3<  > o  52 a  co  h <  a o ^8  -<  H  13 E—  o  H E-  o o  _ o  E— O O O U  <o  o o <_ oo o fe o Cu  <<  J  58  h  ^ o  < o  H E—  5S <  f-  H  3  *8  < o  =2  J  _ o > O  a o o o o o <  P <  ^ E—  > O  rt  < o  2  <  a> O  =2 J3 S cu o  < < „ o  y  o < < <  -o 9o  p.O  H E3 O u E_ •J O  u.  O o  o  O o  < o  S  O  u  o  V  rt EJ  y  <  O o >-y  •s o °  o < <  2  5 O  o  . y  U — Es  o  o o  JC-O  — f H O  O o  E_  o rt ° .2 y < o » o  at H  - y  u  %  CL,  8bi  Ct  ^ -a  3  cd  I  CD  O  oo  03  CU  CU  cr <u  er V)  z  Q  _c cu "5 ° c c 3  <" y ^  H  O o  _ y  rt H  > o  CU  3 00  H  Cu  33  i n F i g u r e s 1.6 and Escherichia  1.7.  C o n s t r u c t i o n o f a P I gene c l o n e and the expression o f P I i n  coli was obtainable.  P r o t e i n p r o d u c e d b y the c l o n e w a s detectable w i t h a n t i - P I  m o n o c l o n a l antibodies and w a s observed to be i d e n t i c a l i n size to native protein I. H o w e v e r , p r o l o n g e d expression o f protein I d u r i n g o v e r n i g h t g r o w t h w a s lethal t o the E. coli cells, p o s s i b l y due to the a c t i v i t y o f the g o n o c o c c a l p o r i n o r alterations i n the structural integrity o f the bacterial outer membrane ( C a r b o n e t t i and S p a r l i n g , 1987).  1.3.3 F u n c t i o n o f protein I Studies  o f outer  membrane  porins  o f Escherichia  coli  have  s h o w n that  these  m o n o m e r s adopt a p r i m a r i l y j3-sheet secondary structure ( G r a v i t o and R o s e n b u s c h , 1980 and G r a v i t o et al,  1983).  In the f o r m a t i o n o f the channel, these m o n o m e r s c o m b i n e to adopt a  quaternary structure to f o r m trimers.  P r o t e i n I o f Neisseria gonorrhoeae  forms trimers ( B l a k e and G o t s c h l i c h , 1982 and D o u g l a s et al,  1981).  is also a p o r i n and  Studies e x a m i n i n g the  i n c o r p o r a t i o n o f p r o t e i n I into l i p i d v e s i c l e s s h o w e d that the protein f o r m e d channels i n the bilayer, increasing the p e r m e a b i l i t y to ions and v a r i o u s m a c r o m o l e c u l e s , such as sugars ( G r e c o et al,  1980, D o u g l a s et al,  protein I functions  as  artificial planar bilayers.  1981).  an anion-selective,  h y d r o p h i l i c channel  when  incorporated  into  Incorporated protein w a s s h o w n to self-associate into a t r i m e r i c  structure f o r m i n g a voltage-dependent have also demonstrated  Y o u n g and c o w o r k e r s (1983) demonstrated that  aqueous pore o f at least 11A i n diameter.  that g o n o c o c c a l p r o t e i n I can be  spontaneously  Studies  transferred  as  functional channels from the bacterial m e m b r a n e into artificial l i p i d bilayers, as w e l l as red b l o o d cells ( B l a k e and G o t s c h l i c h , 1983 and L y n c h et al,  1984).  These results suggest that  the " s p i k i n g " o f host cells b y p r o t e i n I w i t h its associated i o n o p h o r i c properties m a y enable  34  the b a c t e r i u m to i n v a d e n o n - p h a g o c y t i c host e p i t h e l i a l c e l l s as s h o w n b y W a r d et al. (1974) and M c G e e et al. (1978). Infection o f surface e p i t h e l i a l cells facilitates spread o f i n f e c t i o n to u n d e r l y i n g cells and hence further contributes to the pathogenesis o f g o n o c o c c a l disease.  1.3.4 A n t i g e n i c properties o f g o n o c o c c a l protein I T h e g o n o c o c c a l surface structures play a c r i t i c a l role i n the p r i m a r y interaction w i t h the host and facilitate the development o f i n f e c t i o n ( S w a n s o n , 1981).  E a r l y studies s h o w e d  that crude outer membrane preparations c o u l d p r o v i d e some degree o f p r o t e c t i o n against infection i n a g u i n e a p i g m o d e l ( B u c h a n a n and A r k o , 1977). These results suggest that there are antigens o n the c e l l surface capable o f i n d u c i n g i m m u n i t y to g o n o c o c c a l i n f e c t i o n and thus potentially o f great interest i n the research and d e v e l o p m e n t o f a gonorrhea v a c c i n e (Heckels,  1978  and  Gotschlich,  1984).  A n t i b o d i e s against  components have been s h o w n to be b a c t e r i c i d a l and o p s o n i c .  these  outer  membrane  Therefore, antibodies m a y  p r o v i d e protection to host cells b y i n h i b i t i n g bacterial c e l l attachment at the m u c o s a l surface or b y p r o m o t i n g phagocytosis and c o m p l e m e n t - m e d i a t e d serum k i l l i n g ( W a r d et al., and V i r j i ,  1981).  B u c h a n a n and  c o w o r k e r s (1980)  1978  s h o w e d that i n d i v i d u a l s that  experienced an episode o f g o n o c o c c a l p e l v i c i n f l a m m a t o r y disease generated  had  antibodies  against the o r g a n i s m that p r o v i d e d some p r o t e c t i o n against recurrent salpingitis.  These  studies p r o v i d e further e v i d e n c e that protective i m m u n i t y m a y be possible b y u t i l i z i n g an outer membrane constituent i n a v a c c i n e f o r m u l a t i o n . H o w e v e r , the large degree o f antigenic v a r i a t i o n i n m a n y o f these surface antigens has p r o m p t e d a search for a n t i g e n i c a l l y c o n s e r v e d cell components ( Z a k et al., 1984).  35  P r o t e i n I is an a n t i g e n i c a l l y stable protein that has been s h o w n to elicit the p r o d u c t i o n o f b a c t e r i c i d a l and o p s o n i c antibodies ( R i c e et al, 1991).  1980, Sarafian, 1983 and G u l a t i et  al,  In addition, the central role o f p r o t e i n I i n the pathogenesis o f g o n o c o c c a l i n f e c t i o n  b y " s p i k i n g " or transferring into the host e p i t h e l i a l c e l l m e m b r a n e to trigger endocytosis o f the bacterial c e l l makes protein I an attractive target i n the d e v e l o p m e n t o f a g o n o c o c c a l vaccine In vitro studies have s h o w n that m o n o c l o n a l antibodies against outer  membrane  protein I A e x h i b i t e d b a c t e r i c i d a l and o p s o n i c a c t i v i t y and w e r e effective i n protecting epithelial cells f r o m the c y t o t o x i c effects  o f gonococci (Virji  et al,  1987).  Virji  and  c o w o r k e r s also demonstrated that anti-PEB antibodies w e r e also b a c t e r i c i d a l and o p s o n i c w i t h the epitope for antibody b i n d i n g located at, or close to, the c h y m o t r y p s i n cleavage site w i t h i n the surface-exposed l o o p r e g i o n (Fletcher et al,  1986).  A n t i b o d i e s directed t o w a r d s P E B  w e r e observed to be m o r e protective against i n f e c t i o n o f e p i t h e l i a l c e l l s than w e r e a n t i - P I A antibodies ( V i r j i et al,  1986 and V i r j i et al,  1987).  These observations m a y reflect the  greater transferring a b i l i t y o f P I A into host epithelial c e l l membranes  during infection  ( B l a k e , 1985). These protein I characteristics support the p r o p o s i t i o n that protein I w o u l d be an important antigenic c o m p o n e n t o f a potential g o n o c o c c a l subunit v a c c i n e .  36  1.4 I M M U N O L O G I C A L A D J U V A N T S A s mentioned earlier, due to the potential t o x i c i t y o f c o n v e n t i o n a l v a c c i n e s , it w o u l d be preferable to identify and purify a single, c o n s e r v e d antigen or generate r e c o m b i n a n t subunit or synthetic peptides.  H e n c e , the target and specificity o f the i m m u n e  response  w o u l d be c o n t r o l l e d and o p t i m i z e d . U n f o r t u n a t e l y , peptide or protein antigens are often n o n i m m u n o g e n i c or o n l y w e a k l y i m m u n o g e n i c w h e n a d m i n i s t e r e d alone ( R i c h a r d s et al,  1988).  In some cases, the purified antigen does elicit an a n t i b o d y response; h o w e v e r , the i m m u n e response generated protective  m a y be directed t o w a r d s certain determinants  i m m u n i t y against  infection.  This  may  be  a result  that m a y not p r o v i d e o f inadequate  surface  presentation o f the epitope required for a n t i b o d y b i n d i n g and o p s o n i z a t i o n o f the w h o l e o r g a n i s m ( W e t z l e r et al., amplified  when  1988).  T h e i m m u n e response to w e a k i m m u n o g e n s m a y  administered with  an  adjuvant.  A  number  o f adjuvants  have  investigated for i m m u n e potentiating a c t i v i t y for use i n v a c c i n e s ( E d e l m a n , 1980). oils,  such as F r e u n d ' s  adjuvant  ( F A ) , have  been  be  been  Mineral  s h o w n to have a n t i b o d y - s t i m u l a t i n g  properties; h o w e v e r , this is associated w i t h severe side effects, and thus is not acceptable for h u m a n subjects.  A l u m i n u m h y d r o x i d e (or a l u m ) is the o n l y adjuvant that has been a p p r o v e d  b y the F D A for use i n humans.  It is less than i d e a l , as it induces g r a n u l o m a s and painful  i n f l a m m a t i o n at the site o f injection and therefore research has c o n t i n u e d t o d e v e l o p safer and  more  effective  adjuvants.  L i p o s o m e s have  been  i n d i c a t e d to  have  potential  as  i m m u n o l o g i c a l adjuvants and p o s s i b l e alternatives to a l u m and F A ( A l v i n g , 1987, A l v i n g , 1991 and G r e g o r i a d i s , 1990). T h e f o l l o w i n g sections describe the structure and f u n c t i o n o f l i p i d s , methods o f l i p o s o m e preparation and role o f l i p o s o m e s as i m m u n o l o g i c a l adjuvants.  37  1.4.1 L i p o s o m e s : M o d e l b i o m e m b r a n e s B i o l o g i c a l membranes are c o m p o s e d o f a w i d e range o f m o l e c u l e s , o f w h i c h l i p i d s are  major b u i l d i n g b l o c k s .  The plasma membrane  o f the  eukaryotic cell  acts as  a  p e r m e a b i l i t y barrier between the external e n v i r o n m e n t and the internal c e l l u l a r m a c h i n e r y as w e l l as p r o v i d i n g a m a t r i x w i t h w h i c h m e m b r a n e proteins c a n be associated.  Proteins are  often inserted into and t h r o u g h this l i p i d b i l a y e r ( F i g u r e 1.8, adapted from C u l l i s and H o p e , 1991)  T h e a b i l i t y o f l i p i d s to adopt a b i l a y e r c o n f o r m a t i o n is dictated b y the a m p h i p a t h i c  characteristics o f these m e m b r a n e lipids. L i p i d structure, as s h o w n i n F i g u r e 1.9, consists o f a polar head g r o u p that is h y d r o p h i l i c i n nature and a non-polar o r h y d r o p h o b i c tail.  In the  b i l a y e r structure, polar heads are oriented t o w a r d s the aqueous environment, whereas the h y d r o p h o b i c a c y l c h a i n r e g i o n is sequestered  f r o m water.  T h e fluidity o f the r e s u l t i n g  m e m b r a n e is dependent o n the nature o f the a c y l c h a i n c o m p o s i t i o n and other factors such as sterol content. A n important aspect o f membrane f u n c t i o n is the r o l e that integral m e m b r a n e proteins play i n the b i o l o g i c a l a c t i v i t y o f the c e l l . I n order t o e x a m i n e the characteristics o f particular proteins,  they  membranes.  can  be  isolated,  purified  and  inserted  into  well-defined, lipid  model  A variety o f m e m b r a n e proteins have been reconstituted into such m o d e l  membranes w h i c h are termed l i p o s o m e s ( M a d d e n , 1986, M a d d e n , 1988). L i p o s o m e s consist o f a l i p i d b i l a y e r s u r r o u n d i n g an aqueous core.  T h e l i p i d b i l a y e r c a n be constructed w i t h  different l i p i d species d e p e n d i n g o n the desired l i p o s o m e characteristics.  38  Cytoplasm  F i g u r e 1.8: T h e e u k a r y o t i c p l a s m a  membrane.  L i p i d , protein and carbohydrates are c l o s e l y associated i n the e u k a r y o t i c p l a s m a as described b y the f l u i d m o s a i c m o d e l ( T a k e n f r o m C u l l i s and H o p e , 1991).  membrane  39  F i g u r e 1.9: G e n e r a l p h o s p h o l i p i d s t r u c t u r e . P h o s p h o l i p i d s are major b u i l d i n g b l o c k s o f b i o l o g i c a l membranes. bilayer-forming illustrated.  phospholipid,  l-palmitoyl-2-oleoyl  T h e structure o f a  phosphatidylcholine  (POPC),  is  A s a result o f their a m p h i p a t h i c nature, l i p i d s i n a n aqueous e n v i r o n m e n t orient  w i t h their a c y l chains ( R 2 and R 3 ) s h i e l d e d f r o m the water, whereas the polar head g r o u p is directed t o w a r d s the aqueous m e d i u m . group,  glycerol  backbone  and  T h e p o l a r head g r o u p consists o f the  c h o l i n e , the  Rl  substituent.  phosphate  Liposomes o f  defined  characteristics c a n be prepared w i t h v a r i o u s substitutions at groups R l , R 2 and R 3 .  For  example, s h o w n are polar head g r o u p s serine, ethanolamine, and g l y c e r o l that can be substituted to obtain p h o s p h o l i p i d s w i t h different charges and headgroup sizes.  40  41  1.4.2 L i p o s o m e preparation and characterization A  variety o f different  methods generate systems  techniques  have been used to prepare l i p o s o m e s .  d i f f e r i n g i n size and  lamellarity.  These  The classical method  preparing l i p o s o m e s was first described o v e r 30 years ago ( B a n g h a m et al,  1965).  of The  procedure i n v o l v e s the h y d r a t i o n and d i s p e r s i o n o f a d r i e d l i p i d f i l m i n water resulting i n spontaneous v e s i c u l a t i o n .  T h e l i p o s o m e p o p u l a t i o n generated b y this technique is u s u a l l y  heterogeneous i n size (1 u m - 2 0 u m ) ( F i g u r e 1.10) and consists o f v e s i c l e s w i t h several concentric lamellae.  These systems are c a l l e d large m u l t i l a m e l l a r v e s i c l e s ( M L V ) .  Other  methods o f l i p o s o m e preparation have been r e v i e w e d b y S z o k a and Papahadjopoulos (1980). S o m e o f these procedures  i n v o l v e d i s s o l v i n g l i p i d i n organic solvent and then  injecting or infusing the sample into aqueous buffer.  slowly  R e v e r s e phase evaporation has also  been used and i n v o l v e s m i x i n g l i p i d d i s s o l v e d i n o r g a n i c solvent w i t h aqueous  buffer  f o l l o w e d b y r e m o v a l o f the solvent under partial v a c u u m t o f o r m a t h i c k g e l o f hydrated l i p i d w h i c h is then diluted to generate large u n i l a m e l l a r v e s i c l e s ( L U V ) (0.1 um-1 um).  Smaller  u n i l a m e l l a r v e s i c l e s o f 20-50 n m c a n be prepared b y s o n i c a t i o n o f M L V ( H u a n g , 1969). A l t h o u g h the above-mentioned procedures are suitable for generating l i p o s o m e s , the v e s i c l e s are often heterogeneous i n terms o f size, l a m e l l a r i t y and internal trapped v o l u m e s w i t h i n the sample  population.  Vesicle  characteristics,  particularly  size,  lamellarity  and  lipid  c o m p o s i t i o n p l a y an important role i n the f u n c t i o n o f l i p o s o m e s as d r u g d e l i v e r y systems (Ostro and C u l l i s , 1989 and C u l l i s et ai, 1989) and i m m u n o a d j u v a n t s ( A l v i n g , 1987), as w i l l be discussed later.  L i p o s o m e s w i t h h i g h trapped v o l u m e s can be prepared f r o m hydrated  M L V b y subjecting these v e s i c l e s to f i v e c y c l e s o f f r e e z i n g a n d t h a w i n g , w h i c h repeatedly breaks and reforms l i p i d b i l a y e r s y i e l d i n g v e s i c l e s w i t h h o m o g e n e o u s  solute distributions  42  Figure 1.10: Structure of lipid model membranes. T h e structure o f m u l t i l a m e l l a r v e s i c l e s ( M L V ) , c o m p o s e d o f t w o o r m o r e c o n c e n t r i c l a m e l l a e i n an " o n i o n - l i k e " structure, electron  micrograph  (B).  A  is illustrated s c h e m a t i c a l l y i n ( A ) and i n a schematic  representation  and  a  freeze-fracture  freeze-fracture  electron  m i c r o g r a p h d e p i c t i n g the single b i l a y e r structure o f large u n i l a m e l l a r v e s i c l e s ( L U V ) are s h o w n i n ( C ) and ( D ) , respectively. F o r the electron m i c r o g r a p h s , the bar s h o w n represents 2 0 0 n m and a r r o w indicates the d i r e c t i o n o f s h a d o w i n g ( T a k e n from M a d d e n , 1997).  43  and higher trapped v o l u m e s and efficiencies ( M a y e r et al, 1985). H i g h t r a p p i n g efficiencies are required, for example, for o p t i m a l d r u g l o a d i n g i n l i p o s o m a l d r u g d e l i v e r y ( C u l l i s et al, 1989).  systems  F r o z e n and t h a w e d v e s i c l e s ( F A T M L V ) c a n then be size-reduced b y a  rapid extrusion procedure i n w h i c h the F A T M L V are passed ten times t h r o u g h t w o stacked polycarbonate filters o f defined pore size ( H o p e et al,  1985). E x t r u s i o n o f v e s i c l e s t h r o u g h a  100 n m pore size filter has been s h o w n to generate a homogeneous u n i l a m e l l a r v e s i c l e o f defined size (about 90 n m ) ( M a y e r et al, For  protein  incorporation  into  liposomes,  the  p o p u l a t i o n o f large  1986).  freeze-thaw  method  would  be  inappropriate, as the temperature extremes m a y result i n denaturation o f the protein and alterations i n the functional properties o f the reconstituted protein ( M a d d e n et al, Lynch  et  al,  1984).  Similarly,  the  use  o f organic  solvents  i n the  1983 and  preparation  proteoliposomes w o u l d l i k e l y result i n protein p r e c i p i t a t i o n and denaturation.  of  In addition,  due to the v a r i a b l e l a m e l l a r i t y o f M L V , m u c h o f the protein w o u l d l i k e l y be sequestered w i t h i n the inner lamellae and therefore these v e s i c l e s w o u l d not be suitable for e x a m i n i n g the function o f i n t r i n s i c or m e m b r a n e - s p a n n i n g  proteins ( N i c h o l l s et al,  1980).  Furthermore,  Shek and colleagues (1983) s h o w e d that protein entrapped i n L U V e l i c i t e d a greater antibody response than presentation  that  generated  w i t h M L V , i n d i c a t i n g that there w a s  o f protein i n L U V .  Therefore,  a non-denaturing  procedure  a better  surface  is required  for  protein reconstitution into u n i l a m e l l a r l i p o s o m e s . Detergent d i a l y s i s is a c o m m o n technique e m p l o y e d i n the functionally  i n c o r p o r a t i o n o f protein  active state ( M a d d e n et al,  into l i p o s o m e s 1983).  ( M i m m s et al,  Typically,  lipid  1981)  and protein are  in a co-  s o l u b i l i z e d i n detergent, w h i c h is then s l o w l y r e m o v e d b y passive d i a l y s i s against aqueous buffer ( F i g u r e 1.11).  A s the detergent concentration is decreased b e l o w its c r i t i c a l m i c e l l a r  44  Figure 1.11: Protein reconstitution into liposomes. T h e process o f detergent d i a l y s i s d u r i n g p r o t e i n reconstitution into l i p o s o m e s is illustrated. P r o t e i n s o l u b i l i z e d i n detergent is m i x e d w i t h l i p i d i n detergent and then the detergent is slowly removed by dialysis. m i c e l l a r concentration  A s the detergent concentration is l o w e r e d b e l o w the c r i t i c a l  (CMC),  lipid  incorporated i n the l i p i d membrane.  m o l e c u l e s rearrange to f o r m b i l a y e r s w i t h protein  46 concentration, the l i p i d spontaneously vesiculates w i t h l i p o s o m a l p r o t e i n either inserted into the b i l a y e r , trapped w i t h i n the aqueous c o r e o r attached to the l i p o s o m e surface.  Density  gradient centrifugation experiments have s h o w n free protein to migrate i n a peak separate f r o m l i p i d , l i k e l y as denatured protein (Stahn et al, It has  been  1992).  s h o w n that v e s i c l e size can influence the  l i p o s o m e s (Juliano and Stamp, 1975 and S e n i o r et al,  c i r c u l a t i o n lifetimes  of  1985). M L V and S U V , d e p e n d i n g o n  their l i p i d c o m p o s i t i o n , have short h a l f - l i v e s i n c i r c u l a t i o n , whereas L U V generally have longer residence times ( A l l e n et al,  1989).  T h e enhanced clearance rates o f M L V m a y be  due to the greater degree o f interaction between v e s i c l e s and p l a s m a proteins or lipoproteins ( S c h e r p h o f et al,  1978 and F i n k e l s t e i n and W e i s s m a n , 1979). F o r S U V , the b i l a y e r has a  v e r y s m a l l radius o f curvature and the l i p i d s experience a greater degree o f stress, p r o d u c i n g p a c k i n g defects i n the l i p i d b i l a y e r ( C u l l i s and H o p e , 1991).  T h e b i l a y e r defects m a k e the  vesicles unstable and l e a k y and thus susceptible to the insertion or b i n d i n g o f lipoproteins, and apoproteins, facilitating their r a p i d clearance times w o u l d be o f particular importance  for the  from  circulation.  Enhanced circulation  d e l i v e r y o f chemotherapeutic  agents;  however, for a l i p o s o m a l subunit v a c c i n e , clearance o f the v e s i c l e s to the r e t i c u l o e n d o t h e l i a l system ( R E S ) w o u l d enhance antigen d e l i v e r y and u p t a k e b y A P C , such as macrophages, cells and dendritic cells.  B  T a r g e t i n g and d e l i v e r y o f l i p o s o m e s to the R E S w o u l d therefore  lead to an enhanced i m m u n e response. T h e l i p i d c o m p o s i t i o n o f l i p o s o m e s c a n influence the characteristics o f the l i p o s o m a l system.  Studies have s h o w n that a d d i t i o n o f cholesterol results i n a less permeable and m o r e  r i g i d membrane.  T h i s results i n reduced leakage from l i p o s o m e s and decreased b i n d i n g o f  high-density l i p o p r o t e i n ( A l l e n ,  1981), w h i c h translates into extended  c i r c u l a t i o n times  47  ( A l l e n , 1989). These aspects have p a r t i c u l a r l y i m p o r t a n c e i n the encapsulation, retention and d e l i v e r y o f chemotherapeutic  agents.  I n c o r p o r a t i o n o f g l y c o l i p i d s into the b i l a y e r l i p i d  m i x t u r e results i n increased c i r c u l a t i o n h a l f - l i v e s , p o s s i b l y b y p r o v i d i n g a steric hindrance preventing a p o l i p o p r o t e i n f r o m g a i n i n g access to and inserting into the l i p i d b i l a y e r ( A l l e n , 1989).  T h e particular characteristics o f l i p i d b i l a y e r s p l a y an important role i n the fate o f  liposomes  in  vivo,  chemotherapeutic  especially  agents.  regard  to  the  pharmacokinetics  of  encapsulated  S i m i l a r l y , the charges o f the l i p i d s c o m p o s i n g the b i l a y e r can  influence the fate o f l i p o s o m e s . macrophages  with  Studies have s h o w n that cationic l i p o s o m e s are taken up b y  more efficiently than neutral o r a n i o n i c l i p o s o m e s ( N a k a n i s h i et al,  1997).  Therefore, the l i p i d c o m p o s i t i o n o f a l i p o s o m a l subunit v a c c i n e s h o u l d be characterized to determine  the effects  o n protein insertion, orientation w i t h i n the  l i p i d b i l a y e r and  the  influence o n antigenicity.  1.4.3 L i p o s o m e s as d r u g d e l i v e r y systems L i p o s o m e s have been w i d e l y used as carrier systems for v a r i o u s  chemotherapeutic  agents, particularly antifungal and antineoplastic drugs ( M e h t a and L o p e z - B e r e s t e i n , 1989 and N o r t h f e l t et al,  1998). E n c a p s u l a t i o n o f drugs i n l i p o s o m e s has been s h o w n to be w e l l  tolerated w i t h increased efficacy and decreased t o x i c i t y c o m p a r e d to free drug. al,  1989, B o m a n et al,  1994 and C h a n g et al,  ( M a y e r et  1997). A n t i - t u m o r agents are often associated  w i t h cardiac t o x i c i t y ; h o w e v e r , encapsulation i n l i p o s o m e s results i n reduced cardiac u p t a k e and cardiac t o x i c i t y ( R a h m a n et al,  1980 and G a b i z o n et al,  1982).  T h e increased d r u g  efficacy is p r o b a b l y due to decreased clearance b y c e l l s o f the R E S , p r o l o n g e d c i r c u l a t i o n life-time and a c c u m u l a t i o n and release at the t u m o r  site.  L i p o s o m a l encapsulation  of  48  therapeutic agents appears to be an effective a p p r o a c h to therapy and m a y have advantages c o m p a r e d to c o n v e n t i o n a l methods o f chemotherapy.  These studies also demonstrate that  l i p o s o m e s are effective carrier systems that are safe as carriers i n a potential subunit v a c c i n e .  1.4.4 L i p o s o m e s as i m m u n o l o g i c a l adjuvants A d j u v a n t s are thought to f u n c t i o n b y t w o possible m e c h a n i s m s ( A l l i s o n and B y a r s , 1986).  O n e m e c h a n i s m is that the adjuvant creates a depot at the injection site and retards  antigen clearance ( A n t i m i s i a r i s et al, duration  o f release  and  1993).  interaction w i t h  R e d u c e d l o c a l clearance thus p r o l o n g s the  antigen  presenting  cells ( A P C ) .  The  local  i n f l a m m a t i o n generated b y f o r m u l a t i o n s c o n t a i n i n g a l u m or F A m a y result i n the m i g r a t i o n and infiltration o f A P C to the site o f i n o c u l a t i o n , hence f a c i l i t a t i n g an i m m u n e response.  A  second m e c h a n i s m o f a c t i o n is the a c t i v a t i o n o f macrophages, a subset o f the A P C . F o r example, the presence  o f l i p o p o l y s a c c h a r i d e ( L P S ) or m u r a m y l dipeptide ( M D P ) i n the  adjuvant preparation can stimulate macrophages to release i n t e r l e u k i n 1 (EL-1) ( G r e g o r i a d i s , 1990).  T h e c o m b i n a t i o n o f antigen and EL-1 stimulates T cells to p r o d u c e E L - 2 and other  c y t o k i n e factors, w h i c h trigger c e l l - m e d i a t e d ( C M I ) or h u m o r a l i m m u n i t y ( H I ) . V a c c i n a t i o n b y d e l i v e r i n g antigens i n l i p o s o m e s may be advantageous  over other  preparations due to the targeting and uptake o f l i p o s o m e s b y macrophages, dendritic c e l l s and B cells w h i c h are major sites i n the p a t h w a y s o f i m m u n o l o g i c a l antigen p r o c e s s i n g (Unanue, 1984).  Investigators have also s h o w n that macrophages are a major subset o f  antigen-presenting c e l l s capable o f T H 1 c e l l a c t i v a t i o n ( B r e w e r et al, macrophages  resulted  i n the  depletion  suppression o f I g G 2 a antibodies.  of TH1  cell-associated  1994)  cytokines  Depletion o f as  well  as  A s m e n t i o n e d earlier, the l o c a t i o n o f antigen p r o c e s s i n g  49  dictates the type antigen presentation o n the A P C surface  B r i e f l y , endogenous peptides are  presented o n M H C class I m o l e c u l e s , whereas e x o g e n o u s antigens are associated w i t h M H C class II surface molecules. antigen  processing  I m m u n i z a t i o n w i t h s o l u b l e antigens alone results i n exogenous  through  the  endosomal  compartment  and  therefore  insufficient  presentation o n M H C class I m o l e c u l e s and p o o r i n d u c t i o n o f C T L ( H a r d i n g et al,  1991).  H o w e v e r , antigen d e l i v e r y i n l i p o s o m e s t o A P C leads to phagocytosis o f l i p o s o m a l antigen and processing to the t r a n s - G o l g i w h e r e antigen is b o u n d to M H C class I m o l e c u l e s resulting i n a C T L response ( R e d d y et al,  1992, N a i r et al,  1992 and R a o et al,  1999). T h e results o f  these studies indicate that l i p o s o m e s m a y be efficient adjuvants for e l i c i t i n g a c e l l - m e d i a t e d i m m u n e response for protection against intracellular pathogens. M o r g a n and c o w o r k e r s (1984)  s h o w e d that an E p t e i n - B a r r antigen i n c o m p l e t e  F r e u n d ' s adjuvant ( C F A ) i n d u c e d o n l y a w e a k and d e l a y e d response, whereas l i p o s o m a l l y encapsulated antigen e l i c i t e d higher and p r o l o n g e d a n t i b o d y titers.  Studies w i t h a c l o n e d ,  synthetic, m a l a r i a c i r c u m s p o r o z o i t e ( C S ) peptide has been s h o w n to be n o n - i m m u n o g e n i c o n its o w n , but c o u l d be rendered h i g h l y a n t i g e n i c b y i n c o r p o r a t i o n i n t o l i p o s o m e s ( A l v i n g et al.,  1986).  Furthermore,  the  anti-peptide  antibodies  generated  by immunization w i t h  l i p o s o m a l - C S peptide reacted w e l l w i t h intact ( l i v e and k i l l e d ) sporozoite organisms. importantly, antibody titers i n i m m u n i z e d rabbits r e m a i n e d duration f o l l o w i n g i m m u n i z a t i o n ( A l v i n g et al,  elevated  over a  1986 and A l v i n g , 1987).  More  prolonged  I n a d d i t i o n to  e l i c i t i n g protective h u m o r a l i m m u n i t y , a l i p o s o m a l - C S peptide v a c c i n e has also been s h o w n to induce c y t o l y t i c T l y m p h o c y t e ( C T L ) responses w h i c h are c o n s i d e r e d to be important i n i n d u c i n g protective i m m u n i t y ( W h i t e et al,  1993).  50 P r e l i m i n a r y research evaluated and c o m p a r e d the antigenic properties o f g o n o c o c c a l protein I either g i v e n b y i t s e l f or c o m b i n e d w i t h v a r i o u s adjuvants ( W e t z l e r et al, W e t z l e r et al,  1989a, W e t z l e r et al,  1991 and W e t z l e r et al,  1992).  1988,  These studies s h o w e d  that protein I w a s capable o f e l i c i t i n g antibodies i n a rabbit i m m u n i z a t i o n m o d e l either g i v e n alone, i n a f o r m u l a t i o n w i t h F r e u n d ' s liposomes. formulations,  A l t h o u g h antibodies observations  adjuvant,  were  adsorbed  generated  i n d i c a t e d that F r e u n d ' s  to a l u m or incorporated  against the adjuvant  antigen  and  elicited the highest level o f surface reactive, b a c t e r i c i d a l antibodies. from  proteoliposome-immunized  by  liposome  each  of  into the  preparations  I n addition, antisera  rabbits w e r e far superior to protein I - a l u m sera i n the  agglutination and o p s o n o p h a g o c y t o s i s o f w h o l e g o n o c o c c a l o r g a n i s m s ( W e t z l e r et al,  1988).  T h e results o f these studies and those o f W h i t e and c o w o r k e r s (1993) suggest that l i p o s o m e s are effective adjuvants i n e l i c i t i n g b o t h h u m o r a l and c e l l - m e d i a t e d i m m u n i t y and are w e l l tolerated and have f e w adverse effects.  A s p e c t s such as these m a k e l i p o s o m e s  attractive  alternatives to a l u m for a potential g o n o c o c c a l subunit v a c c i n e . T h e rise i n the n u m b e r o f antibiotic-resistant strains and the e n o r m o u s cost associated w i t h the treatment o f g o n o r r h e a and its c o m p l i c a t i o n s have p r o m p t e d research into methods o f disease prevention, n a m e l y the development o f a v a c c i n e against the g o n o c o c c a l o r g a n i s m . P r e l i m i n a r y research b y W e t z l e r and c o w o r k e r s (1988) s h o w e d that a l i p o s o m a l g o n o c o c c a l subunit  vaccine  exhibited  immunogenic  activity  in  the  rabbit  immunization  model.  H o w e v e r , the l i p o s o m a l f o r m u l a t i o n needs to be further characterized w i t h regard to the protein i n c o r p o r a t i o n and orientation, l i p i d c o m p o s i t i o n and detergent r e m o v a l d u r i n g the reconstitution process.  I n addition, the a c t i v i t y o f the p r o t e o l i p o s o m e f o r m u l a t i o n i n e l i c i t i n g  h u m o r a l and c e l l - m e d i a t e d i m m u n i t y needs to be evaluated.  T h e research presented i n this  51 thesis deals w i t h the d e v e l o p m e n t and c h a r a c t e r i z a t i o n o f a subunit v a c c i n e against Neisseria gonorrhoeae.  52  1.5 RESEARCH HYPOTHESES  T h e h i s t o r i c a l basis o f v a c c i n a t i o n has b e e n to use organisms, w h i c h are therefore  non-pathogenic,  to  k i l l e d o r attenuated  elicit protective  immune  whole  responses.  H o w e v e r , these preparations c o n t a i n a large n u m b e r o f antigens that m a y g i v e rise to adverse reactions to the v a c c i n e itself.  In order to a v o i d these c o m p l i c a t i o n s , it w o u l d be preferable  to develop a subunit v a c c i n e based o n a single, conserved protein rather than the w h o l e organism.  Unfortunately,  separation  o f proteins  from  m e m b r a n e s renders t h e m  i m m u n o g e n i c or o n l y w e a k l y i m m u n o g e n i c and unable, therefore, i m m u n e response.  to elicit a  non-  protective  A d d i n g an i m m u n o a d j u v a n t to the f o r m u l a t i o n , h o w e v e r , can restore the  antigenicity o f these isolated proteins.  It is p r o p o s e d that i n c o r p o r a t i n g a p u r i f i e d bacterial  antigen into l i p o s o m e s c o u l d restore its native p r o t e i n orientation and, therefore, restore and enhance the antigenicity o f the membrane protein to elicit an effective i m m u n e response.  1.6 SPECIFIC RESEARCH OBJECTIVES  T h e objectives o f the research w e r e to: 1.  Characterize g o n o c o c c a l m e m b r a n e p r o t e i n i n c o r p o r a t i o n into l i p o s o m e s and  the factors i n f l u e n c i n g protein reconstitution.  determine  F o r a c o m p a r i s o n , evaluate the e f f i c i e n c y o f  m e n i n g o c o c c a l outer membrane proteins ( M O M P ) i n c o r p o r a t i o n into l i p o s o m e s . 2. D e t e r m i n e the b i o p h y s i c a l characteristics o f l i p o s o m a l g o n o c o c c a l P o r v a c c i n e i n terms o f protein orientation, v e s i c l e size and v e s i c l e m o r p h o l o g y .  53  3.  Characterize in vitro antibody b i n d i n g a c t i v i t y t o reconstituted P o r p r o t e o l i p o s o m e as a  function o f l i p i d c o m p o s i t i o n . 4. D e t e r m i n e antigenicity o f P o r p r o t e o l i p o s o m e s in vivo u s i n g a m u r i n e m o d e l .  Compare  antibody titers for proteoliposomes and free protein. 5. E v a l u a t e the nature o f the a n t i b o d y response i n a m u r i n e m o d e l , h u m o r a l o r c e l l - m e d i a t e d , based o n i m m u n o g l o b u l i n serotypes.  54  CHAPTER 2 MATERIALS AND METHODS  2.1. L i p i d s , c h e m i c a l s and reagents l-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine  (POPC)  (MW  760.1)  was  purchased from N o r t h e r n L i p i d s Inc., V a n c o u v e r , B . C . 1-Palmitoyl, 2 - o l e o y l - s n - g l y c e r o - 3 p h o s p h o e t h a n o l a m i n e ( P O P E ) ( M W 718), 1-Palmitoyl,  2-oleoyl-sn-glycero-3-phosphoserine  ( P O P S ) ( M W 784) and 1-Palmitoyl, 2 - o l e o y l - s n - g l y c e r o - 3 - p h o s p h o g l y c e r o l 771)  were  purchased  dimethylammonium Pharmaceuticals,  from  Avanti Polar  chloride  (DODAC)  Vancouver, B . C .  Lipids, (MW  Alabaster, 582.5)  was  N-Octyl-P-D-glucopyranoside  hydroxyethyl]piperazine-N'-[2-ethanesulfonic  AL.  (POPG) ( M W  N,N-dioleyl-N,N-  obtained  from  INEX  (OGP), H E P E S , (N-[2-  acid]) ( M W 238.3), c h o l i c a c i d ( 3 a , 7 a ,  12a-  T r i h y d r o x y - 5 p - c h o l a n - 2 4 - o i c acid) ( M W 430.6) ( s o d i u m salt), b o v i n e serum a l b u m i n ( B S A ) and o - p h e n y l e n e d i a m i n e  ( 1 , 2 - b e n z e n e d i a m i n e ) d i h y d r o c h l o r i d e ( M W 181.1) w e r e obtained  f r o m S i g m a C h e m i c a l C o . , St. L o u i s , M O . was  purchased  from  [ C]-N-Octyl-P-D-glucopyranoside 14  American Radiolabeled  Chemicals,  dipalmitoyl-[2-palmitoyl-9,10- H(N)]-phosphatidylcholine 3  w e r e obtained  from Dupont,  Boston, M A .  Inc.,  St.  ( C-OGP) 1 4  Louis, M O .  L-a-  ( H - D P P C ) and [ H ] - c h o l i c a c i d 3  3  T h e e n z y m e s used, a - c h y m o t r y p s i n  (EC.  3.4.21.1) and trypsin ( E C . 3.4.21.4), w e r e purchased from C a l b i o c h e m , L a J o l l a , C A . F i c o l l 4 0 0 w a s obtained f r o m P h a r m a c i a , U p p s a l a , Sweden.  T h e reagents for the protein assay  w e r e purchased from Pierce, R o c k f o r d , I L . T h e reagents used i n the s o d i u m d o d e c y l sulfate polyacrylamide  gel  electrophoresis  and  S M - 2 Biobeads  were  obtained  from  Biorad,  H e r c u l e s , C A . T h e reagents for the B C A protein assay and N H S - L C - B i o t i n w e r e purchased  55  f r o m P i e r c e , R o c k f o r d , D L L . T h e peroxidase-conjugated streptavidin, goat anti-mouse I g G , rabbit  anti-mouse  IgG  and  mouse  anti-rabbit  IgG  were  obtained  from  Rockland,  G i l b e r t s v i l l e , P A . T h e g o n o c o c c a l m e m b r a n e p r o t e i n I B ( P o r ) preparations used i n this study w e r e i n purified f o r m (free o f R m p and L O S ) ( W e t z l e r et al,  1989b) and w e r e s u p p l i e d b y  D r . M i l a n S. B l a k e , R o c k e f e l l e r U n i v e r s i t y , N e w Y o r k , N Y t h r o u g h D y n C o r p P R I . T w o P o r samples w e r e used i n the course o f these studies: A s o l u t i o n o f P o r (4 m g / m l ) i n 0.1 M T r i s H C l , 0.2 M N a C l , 10 m M E D T A , 0 . 0 5 % Z 3 , 1 4 , 0 . 0 2 % A z i d e at p H 8.0 and a l y o p h i l i z e d sample prepared made under G o o d M a n u f a c t u r i n g P r a c t i c e s ( G M P ) .  The meningococcal  outer m e m b r a n e proteins c o n t a i n i n g d e t o x i f i e d l i p o o l i g o s a c c h a r i d e (2:3 w t . / v o l . ) ( M O M P ) and E m p i g e n B B ( N - d o d e c y l - N , N - d i m e t h y l g l y c i n e )  ( M W 272) were  supplied by D r .  W e n d e l l Z o l l i n g e r , W a l t e r R e e d A r m y Institute, W a s h i n g t o n , D C .  2.2. R e c o n s t i t u t i o n o f g o n o c o c c a l p r o t e i n I ( P o r ) into l i p o s o m e s 2.2.1 R e c o n s t i t u t i o n o f s o l u b l e P o r P o r protein w a s reconstituted at different p r o t e i n - t o - l i p i d ratios f r o m O G P o r cholate to determine the effect o f protein c o n c e n t r a t i o n o n protein i n c o r p o r a t i o n efficiency.  In  addition, the k i n e t i c s o f detergent r e m o v a l w a s m o n i t o r e d d u r i n g the reconstitution process. P o r protein w a s d i l u t e d to 0.5 m g / m l o r 1.0 m g / m l p r o t e i n i n either 4 0 0 m M O G P , 2 0 m M H E P E S p H 7.4 or 2 0 0 m M s o d i u m cholate, 2 0 m M H E P E S p H 7.4. These solutions (3 m l ) w e r e then added to 150 m g o f P O P C and the p h o s p h o l i p i d w a s d i s s o l v e d b y gentle v o r t e x i n g at 2 5 ° C . T h e r a d i o l a b e l [ H ] - D P P C (0.03 p C i / m g p h o s p h o l i p i d ) w a s i n c l u d e d as a 3  l i p i d marker. A l i q u o t s (1 m l ) o f the O G P solutions w e r e set aside as the 0 h o u r d i a l y s i s t i m e point o r w e r e transferred to d i a l y s i s t u b i n g and d i a l y z e d for either 2 0 hours or 125 hours at  56  4 ° C against 500 v o l u m e s o f 150 m M N a C l , 2 0 m M H E P E S , p H 7.4. E x t e r n a l buffer changes w e r e made at 20 and 50 hours.  I n some experiments, r e m o v a l o f O G P w a s f o l l o w e d u s i n g  [ C ] - O G P (2 p C i / m l ) . D u r i n g d i a l y s i s , samples w e r e taken from the d i a l y s i s tubes after 0, 1, 1 4  2, 4, 8, 16, 24 and 36 hours o f d i a l y s i s .  S i m i l a r l y , a l i q u o t s (1 m l ) o f the c h o l a t e - c o n t a i n i n g  sample w e r e set aside as the 0 h o u r d i a l y s i s t i m e p o i n t and w e r e transferred to d i a l y s i s t u b i n g and d i a l y z e d for either 60 hours or 125 hours at 4 ° C against 500 v o l u m e s o f 150 m M N a C l , 20 m M H E P E S , p H 7.4.  E x t e r n a l buffer w a s c h a n g e d at 20, 50 and 100 hours. In some  cholate-mediated reconstitution experiments, the r a d i o l a b e l [ H ] - c h o l i c a c i d (3.3 p C i / m l ) w a s 3  i n c l u d e d i n the reconstitution mixture. D u r i n g d i a l y s i s , a l i q u o t s w e r e taken from the d i a l y s i s tubes at 0, 2.5, 5, 10, 20, 30, 50, 7 0 , 1 3 0 and 150 h o u r time points. R e s i d u a l O G P and cholate concentrations w e r e determined based o n s c i n t i l l a t i o n c o u n t i n g o f [ C ] - O G P and [ H ] - c h o l i c I 4  3  acid, respectively, i n a B e c k m a n L S 3801 instrument ( F u l l e r t o n , C A ) .  2.2.2 R e c o n s t i t u t i o n o f l y o p h i l i z e d P o r L y o p h i l i z e d P o r protein w a s s o l u b i l i z e d i n 4 0 0 m M O G P , 2 0 m M H E P E S , p H 7.4 b y gentle h o m o g e n i z a t i o n u s i n g a glass/teflon h o m o g e n i z e r .  S o m e batches o f p u r i f i e d P o r  contained s m a l l quantities o f i n s o l u b l e material w h i c h w a s r e m o v e d b y first centrifuging the sample at 2 0 0 0 r p m o n a B a x t e r M e g a f u g e 1.0 (Heraeus Instruments) for 3 minutes and then passing the  supernatant t h r o u g h a 0.2 m i c r o n c e l l u l o s e acetate filter ( M i c r o  Systems) to y i e l d a clear solution.  Filtration  T h e protein c o n c e n t r a t i o n w a s adjusted to 1 m g / m l and  aliquots (1 m l ) o f this s o l u t i o n w e r e then added to either 50 m g P O P C or 50 m g P O P C P O P E (1:1 w e i g h t ratio).  T h e p h o s p h o l i p i d s w e r e d i s s o l v e d b y gentle v o r t e x i n g at 2 5 ° C and the  samples w e r e then p l a c e d i n S p e c t r a P o r II d i a l y s i s t u b i n g (6.4 m m diameter) and d i a l y z e d at  57  4 ° C against 500 v o l u m e s o f 150 m M N a C l , 20 m M H E P E S , p H 7.4 for 36 hours w i t h one change  of  external  buffer  at  16  hours.  The  radiolabel  [ H ] - D P P C (0.03 3  uCi/mg  p h o s p h o l i p i d ) w a s i n c l u d e d as a p h o s p h o l i p i d marker.  2.2.3 R e c o n s t i t u t i o n o f P o r into a n i o n i c and c a t i o n i c p r o t e o l i p o s o m e s T o assess the effect o f l i p i d species and charge o n protein i n c o r p o r a t i o n efficiency, Por  protein w a s  reconstituted  into l i p o s o m e s  o f varying lipid  content.  Briefly,  dry  p h o s p h o l i p i d , P O P C , was w e i g h e d w i t h 5, 10 or 2 5 % (by w t . ) o f P O P G or P O P S i n a total o f 50 m g l i p i d .  M i x t u r e s o f P O P C and 5, 10 or 2 5 % (by wt.) (50 m g total l i p i d ) D O D A C  (23.28 m g / m l ) i n benzene:methanol  (95:5 v / v ) w e r e prepared  by co-lyophilization from  benzene:methanol (95:5 v / v ) ( V i r t i s , G a r d i n e r , N Y ) under h i g h v a c u u m (60-100 mtorr) for a m i n i m u m o f 5 hours.  A s o l u t i o n o f 4 0 0 m M O G P , 20 m M H E P E S at p H 7.4 w a s added to  the dry l i p i d mixtures.  T h e r a d i o l a b e l [ H ] - D P P C (0.03 u C i / m g p h o s p h o l i p i d ) w a s i n c l u d e d  as a l i p i d marker.  3  L i p i d s w e r e s o l u b i l i z e d b y gentle v o r t e x i n g and s o n i c a t i o n at 2 5 ° C .  Por  protein f r o m the stock solution (4 m g / m l ) w a s added to the l i p i d m i x t u r e to g i v e p r o t e i n and l i p i d concentrations  o f 1.0 m g / m l and 50 m g / m l , respectively, i n 4 0 0 m M O G P , 20 m M  H E P E S p H 7.4. F o r control vesicles, H B S , p H 7.4 w a s added to the detergent/lipid m i x t u r e instead o f P o r protein.  E a c h sample w a s transferred to SpectraPor d i a l y s i s t u b i n g (6.4 m m  diameter) and d i a l y z e d for 125 hours at 4 ° C against 500 v o l u m e s o f 150 m M N a C l , 20 m M H E P E S , p H 7.4 w i t h external buffer changes at 20 and 50 hours.  F o l l o w i n g dialysis,  reconstituted samples w e r e a n a l y z e d b y i s o p y c n i c density gradient centrifugation, Q E L S size analysis, protease digestion and c r y o - e l e c t r o n m i c r o s c o p y , as described b e l o w .  58  2.3 R e c o n s t i t u t i o n o f m e n i n g o c o c c a l proteins ( M O M P ) M e n i n g o c o c c a l proteins w e r e reconstituted f r o m O G P and E m p i g e n B B to determine the rate o f detergent r e m o v a l and e f f i c i e n c y o f p r o t e i n i n c o r p o r a t i o n . observed  that E m p i g e n  reconstitution.  B B alone  was  insufficient  Therefore, proteoliposomes  to  dissolve  w e r e reconstituted  H o w e v e r , it w a s  phospholipid  from  prior  to  a mixture o f sodium  cholate and E m p i g e n B B , as described b e l o w . M O M P (0.5 m g / m l ) i n 4 0 0 m M O G P , 20 m M H E P E S , p H 7.4 w a s prepared and added (2 m l ) to 50 m g P O P C . detergent marker.  T h e r a d i o l a b e l [ C ] - O G P (2 p C i / m l ) w a s i n c l u d e d as a 1 4  T h e p h o s p h o l i p i d w a s d i s s o l v e d b y gentle v o r t e x i n g at 2 5 ° C and samples  w e r e then placed i n S p e c t r a P o r II d i a l y s i s t u b i n g (6.4 m m diameter) and d i a l y z e d for 140 hours at 4 ° C against 500 v o l u m e s o f 150 m M N a C l , 20 m M H E P E S , p H 7.4 i n the presence or absence o f S M - 2 B i o b e a d s (2 g).  E x t e r n a l buffer changes w e r e made at 20, 50 and 116  hours. D u r i n g d i a l y s i s , aliquots w e r e taken from the d i a l y s i s tubes at 0, 2.5, 5, 10, 20, 30, 50, 116 and 140 hour t i m e points. M O M P (0.5 m g / m l ) i n 2 0 0 m M s o d i u m cholate, 2 0 m M H E P E S , p H 7.4 w i t h 5.6% E m p i g e n B B w a s prepared and added to dry P O P C  (50 m g / m l ) .  The phospholipid  was  d i s s o l v e d b y gentle v o r t e x i n g at 2 5 ° C and the samples w e r e then p l a c e d i n d i a l y s i s t u b i n g and d i a l y z e d for 12 days at 4 ° C against 500 v o l u m e s o f 150 m M N a C l , 20 m M H E P E S , p H 7.4 w i t h external buffer changes at 36, 82, 142, 214 and 2 5 0 hours.  F o l l o w i n g dialysis,  reconstituted samples w e r e a n a l y z e d b y i s o p y c n i c density gradient centrifugation and Q E L S size analysis.  59  2.4 A n a l y t i c a l procedures 2.4.1 I s o p y c n i c density gradient centrifugation A c o n t i n u o u s F i c o l l gradient w a s prepared ( 0 - 1 0 % F i c o l l ) i n 150 m M N a C l , 2 0 m M , p H 7.4 u s i n g a G r a d i e n t M a k e r ( H o e f e r S c i e n t i f i c Instruments, S a n F r a n c i s c o , C A ) . T h e reconstituted p r o t e o l i p o s o m e s ( 5 0 0  u l ) w e r e l o a d e d o n the  centrifuged  T i swinging bucket  in a B e c k m a n  ultracentrifuge at 1 1 0 , 0 0 0 g  S W 41  av  gradient w h i c h w a s  rotor o n a B e c k m a n  then  L2-65B  for 2 4 hours at 4 ° C . T h e gradients w e r e then fractionated into  500 u l fractions (see F i g u r e L e g e n d s for details) a n d a n a l y z e d for protein and l i p i d content. T h e density gradient profiles w e r e representative o f at least t w o separate experiments.  2.4.2 P r o t e i n and p h o s p h o l i p i d quantitation P h o s p h o l i p i d concentrations and specific a c t i v i t i e s w e r e determined b y a s s a y i n g l i p i d phosphorus content ( F i s k e and S u b b a r o w , 1925) and b y l i q u i d s c i n t i l l a t i o n c o u n t i n g o f the samples o n a B e c k m a n L S 3801 l i q u i d s c i n t i l l a t i o n counter P r o t e i n concentrations w e r e determined b y a m o d i f i e d P i e r c e B C A p r o t e i n assay for microtiter plates (Pierce, R o c k f o r d , I L ) . A 50 u l a l i q u o t o f each standard, b l a n k o r d i l u t e d u n k n o w n sample w a s pipetted into the appropriate m i c r o t i t e r plate w e l l s .  T h e n , 50 u l o f  0 . 5 % S D S w a s added, f o l l o w e d b y the a d d i t i o n o f 100 u l o f w o r k i n g reagent to each w e l l . T h e m i c r o t i t e r plates w e r e incubated at 3 7 ° C for 2 hours.  A b s o r b a n c e w a s measured at 540  n m o n a B i o t e k 96 w e l l m i c r o t i t e r plate reader ( B i o T e k Instruments, W i n o o s k i , V T ) . P r o t e i n concentrations i n the samples w e r e determined f r o m a b o v i n e serum a l b u m i n ( B S A ) standard curve.  60  2.5 Protease d i g e s t i o n T h e s u s c e p t i b i l i t y o f reconstituted P o r protein to p r o t e o l y t i c cleavage b y trypsin  or a - c h y m o t r y p s i n w a s  determined  for the  f o l l o w i n g i s o p y c n i c density gradient centrifugation.  proteoliposome  fractions  either  obtained  In the case o f reconstituted  systems  prepared w i t h either P O P C or P O P C P O P E (1:1), aliquots (25 p i c o n t a i n i n g 17.5 p g protein, POPC  systems  o r 30 p g protein, P O P C : P O P E  systems)  w e r e treated w i t h  c h y m o t r y p s i n (0.5 p g ) o r t r y p s i n (0.2 p g ) at 37 ° C for 15 minutes.  either  a-  Following enzyme  digestion, the p r o t e o l i p o s o m e s w e r e subjected to a d e l i p i d a t i o n procedure (see b e l o w ) p r i o r to S D S - p o l y a c r y l a m i d e gel electrophoresis.  2.6 S D S - p o l y a c r y l a m i d e gel electrophoresis ( S D S - P A G E ) P r i o r to S D S - P A G E , reconstituted p r o t e o l i p o s o m e s w e r e first delipidated.  T o each  sample, 4 0 0 p i methanol, 2 0 0 p i c h l o r o f o r m and 3 0 0 p i d i s t i l l e d water w e r e added.  Samples  were v o r t e x e d and centrifuged at 2 0 0 0 r p m for 10 minutes.  T h e top layer above the p r o t e i n  interface was carefully r e m o v e d and discarded. A n a d d i t i o n a l 3 0 0 p i o f m e t h a n o l w a s added to each sample f o l l o w e d b y v o r t e x i n g and centrifugation at 2 5 0 0 r p m for 10 minutes. supernatant was r e m o v e d and discarded.  The  T h e p r o t e i n pellets w e r e then d r i e d under n i t r o g e n  gas and resuspended i n 25 p i o f 1% S D S . S D S - p o l y a c r y l a m i d e gel electrophoresis w a s undertaken buffer system ( L a e m m l i ,  u s i n g the  discontinuous  1970) e m p l o y i n g a M i n i P r o t e a n I f D u a l S l a b C e l l  ( B i o r a d ) w i t h a 1 9 . 5 % ( w / v ) separating g e l and a 4 . 5 % ( w / v ) s t a c k i n g g e l .  apparatus  Samples were  dissociated i n 0.062 M T r i s / H C l buffer, p H 6.8 c o n t a i n i n g 2 % ( w / v ) S D S , 1 0 % ( w / v ) sucrose, 5 % ( v / v ) 2-f3-mercaptoethanol and 0 . 0 0 1 % ( w / v ) b r o m o p h e n o l b l u e at 9 5 ° C for 4 minutes.  61  Samples w e r e  l o a d e d o n the  stacking g e l a l o n g w i t h m o l e c u l a r w e i g h t standards and  electrophoresis w a s conducted at a constant voltage o f 80 V t h r o u g h the s t a c k i n g g e l and 130 V t h r o u g h the r e s o l v i n g g e l u n t i l the b r o m o p h e n o l b l u e t r a c k i n g d y e w a s a p p r o x i m a t e l y 5 m m f r o m the g e l bottom.  Proteins w e r e detected b y 0 . 1 % C o o m a s s i e B l u e stain i n f i x a t i v e  ( 4 0 % methanol, 1 0 % acetic acid) and destained w i t h 4 0 % methanol, 1 0 % acetic acid.  Gels  w e r e then silver stained u s i n g the B i o r a d silver s t a i n i n g kit w i t h o u t m o d i f i c a t i o n .  2.7 G e l scanning densitometry T o quantitate proteolytic digestion, S D S gels w e r e scanned u s i n g the L K B - B r o m m a Ultrascan X L Laser Densitometer.  T h e relative amounts o f each peptide  band  were  determined b y w e i g h i n g peaks f r o m the densitometer scan p r o f i l e and c a l c u l a t i n g percentage relative to the total peak w e i g h t s for that lane.  2.8 Size r e d u c t i o n o f reconstituted p r o t e o l i p o s o m e s b y e x t r u s i o n F o l l o w i n g reconstitution, p r o t e o l i p o s o m e s procedure ( H o p e et al,  1985 and M a y e r et al,  were  1986a).  size-reduced  u s i n g an  extrusion  B r i e f l y , reconstituted systems w e r e  placed i n an E x t r u d e r ( L i p e x B i o m e m b r a n e s , V a n c o u v e r , B . C . , Canada) and extruded  10  times t h r o u g h t w o (stacked) polycarbonate filters (Costar, C a m b r i d g e , M A ) o f defined pore size ( 1 0 0 - 6 0 0 n m ) under n i t r o g e n pressures o f 100-400 psi at 3 7 ° C .  F o l l o w i n g extrusion,  v e s i c l e size distributions w e r e determined u s i n g quasi-elastic l i g h t scattering ( Q E L S ) . I n addition, samples w e r e a n a l y z e d for protein and l i p i d concentration.  62  2.9 Quasi-elastic light scattering ( Q E L S ) R e c o n s t i t u t e d systems w e r e a n a l y z e d to determine v e s i c l e size distributions b y q u a s i elastic light scattering ( Q E L S ) analysis u s i n g a N i c o m p M o d e l 2 7 0 S u b m i c r o n P a r t i c l e S i z e r as described p r e v i o u s l y ( K o l c h e n s et al, 1993).  2.10 C r y o - t r a n s m i s s i o n electron m i c r o s c o p y ( c r y o - T E M ) Reconstituted electron m i c r o s c o p y .  vesicles were  a n a l y z e d u s i n g the technique  o f cryo-transmission  B r i e f l y , sample f i l m s w e r e prepared i n a c u s t o m - b u i l t e n v i r o n m e n t a l  chamber under c o n t r o l l e d temperature ( 2 5 ° C ) and h u m i d i t y c o n d i t i o n s . T h e films w e r e then v i t r i f i e d b y r a p i d freezing i n l i q u i d ethane and transferred t o a Z e i s s E M 9 0 2 t r a n s m i s s i o n electron m i c r o s c o p e for analysis. T h e specimens w e r e kept b e l o w 108 ° K d u r i n g the transfer and v i e w i n g procedures t o prevent sample perturbation and ice formation.  The microscope  w a s operated i n zero-loss, b r i g h t - f i e l d m o d e and at an accelerating voltage o f 80 k V ( B e l l a r e etal, 1988 and D u b o c h e t etal, 1988).  2.11 A n t i b o d y b i n d i n g e v a l u a t i o n 2.11.1 S a m p l e preparation for antigenicity tests T h e antigenic properties o f five reconstituted P o r p r o t e o l i p o s o m e f o r m u l a t i o n s w e r e c o m p a r e d u s i n g a n antibody b i n d i n g assay.  I n this experiment, t w o protein batches w e r e  e x a m i n e d ( G M P and M S l l j e A r m p ) and these w e r e reconstituted w i t h either P O P C alone o r P O P C P O P E (1:1) (as described above). reduced b y extrusion as described above.  F o l l o w i n g reconstitution, these samples w e r e s i z e A fifth sample contained P O P C P O P E (1:4) and  was prepared w i t h P o r MSIIJBA/TM/J a c c o r d i n g to a procedure described p r e v i o u s l y ( W e t z l e r  63  et al,  1988, W e t z l e r et al,  1992). B r i e f l y , the d r y l i p i d w a s s o l u b i l i z e d i n a s o l u t i o n  c o n t a i n i n g MS11JBA/TM£> (2 m g / m l ) i n 4 0 0 m M O G P , 2 0 m M H E P E S , p H 7.4 and then d i a l y z e d against 500 v o l u m e s o f 150 m M N a C l , 2 0 m M H E P E S , p H 7.4 at 4 ° C for 36 hours with  changes o f external buffer after 10 and 2 0 hours.  F o l l o w i n g d i a l y s i s , the sample w a s  sonicated i n a water bath at 3 7 ° C for 2 0 minutes to p r o d u c e s m a l l u n i l a m e l l a r v e s i c l e s .  2.11.2 P o r i n m o n o c l o n a l antibodies T h e m o n o c l o n a l antibodies ( M A b ) u t i l i z e d characterized  u s i n g p u r i f i e d p o r i n s ( W e t z l e r et  i n these studies  al,  were  1988) f r o m v a r i o u s  produced  and  strains.  The  h y b r i d o m a cells p r o d u c i n g these M A b s w e r e c l o n e d b y l i m i t i n g d i l u t i o n and a c c l i m a t i z e d for g r o w t h i n serumless H y b r i m a x m e d i a ( B R I , B a l t i m o r e , M D ) .  The M A b s were purified  using a column packed with H A - U l t r a m a x  ( S i g m a C h e m i c a l C o . , St. L o u i s , M O . ) as  p r e v i o u s l y described (Stanker et al,  T h e a n t i b o d y fractions w e r e p o o l e d and the  1985).  antibodies precipitated b y the a d d i t i o n o f an equal v o l u m e o f 3 0 % ( w t . / v o l . ) p o l y e t h y l e n e glycol  8000  centrifugation  (JT.  Baker,  Phillipsburg,  NJ).  at 3 0 , 0 0 0 g for 2 0 minutes, the  The  precipitates  were  collected  supernatant discarded and the  by  antibodies  resuspended i n P B S . T h e p u r i f i e d M A b s w e r e stored at 4 ° C until used.  2.11.3 E L I S A and i n h i b i t i o n E L I S A assays M i c r o t i t e r plates ( N u n c - I m m u n o P l a t e I I F , V a n g a r d International, N e p t u n e , N . J . ) w e r e sensitized b y a d d i n g 0.1 m l per w e l l o f the p u r i f i e d p o r i n from the strain M S I ljeAr/wp, 2.0 p g / m l i n 0.1 s o d i u m bicarbonate buffer, p H 9.6.  T h e plates w e r e incubated o v e r n i g h t at  r o o m temperature. T h e plates w e r e w a s h e d five times w i t h 0 . 9 % N a C l , 0 . 0 5 % T w e e n 2 0 , 10  64  mM  s o d i u m acetate, p H 7.0, 0 . 0 2 %  s o d i u m azide.  The Neisserial porin monoclonal  antibodies were d i l u t e d i n P B S and added to the plate and incubated for 2 hours at r o o m temperature.  T h e plates w e r e again w a s h e d as before and the secondary antibody, a l k a l i n e  phosphatase-conjugated  goat anti-mouse I g G and I g M ( T a g o Inc., B u r l i n g a m e , C A ) , w a s  d i l u t e d i n P B S / 0 . 5 % T w e e n 20, added to the plates and incubated for 1 h o u r at r o o m temperature. Phosphatase  T h e plates w e r e w a s h e d Substrate  as before  and / ? - n i t r o p h e n y l phosphate ( S i g m a  104) (1 m g / m l ) i n 0.1 M diethanolamine, 1 m M M g C ^ , 0.1 m M  ZnCl2, 0 . 0 2 % s o d i u m azide, p H 9.8 w a s added.  T h e plates w e r e  incubated at  room  temperature for 1 h o u r and the absorbance at 4 0 5 n m w a s determined u s i n g an E L 3 1 1 s x A u t o m a t e d M i c r o p l a t e R e a d e r ( B i o - T e k Instruments, Inc., W i n o o s k i , V T ) . C o n t r o l w e l l s l a c k e d either the p r i m a r y and/or secondary antibody. T h i s w a s done to obtain a titre for each m o n o c l o n a l antibody w h i c h w o u l d g i v e a h a l f m a x i m a l r e a d i n g i n the E L I S A assay.  This  titer for each p o r i n m o n o c l o n a l antibody w a s then used i n an i n h i b i t i o n E L I S A .  The  microtiter plate w a s sensitized and w a s h e d as before.  L i p o s o m e s c o n t a i n i n g the i n d i c a t e d  amount o f p o r i n s (measured i n | i g protein), as w e l l as w h o l e bacteria, w e r e added to a separate V - b o t t o m e d m i c r o t i t e r plate ( N u n c - I m m u n o P l a t e 9 6 V , V a n g a r d N e p t u n e , N . J . ) and d i l u t e d i n P B S  International,  A n e q u a l v o l u m e o f e a c h o f the p o r i n m o n o c l o n a l  antibodies, d i l u t e d i n P B S , w a s added to the w e l l s s u c h that the final concentration o f the antibody represented their s p e c i f i c " h a l f m a x i m a l " titers. 150 u.1.  T h e total v o l u m e i n each w e l l w a s  T h e plates w e r e incubated for 2 hours o n a h o r i z o n t a l p l a t f o r m shaker.  The  microtiter plates w e r e then centrifuged at 3 0 0 0 r p m for 5 minutes i n a R C 5 Superspeed refrigerated centrifuge ( D u p o n t Instruments, W i l m i n g t o n , D E ) u s i n g a S H - 3 0 0 0 rotor w i t h m i c r o p l a t e carriers. A n aliquot (100 u l ) f r o m each w e l l o f the V - b o t t o m m i c r o t i t e r plate w a s  65  transferred to the pre-washed p o r i n - s e n s i t i z e d m i c r o t i t e r plate.  T h i s plate w a s incubated for  2 hours, w a s h e d and the conjugated second a n t i b o d y added as stated.  T h e plate w a s then  processed and read as described. T h e i n h i b i t i o n w a s c a l c u l a t e d as f o l l o w s : 1 - ( E L I S A v a l u e after absorption) ( E L I S A v a l u e o f unabsorbed a n t i b o d y )  E a c h o f the assays w a s done i n t r i p l i c a t e o n different days.  T h e i n h i b i t i o n values represent  the m e a n o f these assays.  2.12 A n t i b o d y b i n d i n g a c t i v i t y o f a n i o n i c and c a t i o n i c P o r p r o t e o l i p o s o m e s 2.12.1 B i o t i n y l a t i o n o f goat anti-mouse I g G In order to measure a n t i b o d y b i n d i n g , b i o t i n w a s conjugated to the detecting a n t i b o d y as described b e l o w .  G o a t anti-mouse I g G (5 m g ) w a s resuspended i n 100 u l P B S .  The  antibody was then passed d o w n 1 m l S e p h a d e x G - 5 0 s p i n c o l u m n s hydrated i n P B S that had been pre-spun at 1000 r p m for 3 minutes i n a L a b o f u g e 4 0 0 table top centrifuge (Heraeus, Germany).  F o l l o w i n g centrifugation, 1 u l o f freshly prepared N F f S - L C - b i o t i n (156 m M i n  d i m e t h y l sulfoxide) w a s added to the a n t i b o d y m i x t u r e and incubated for 3 0 minutes at ambient temperature.  T h e sample w a s then centrifuged d o w n pre-spun 1 m l S e p h a d e x G - 5 0  spin c o l u m n s hydrated i n P B S . T h e final sample w a s d i l u t e d 3-fold and stored at 4 ° C .  2.12.2 E L I S A a n t i b o d y b i n d i n g assays M i c r o t i t e r plates ( N u n c - I m m u n o Plate, D e n m a r k ) w e r e sensitized b y a d d i n g  0.1  m l / w e l l o f the purified P o r protein f r o m the strain M S l l j B A r m p , 2 u g / m l i n 0.1 M s o d i u m bicarbonate buffer, p H 9.6.  T h e plates w e r e incubated o v e r n i g h t at 4 ° C .  Meanwhile,  66  reconstituted samples w e r e d i l u t e d i n H B S / B S A a n d aliquots between 0-150 u l pipetted into E p p e n d o r f tubes and the total v o l u m e w a s made up to 150 u l w i t h H B S / B S A .  Neisserial  p o r i n m o n o c l o n a l a n t i b o d y w a s d i l u t e d i n H B S / B S A and 0.1 m l w a s added to each tube. samples w e r e gently v o r t e x e d and i n c u b a t e d overnight at 4 ° C .  The  A f t e r 24 hours, sensitized  plates w e r e w a s h e d once w i t h P B S and then w e r e b l o c k e d w i t h b l o c k i n g buffer ( P B S / B S A ) for 30 minutes at r o o m temperature.  T h e b l o c k i n g buffer w a s r e m o v e d and 0.1 m l o f each  p r o t e o l i p o s o m e / M A b i n c u b a t i o n m i x t u r e w a s plated ( i n duplicate).  A 100 u l a l i q u o t o f  H B S / B S A w a s added to each w e l l to m a k e the total v o l u m e to 2 0 0 u l . Plates w e r e incubated overnight at 4 ° C . F o l l o w i n g the p r o t e o l i p o s o m e p l a t i n g , plates w e r e w a s h e d w i t h P B S and then w e r e b l o c k e d w i t h P B S / B S A f o l l o w e d b y the a d d i t i o n o f 2 0 0 u l / w e l l o f a 1/1000 d i l u t i o n o f b i o t i n y l a t e d goat anti-mouse I g G and incubated for 1 hour at ambient temperature. F o l l o w i n g reaction w i t h b i o t i n - c o n j u g a t e d i m m u n o g l o b u l i n , plates w e r e w a s h e d and b l o c k e d and  then 200 u l / w e l l o f a 1/10000 d i l u t i o n o f streptavidin horse r a d i s h peroxidase w e r e  added to the plates and incubated for 60 minutes at ambient temperature. three  times  with  PBS  and  blocked  with  blocking  buffer  and  Plates w e r e w a s h e d 0.2  ml/well  o f o-  p h e n y l e n e d i a m i n e i n 100 m M N a C l , 50 m M citrate, p H 5.0 w a s added. C o l o r w a s d e v e l o p e d and reaction w a s terminated w i t h the a d d i t i o n o f 50 u l / w e l l o f 2 M H C 1 . T h e absorbance w a s measured at 4 9 0 n m o n a 96 w e l l m i c r o t i t e r plate reader ( B i o T e k Instruments, VT).  Winooski,  67 2.13 7/7 vivo antigenicity of Por proteoliposomes 2.13.1 Immunization B A L B / c mice (females; 8 weeks old) were immunized intraperitoneally (200 pi) and intradermally (30 ul) with neutral, anionic and cationic proteoliposome formulations at an antigen dose of 1 pg Por. Injections were made at 0, 3 and 7 weeks and blood was collected via the tail vein at 2, 5 and 9 weeks. Samples were centrifiiged and sera were collected and analyzed by E L I S A assays (see below).  2.13.2 E L I S A antibody titer assays Microtiter plates (Nunc-Immuno Plate, Denmark) were sensitized by adding 0.1 ml/well of the purified Por protein from the strain M S I IJBAT/W/?, 2 pg/ml in 0.1 M sodium bicarbonate buffer, pH 9.6. The plates were incubated overnight at 4 °C. After 24 hours, sensitized plates were washed once with PBS and then were blocked with blocking buffer (PBS/BSA) for 30 minutes at room temperature.  After the plates were blocked, sera from  immunized mice were serially diluted in H B S / B S A and added to Por-sensitized plates and incubated overnight at 4°C. After the incubation, plates were washed with PBS and then were blocked with PBS/BSA followed by the addition of 200 pl/well of a 1/1000 dilution of biotinylated goat anti-mouse IgG and incubated for 1 hour at ambient temperature. Following reaction with biotin-conjugated antibody, plates were washed and blocked and then 200 pl/well of a 1/10000 dilution of streptavidin horse radish peroxidase were added to the plates and incubated for 60 minutes at ambient temperature. Plates were washed three times with PBS and blocked with blocking buffer and 0.2 ml/well of o-phenylenediamine in 100 m M NaCl, 50 m M citrate, pH 5.0 was added. Color was developed and the reaction was  68  terminated w i t h the a d d i t i o n o f 50 u l / w e l l o f 2 M H C 1 .  T h e absorbance w a s measured at 4 9 0  n m o n a 96 w e l l m i c r o t i t e r plate reader.  2.13.3 E L I S A a n t i b o d y i s o t y p i n g assays M i c r o t i t e r plates ( N u n c - I m m u n o Plate, D e n m a r k ) w e r e sensitized b y a d d i n g 0.1 m l / w e l l o f the purified P o r protein f r o m the strain MS11JBA/7W£>, 2 u g / m l i n 0.1 M s o d i u m bicarbonate buffer, p H 9.6.  T h e plates w e r e incubated o v e r n i g h t at 4 ° C .  A f t e r 24 hours,  sensitized plates w e r e w a s h e d once w i t h P B S and then w e r e b l o c k e d w i t h b l o c k i n g buffer ( P B S / B S A ) for 30 minutes at r o o m temperature. A f t e r the plates w e r e b l o c k e d , sera w e r e diluted and plated o n P o r - s e n s i t i z e d plates and incubated o v e r n i g h t at 4 ° C .  Plates w e r e  w a s h e d w i t h P B S and w e r e then b l o c k e d w i t h P B S / B S A f o l l o w e d b y 1 h o u r i n c u b a t i o n w i t h 200  u l / w e l l o f rabbit anti-mouse I g G ( s p e c i f i c for isotypes G I and G 2 a ) . T h i s reaction w a s  f o l l o w e d b y a 1 h o u r i n c u b a t i o n w i t h 2 0 0 u l / w e l l o f 1/1000 d i l u t i o n o f b i o t i n y l a t e d m o u s e anti-rabbit I g G . Plate incubations w e r e p e r f o r m e d at r o o m temperature.  F o l l o w i n g reaction  w i t h biotin-conjugated i m m u n o g l o b u l i n , a l l plates w e r e w a s h e d and b l o c k e d as before and then 200 u l / w e l l o f a 1/10000 d i l u t i o n o f streptavidin horse r a d i s h p e r o x i d a s e w e r e added to the plates and incubated for 6 0 minutes at ambient temperature.  Plates w e r e w a s h e d three  times w i t h P B S and b l o c k e d w i t h b l o c k i n g buffer and 0.2 m l / w e l l o f o - p h e n y l e n e d i a m i n e i n 100 m M N a C l , 50 m M citrate, p H 5.0 w a s added.  C o l o r w a s d e v e l o p e d and r e a c t i o n w a s  terminated w i t h the a d d i t i o n o f 50 u l / w e l l o f 2 M H C 1 . n m o n a 96 w e l l m i c r o t i t e r plate reader.  T h e absorbance w a s measured at 4 9 0  69 2.14 T i s s u e h i s t o l o g y M i c e s h o w i n g side effects due to the P o r p r o t e o l i p o s o m e s e x h i b i t e d i n f l a m m a t i o n at the site o f injection. formalin.  Therefore, the tissue at the injection site w a s e x c i s e d and f i x e d w i t h  W h e n the tissue had been fixed, the fixative w a s w a s h e d out and the tissue w a s  then dehydrated  and embedded  i n paraffin.  T h e hardened  paraffin w a s m o u n t e d o n a  m i c r o t o m e and cut into five m i c r o n slices. T h e thin sections w e r e then p l a c e d o n slides and paraffin w a s r e m o v e d f r o m the tissue w i t h x y l e n e . W h e n the paraffin w a s d i s s o l v e d out, the tissue  was  dehydrated.  re-hydrated,  stained  with  h e m a t o x y l i n and  T h e sample w a s permanently  fixed  eosin  ( H & E stain)  and  again  i n p l a c e w i t h a m o u n t i n g m e d i u m and  c o v e r e d w i t h a c o v e r s l i p . T h e tissue samples w e r e then v i e w e d i n n o r m a l bright field m o d e with a Zeiss A x i o p h o t microscope (West Germany).  2.15 Statistical methods T h e statistical s i g n i f i c a n c e o f the a n t i b o d y titer data w a s determined b y a single factor A N O V A followed by a  post hoc Scheffe's test for m u l t i p l e c o m p a r i s o n s .  isotype data w e r e a n a l y z e d w i t h the Student considered to be statistically significant.  Mest.  The antibody  A P value o f less than 0.05  was  70  CHAPTER 3 FACTORS INFLUENCING PROTEIN INCORPORATION INTO LIPOSOMES  F a c t o r s i n f l u e n c i n g protein i n c o r p o r a t i o n into l i p o s o m e s are addressed i n this chapter. Comparison  was  made  o f protein  reconstitution  efficiency  d u r i n g the  preparation  of  proteoliposomes f r o m a m i x t u r e o f m e m b r a n e proteins ( M O M P ) or f r o m a single protein (Por).  A d d i t i o n a l l y , the rate o f detergent r e m o v a l and protein i n c o r p o r a t i o n e f f i c i e n c y w e r e  e x a m i n e d for different detergents. F i n a l l y , the effects o f p r o t e i n - t o - l i p i d ratio, reconstitution t i m e and protein species o n i n c o r p o r a t i o n e f f i c i e n c y are outlined.  C h a r a c t e r i z a t i o n o f the  proteoliposomes is c o v e r e d i n C h a p t e r 4.  3.1 INTRODUCTION T h e technique o f protein reconstitution has been useful i n s t u d y i n g v a r i o u s membrane proteins.  Studies have investigated the functions o f m e m b r a n e receptor proteins such as  b e n z o d i a z e p i n e and i n s u l i n receptors b y s o l u b i l i z a t i o n and subsequent i n c o r p o r a t i o n into artificial l i p i d b i l a y e r s ( A n h o l t et al, various  membrane-bound  enzymes  1986 and T r a n u m - J e n s e n et al, have  been  reconstituted  into  1994).  In addition,  liposomes with  good  r e c o v e r y o f e n z y m e a c t i v i t y ( A n h o l t , 1988 and D r i e s s e n and W i c k n e r , 1990). B y i s o l a t i o n o f i n d i v i d u a l proteins into planar b i l a y e r s , the structure, f u n c t i o n and orientation o f specific proteins i n their native, m e m b r a n e - b o u n d state can be determined. Isolation  and  reconstitution  reconstitution o f antigens  o f individual  membrane  i n potential v a c c i n e preparations.  proteins  is useful  in  the  Conventional vaccines  use  attentuated or k i l l e d w h o l e organisms that m a y cause m i l d to severe reactions ( L a u t e r i a et  al,  71  1974, B a r k i n and P i c h i c h e r o , 1979 and R o i t t et al, 1996).  T h i s p r o b l e m has p r o m p t e d the  development o f subunit a n d peptide v a c c i n e s that are based o n single, conserved  antigens.  U n f o r t u n a t e l y , these v a c c i n e s are often p o o r i m m u n o g e n s f o r p r o v i d i n g protective i m m u n i t y i n the absence o f an adjuvant ( R i c h a r d s et al, 1996 and W e t z l e r et al, 1992). T h e use o f l i p o s o m e s as potential i m m u n o a d j u v a n t s extensively  reviewed  incorporation  (Alving,  o f purified  1987, G r e g o r i a d i s ,  antigens  i m m u n o g e n i c i t y o f the antigen.  into  liposomes  a n d v a c c i n e carriers has been  1990 a n d A l v i n g , has been  shown  1991).  The  to restore the  L o c a l i z a t i o n o f the antigen into the l i p o s o m a l m e m b r a n e  a l l o w s f o r surface presentation o f antigen to B cells f o r i n d u c t i o n o f h u m o r a l i m m u n i t y a n d to antigen presenting cells f o r c e l l - m e d i a t e d i m m u n i t y . L i p o s o m e s have the a b i l i t y t o elicit b o t h a c e l l u l a r - m e d i a t e d i m m u n e response ( G a r c o n a n d S i x , 1991) a n d a h u m o r a l i m m u n e response  (Therien  et al,  1990).  Studies  have  shown  liposomes  immunopotentiators i n hepatitis A (Just et al, 1992 a n d G l i i c k et al,  t o be  effective  1992) a n d i n f l u e n z a  v a c c i n e s ( G l i i c k , 1992 and Stahn et al, 1992). I n addition, l i p o s o m e s have adjuvant a c t i v i t y i n v a c c i n e s against p r o t o z o a n ( W h i t e et al, 1993) and bacterial o r g a n i s m s ( M u t t i l a i n e n et al, 1995). In a d d i t i o n to Neisseria gonorrhoeae, another N e i s s e r i a l o r g a n i s m  for w h i c h a  v a c c i n e is currently b e i n g sought is Neisseria meningitidis. M e n i n g i t i s ( m e n i n g o c o c c a l ) disease arises  from  various serogroups  a n d varies f r o m country t o country.  p r o p o r t i o n o f this disease is caused b y strains A , B , C , Y a n d W 1 3 5 .  Highly  A large effective  capsular p o l y s a c c h a r i d e v a c c i n e s against strains A , C , Y a n d W 1 3 5 have been d e v e l o p e d . Unfortunately,  group  nonimmunogenic.  B capsular  polysaccharide  vaccines  are f o u n d  T h e fact that serogroup B , the p r e d o m i n a n t  to be essentially  cause o f m e n i n g o c o c c a l  72  disease i n m a n y temperate countries, lacks p o l y s a c c h a r i d e i m m u n o g e n i c i t y has p r o m p t e d the research and d e v e l o p m e n t o f alternative v a c c i n e s based o n m e m b r a n e proteins.  Studies have  s h o w n adjuvant-protein v a c c i n e s to have m a r k e d l y i m p r o v e d antibody responses ( W a n g and F r a s c h , 1984 and F r a s c h et al,  1987).  F u r t h e r m o r e , proteosome-based v a c c i n e s have been  s h o w n to be i m m u n o g e n i c ( R u e g g et al., 1990) and outer m e m b r a n e v a c c i n e s c a n increase i m m u n i t y to g r o u p B m e n i n g o c o c c i ( Z o l l i n g e r et al, Z o l l i n g e r and M o r a n , 1991).  1987, R o s e n q v i s t et al,  1988 and  B a s e d o n these studies, the reconstitution o f a m i x t u r e o f  m e n i n g o c o c c a l membrane proteins into l i p o s o m e s w a s characterized. T h e d e v e l o p m e n t o f a potential l i p o s o m e v a c c i n e requires that the c o n d i t i o n s for o p t i m a l protein i n c o r p o r a t i o n be determined.  Therefore, the research i n the present chapter  deals w i t h the characterization o f the process o f detergent-mediated  protein reconstitution  and addresses the factors i n f l u e n c i n g protein i n c o r p o r a t i o n into l i p o s o m e s . I n particular, the kinetics  of  detergent  removal  and  residual  p r o t e o l i p o s o m e sample must be evaluated.  detergent  levels  in  the  reconstituted  T h e detergent concentration i n the sample has  relevance w i t h regard to sample stability and v a c c i n e safety.  I n addition, the characterization  o f protein i n c o r p o r a t i o n d u r i n g d i a l y s i s and the effect o f p r o t e i n - t o - l i p i d ratio are essential for o p t i m i z i n g the c o n d i t i o n s for efficient protein i n c o r p o r a t i o n .  T h e protein i n c o r p o r a t i o n  efficiencies d u r i n g the preparation o f p r o t e o l i p o s o m e s f r o m a m i x t u r e o f membrane proteins ( M O M P ) and a single protein ( P o r ) were e x a m i n e d .  73  3.2 R E S U L T S  3.2.1 Detergent r e m o v a l d u r i n g reconstitution o f P o r protein T h e n o n - i o n i c surfactant o c t y l g l u c o p y r a n o s i d e ( O G P ) w a s selected for these studies because it is reported to be a r e l a t i v e l y m i l d , non-denaturing detergent and has a r e l a t i v e l y h i g h c r i t i c a l m i c e l l a r concentration ( C M C ) o f 21 m M ( G o u l d et al,  1981).  T h e rate o f  r e m o v a l o f O G P (initial concentration 4 0 0 m M ) w a s m o n i t o r e d b y a s s a y i n g for [ C ] - O G P I 4  d u r i n g d i a l y s i s . A s s h o w n i n F i g u r e 3.1, detergent r e m o v a l is r a p i d , p a r t i c u l a r l y d u r i n g the first 16 hours, w h i c h l i k e l y represents the r e m o v a l o f m o n o m e r i c or m i c e l l a r detergent. W h e n P o r p r o t e o l i p o s o m e f o r m a t i o n has o c c u r r e d , it is l i k e l y that subsequent O G P r e m o v a l f r o m the v e s i c l e s w i l l be constrained b y the rate o f O G P " f l i p - f l o p " f r o m the inner m o n o l a y e r to the outer m o n o l a y e r .  A f t e r 36 hours o f d i a l y s i s , h o w e v e r , O G P r e m a i n i n g i n the P O P C  and P O P C P O P E systems w a s 1.5% and 3 . 3 % o f the starting concentration, respectively. T h i s corresponds to 6.0 m M and 13.3 m M O G P , o r 9 % and 2 0 % relative to the l i p i d concentration, respectively. These concentrations are w e l l b e l o w the C M C o f 21 m M . R e m o v a l o f the i o n i c surfactant, cholate, d u r i n g P o r protein reconstitution w a s also determined.  Interestingly, cholate r e m o v a l w a s m u c h s l o w e r than the r e m o v a l o f O G P  ( F i g u r e 3.2). O n l y after about 50 hours o f d i a l y s i s is the r e s i d u a l cholate concentration at o r b e l o w the C M C , w h i c h is reported to be 13-16 m M ( C h a t t o p a d h y a y and L o n d o n , 1984 and Z h a n g et al,  1996).  After  140 hours o f d i a l y s i s , r e s i d u a l cholate i n the reconstitution  m i x t u r e w a s 1.3% o f initial, w h i c h corresponds to 2.6 m M , w e l l b e l o w its C M C . O G P levels are w e l l b e l o w the C M C after o n l y 20 hours o f d i a l y s i s .  I n contrast,  74  0.45  Time (hours)  Figure 3.1: N - O c t y l - ( 3 - D - g l u c o p y r a n o s i d e m o n i t o r e d b y assaying for  1 4  levels  during  dialysis.  C - O G P d u r i n g the d i a l y s i s procedure.  levels for P O P C proteoliposomes ( • ) and P O P C : P O P E  Residual  OGP  was  S h o w n are detergent  proteoliposomes(T).  75  Figure 3.2:  S o d i u m cholate levels d u r i n g d i a l y s i s .  T h e levels o f s o d i u m cholate  measured b y assaying for [ H ] - c h o l i c a c i d d u r i n g the d i a l y s i s procedure. 3  were  76  3.2.2 Influence o f P o r p r o t e i n / l i p i d ratio o n reconstitution e f f i c i e n c y T h e influence o f p r o t e i n / l i p i d ratio o n e f f i c i e n c y o f protein i n c o r p o r a t i o n into liposomes  during  reconstitution  was  examined.  Proteoliposome  samples  containing  g o n o c o c c a l protein I (Por) and P O P C w e r e prepared f r o m O G P (See section 2.2) at initial p r o t e i n / l i p i d ratios (wt/wt) o f either 0.02 or 0.01.  S a m p l e s taken p r i o r to d i a l y s i s , at b o t h  p r o t e i n / l i p i d ratios, s h o w a b r o a d band i n the top h a l f o f the gradient (Figures 3 . 3 A and 3.4A).  A large p r o p o r t i o n o f the p r o t e i n is seen to migrate to the b o t t o m o f the gradient.  F o l l o w i n g d i a l y s i s for 20 hours, a p r o t e i n / l i p i d b a n d e x t e n d i n g over a r e l a t i v e l y n a r r o w density range (gradient depth 3-5 m l ) is observed.  H o w e v e r , there is still a significant  p r o p o r t i o n o f free P o r protein, w h i c h migrates to the b o t t o m o f the gradient.  T h i s represents  3 0 % and 5 0 % o f total protein for P / L ratios o f 0.01:1 and 0.02:1, respectively. observation indicates protein i n c o r p o r a t i o n is i n c o m p l e t e after 2 0 hours.  This  A f t e r 125 hours o f  dialysis, a further n a r r o w i n g o f the p h o s p h o l i p i d b a n d is seen w i t h essentially a l l o f the P o r protein associated w i t h this l i p i d b a n d at the l o w e r p r o t e i n - t o - l i p i d ratio (0.01:1) ( F i g u r e 3.3C).  It is important to note, h o w e v e r , that at the higher p r o t e i n - t o - l i p i d ratio (0.02:1),  a p p r o x i m a t e l y 1 7 % o f P o r protein remains un-associated w i t h l i p i d and migrates to the b o t t o m o f the gradient (Figure 3 . 4 C ) .  T h i s suggests that protein i n c o r p o r a t i o n is saturable  and l i m i t e d b y the p h o s p h o l i p i d concentration. P r o t e i n reconstitution w a s also characterized u s i n g the i o n i c surfactant,  cholate.  I s o p y c n i c density gradient profiles for P o r / P O P C ( P / L = 0 . 0 1 ) mixtures reconstituted for either 0, 60 or 125 hours from cholate are s h o w n i n F i g u r e 3.5.  T h e i n i t i a l sample (time z e r o )  shows the l i p i d band dispersed over a b r o a d density range w i t h i n the top h a l f o f the gradient,  77  Figure 3.3: O G P - m e d i a t e d P o r reconstitution into P O P C l i p o s o m e s ( P / L = 0 . 0 1 ) . I s o p y c n i c density gradient centrifugation profiles for g o n o c o c c a l P r o t e i n I l i p o s o m e s reconstituted O G P : A ) 0 hours, B ) 20 hours and C ) 125 hours.  4 ° C for 24 hours o n a continuous F i c o l l gradient P h o s p h o l i p i d (open circles).  from  S a m p l e s w e r e centrifuged at 1 1 0 , 0 0 0 g (0-10%).  P r o t e i n (filled  av  at  circles).  0  1 2 3 4 5 6 7 8 9 10 11 12 13 Gradient Depth (ml)  79  F i g u r e 3.4:  O G P - m e d i a t e d P o r reconstitution into P O P C l i p o s o m e s ( P / L = 0 . 0 2 ) .  Isopycnic  density gradient centrifugation profiles for g o n o c o c c a l P r o t e i n I l i p o s o m e s reconstituted O G P : A ) 0 hours, B ) 20 hours and C ) 125 hours. 4 ° C for 24 hours  o n a continuous F i c o l l gradient  P h o s p h o l i p i d (open circles).  from  Samples w e r e centrifiiged at 1 1 0 , 0 0 0 g (0-10%).  P r o t e i n (filled  av  at  circles).  81  Figure 3.5: C h o l a t e - m e d i a t e d P o r reconstitution I s o p y c n i c density  gradient  centrifugation  profiles  into for  POPC  liposomes  gonococcal Protein I  reconstituted f r o m cholate: A ) 0 hours, B ) 60 hours and C ) 125 hours. centrifuged at 1 1 0 , 0 0 0 g  av  (P/L=0.01).  at 4 ° C for 24 hours o n a continuous F i c o l l gradient  P r o t e i n (filled circles). P h o s p h o l i p i d (open circles).  liposomes  Samples w e r e (0-10%).  83  whereas P o r protein is observed to migrate near the b o t t o m o f the gradient.  A f t e r 60 and 125  hours o f dialysis, there are increases, 7 0 % and 7 7 % , respectively, i n p r o t e i n i n c o r p o r a t i o n as the l i p i d and protein are observed to c o - m i g r a t e d o w n the gradient.  H o w e v e r , for this  p r o t e i n / l i p i d ratio there is a significant fraction ( a p p r o x i m a t e l y 2 3 % ) o f u n i n c o r p o r a t e d P o r , i n contrast to proteoliposomes prepared f r o m O G P at the same p r o t e i n / l i p i d ratio ( F i g u r e 3.3).  These  results  indicate  that  Por  protein  incorporation  is  less  efficient  during  reconstitution f r o m cholate.  3.2.3 P r o t e o l i p o s o m e size R e c o n s t i t u t e d systems w e r e e x a m i n e d u s i n g quasi-elastic l i g h t scattering ( Q E L S ) to determine  v e s i c l e size distributions.  analysis  R e c o n s t i t u t i o n f r o m O G P resulted i n  p r o t e o l i p o s o m e s e x h i b i t i n g a r e l a t i v e l y b r o a d size d i s t r i b u t i o n w i t h m e a n v e s i c l e diameter about 317 n m and standard d e v i a t i o n o f 180 n m ( T a b l e 3.1).  It has been reported that  vesicles reconstituted f r o m cholate have m e a n diameters o f about 50 n m , depending o n l i p i d c o m p o s i t i o n ( M a d d e n et al. 1983 and M a d d e n , 1986).  V e s i c l e s o f this size w o u l d be i d e a l  for sterile filtration o f a potential v a c c i n e candidate p r i o r to use i n a c l i n i c a l setting.  It w a s  observed, h o w e v e r , that reconstitution o f P o r p r o t e o l i p o s o m e s f r o m cholate resulted i n systems w i t h mean v e s i c l e diameter greater than 500 n m e x h i b i t i n g a C h i  2  o f 5 and thus  w o u l d have to be size-reduced p r i o r to s t e r i l i z a t i o n b y t e r m i n a l filtration t h r o u g h 0.2 m i c r o n filters.  This C h i  2  v a l u e is an i n d i c a t o r o f the goodness-of-fit  distribution and a c a l c u l a t e d G a u s s i a n d i s t r i b u t i o n .  A Chi  2  b e t w e e n the actual size  v a l u e o f less than 2 w o u l d  indicate g o o d agreement o f the r a w data to a G a u s s i a n fit. T h e observed C h i  2  o f 5, therefore,  suggests that the d i s t r i b u t i o n profile m a y be s k e w e d t o w a r d s larger or smaller v e s i c l e sizes or  84  Reconstitution  M e a n V e s i c l e D i a m e t e r (nm)  Detergent  Standard D e v i a t i o n (nm)  OGP  317  180  Cholate  543  277  OGP/Extruded Vesicles  96  21  Table 3.1: Q E L S Size analysis o f P o r p r o t e o l i p o s o m e s reconstituted f r o m O G P and cholate.  85  a b i m o d a l d i s t r i b u t i o n m a y exist. P o r p r o t e o l i p o s o m e s are further characterized b y C T E M i n the next chapter to e x a m i n e m o r p h o l o g y and size distributions.  3.2.4 R e c o n s t i t u t i o n o f m e n i n g o c o c c a l outer m e m b r a n e protein ( M O M P ) : R e s i d u a l detergent levels. R e c o n s t i t u t i o n experiments w e r e c o n d u c t e d u s i n g t w o different detergent preparations to determine the effect o f detergent properties o n p r o t e o l i p o s o m e formation. T h e surfactant solutions under i n v e s t i g a t i o n w e r e o c t y l g l u c o s i d e and a c o m b i n a t i o n o f s o d i u m cholate and E m p i g e n B B . Differences i n i o n i c character and c r i t i c a l m i c e l l a r concentrations detergents m a y influence m e m b r a n e p r o t e i n i n c o r p o r a t i o n into liposomes.  o f these  T h e effects  of  these detergents o n protein i n c o r p o r a t i o n efficiency and residual detergent levels d u r i n g the dialysis were examined. In previous reconstitution  experiments,  O G P removal during gonococcal  protein  reconstitution was quite r a p i d and residual detergent levels w e r e found to be w e l l b e l o w the critical m i c e l l e concentration.  R e m o v a l o f O G P d u r i n g m e n i n g o c o c c a l protein reconstitution  i n the presence and absence o f S M - 2 B i o b e a d s ( B i o r a d ) w a s m o n i t o r e d b y a s s a y i n g for [ C ] 1 4  O G P during dialysis.  A s s h o w n i n F i g u r e 3.6, detergent r e m o v a l f r o m the  m i x t u r e is r a p i d , p a r t i c u l a r l y d u r i n g the first 15 hours.  reconstitution  It w a s anticipated that B i o b e a d s ,  polystyrene p o l y m e r s , w h e n added to the external buffer, w o u l d b i n d to detergent m o l e c u l e s that have already been r e m o v e d f r o m the reconstitution  mixture.  b i n d i n g to the B i o b e a d s i n the external buffer w o u l d further  Detergent  increase the  molecules  concentration  gradient and lead to an increase i n the rate o f O G P r e m o v a l f r o m the reconstitution mixture. H o w e v e r , the rate o f r e m o v a l w a s s i m i l a r w i t h and w i t h o u t the a d d i t i o n o f B i o b e a d s .  This  86  0  20  40  60  80  100 120 140  Time (hours)  Figure 3.6: O G P levels glucopyranoside(OGP) process.  during  were  MOMP  measured  by  reconstitution. assaying  for  The  levels  [ C]-OGP 1 4  of  N-octyl-f3-D-  d u r i n g the  dialysis  O G P levels i n absence o f S M - 2 B i o b e a d s (open circles) and i n presence o f S M - 2  B i o b e a d s (open triangles).  87  suggests that either the rate l i m i t i n g step i n O G P r e m o v a l w a s not the concentration gradient across the d i a l y s i s m e m b r a n e or that the B i o b e a d s d i d not b i n d O G P to any great extent. W h e n p r o t e o l i p o s o m e f o r m a t i o n has o c c u r r e d , it is l i k e l y that a d d i t i o n a l O G P r e m o v a l from the v e s i c l e s w i l l be determined b y the rate o f O G P exchange from the inner m o n o l a y e r to the outer m o n o l a y e r . A f t e r 140 hours o f d i a l y s i s , O G P r e m a i n i n g i n b o t h samples w a s 0 . 2 5 % o f the initial concentration. a p p r o x i m a t e l y 21 meningococcal  T h i s corresponds to 1 m M O G P w h i c h is w e l l b e l o w the C M C o f  m M (Gould  protein  et  al,  reconstitution  1981).  The  followed  very  kinetics o f O G P removal  during  c l o s e l y the  during  rate observed  g o n o c o c c a l protein reconstitution.  3.2.5 C h a r a c t e r i z a t i o n o f M O M P i n c o r p o r a t i o n d u r i n g reconstitution H a v i n g determined the rates o f r e m o v a l o f O G P , the reconstitution process characterized b y m o n i t o r i n g protein i n c o r p o r a t i o n into l i p o s o m e s as the dialyzed  away.  M e n i n g o c o c c a l outer  membrane  proteins  (MOMP)  detergent  and P O P C  d i s s o l v e d i n O G P and d i a l y z e d for 20 and 125 hours (as described under M e t h o d s ) . aliquot o f the o r i g i n a l sample was also retained. was  then  used  to  monitor  reconstitution process.  vesicle  The initial  formation sample  was was were An  I s o p y c n i c density gradient centrifugation and  protein  (time zero)  p h o s p h o l i p i d i n the top h a l f o f the gradient ( F i g u r e 3.7).  shows  incorporation a broad  Some M O M P  during  the  distribution o f is also f o u n d  associated w i t h the l i p i d band, l i k e l y due to spontaneous v e s i c u l a t i o n w h e n the sample is diluted o n the gradient.  F o r this i n i t i a l sample, the r e m a i n i n g protein is seen to migrate as a  peak near the b o t t o m o f the gradient.  A f t e r 20 or 125 hours o f d i a l y s i s , h o w e v e r , it can be  seen that c o - m i g r a t i o n o f p h o s p h o l i p i d and p r o t e i n o c c u r s to a p o s i t i o n a p p r o x i m a t e l y a t h i r d  88  Figure 3 . 7 :  O G P - m e d i a t e d M O M P reconstitution i n t o P O P C l i p o s o m e s .  I s o p y c n i c density  gradient centrifugation profiles for m e n i n g o c o c c a l O M P l i p o s o m e s reconstituted f r o m O G P : A ) 0 hours, B ) 20 hours and C ) 125 hours.  S a m p l e s w e r e centrifuged at 1 1 0 , 0 0 0 g  av  at 4 ° C  for 2 4 hours o n a continuous F i c o l l gradient ( 0 - 1 0 % ) . P r o t e i n ( f i l l e d c i r c l e s ) . P h o s p h o l i p i d (open circles).  0  1 2 3 4 5 6 7 8 9 10 11 12 13 Gradient Depth (ml)  90  o f the w a y d o w n the gradient. n a r r o w density range.  F u r t h e r m o r e , the p r o t e i n / l i p i d band extends over a r e l a t i v e l y  A p p r o x i m a t e l y 8 0 % o f the protein associated w i t h the l i p i d , w i t h 2 0 %  free protein at the b o t t o m o f the gradient.  T h i s p r o f i l e indicates a h i g h M O M P i n c o r p o r a t i o n  efficiency d u r i n g the reconstitution procedure, but i n c o m p l e t e c o m p a r e d to O G P - m e d i a t e d g o n o c o c c a l protein i n c o r p o r a t i o n at the same p r o t e i n / l i p i d ratio.  3.2.6 E m p i g e n B B - m e d i a t e d M O M P reconstitution into l i p o s o m e s P r o t e i n i s o l a t i o n studies o f m e n i n g o c o c c a l m e m b r a n e proteins have i n d i c a t e d that i n c l u s i o n o f a z w i t t e r i o n i c detergent d u r i n g protein s o l u b i l i z a t i o n and p u r i f i c a t i o n c a n restore the  antibody b i n d i n g capacity o f m e m b r a n e  Therefore,  Empigen  BB,  a  commonly  proteins  used  ( M a n d r e l l and Z o l l i n g e r ,  zwitterionic  detergent,  was  1984).  used  m e n i n g o c o c c a l protein reconstitution experiments and the results w e r e then c o m p a r e d  in to  reconstitution w i t h o c t y l g l u c o s i d e . F o l l o w i n g d i a l y s i s for 12 days, the p r o t e i n - l i p i d m i x t u r e s w e r e observed to be transparent, suggesting that either any v e s i c l e s present w e r e v e r y s m a l l , or that no v e s i c u l a t i o n had o c c u r r e d .  D e n s i t y gradient centrifugation s h o w e d little o r no  protein associated w i t h the p h o s p h o l i p i d b a n d as the p h o s p h o l i p i d c o m p o n e n t w a s observed to migrate i n the top h a l f o f the gradient.  M o s t o f the p r o t e i n w a s seen i n a peak fraction at  the b o t t o m o f the gradient ( F i g u r e 3.8). T h e separation o f the t w o peaks indicates that v e s i c l e f o r m a t i o n d i d not o c c u r during d i a l y s i s ; therefore, protein i n c o r p o r a t i o n c o u l d not o c c u r .  91  Figure gradient  3 . 8 : Empigen BB/cholate-mediated M O M P  centrifugation  Empigen BB/cholate.  profile for  reconstitution.  meningococcal O M P liposomes  S a m p l e as centrifuged at 1 1 0 , 0 0 0 g  av  Isopycnic  density  reconstituted  from  at 4 ° C for 24 hours o n a  continuous F i c o l l gradient (0-10%). P r o t e i n ( f i l l e d c i r c l e s ) . P h o s p h o l i p i d (open c i r c l e s ) .  92  3.3  DISCUSSION  The  subject  of  phospholipid  and  protein  solubilization  and  subsequent  p r o t e o l i p o s o m e reconstitution has been r e v i e w e d e x t e n s i v e l y ( H e l e n i u s and S i m o n s , 1975, A l l e n et al,  1980, L i c h t e n b e r g et al,  1983, H j e l m e l a n d , 1990 and M a d d e n , 1988).  o f detergents and l i p o s o m e s have suggested interaction (Paternostre et al,  a three-stage m o d e l o f  1988 and A n g r a n d et al,  1997).  Studies  detergent-liposome  During  detergent-induced  s o l u b l i z a t i o n , the first stage i n v o l v e s the p a r t i t i o n i n g o f n o n - m i c e l l a r detergent into the liposomal  b i l a y e r resulting i n increased  Stage t w o corresponds  membrane  permeability without solubilization.  to the gradual d i s r u p t i o n o f the b i l a y e r and the  detergent-lipid m i x e d m i c e l l e s and detergent saturation o f the l i p o s o m e s . constitutes the complete micelles.  It  is  s o l u b i l i z a t i o n and c o n v e r s i o n o f l i p o s o m e s into  assumed  that  detergent-mediated  reconstitution  of  emergence o f T h e final stage detergent-lipid liposomes  proteoliposomes f o l l o w s the reverse o f the s o l u b i l i z a t i o n process ( R i g a u d et al, Initially, lipid/detergent and/or lipid/protein/detergent  or  1988).  are present as m i x e d m i c e l l e s .  As  detergent is r e m o v e d , m i x e d m i c e l l e s b e c o m e unstable l e a d i n g to the f o r m a t i o n o f structures c o m p o s e d o f l i p i d s and proteins that eventually f o r m vesicles. intrabilayer detergent is r e m o v e d .  I n the final phase, residual  T h e i n c o r p o r a t i o n o f p r o t e i n into the b i l a y e r is thought to  o c c u r v i a t w o possible m e c h a n i s m s . inserts into these preformed v e s i c l e s .  E i t h e r l i p i d v e s i c l e s are f o r m e d and the protein then A l t e r n a t i v e l y , protein m a y b e c o m e incorporated into  the b i l a y e r d u r i n g the v e s i c u l a t i o n process.  R e c o n s t i t u t i o n studies have i n d i c a t e d that the  presence o f m i x e d m i c e l l e s is required for protein i n c o r p o r a t i o n t o o c c u r ( R i g a u d et 1988).  I n addition, these studies have suggested that for i n i t i a l detergent  al,  concentrations  above the c r i t i c a l m i c e l l a r concentration, c o m p l e t e protein i n c o r p o r a t i o n c o u l d be obtained.  93  Researchers  will  be  determined b y the relative i n i t i a l concentrations o f l i p i d , protein and detergent ( M i m m s et  al,  1981).  have  suggested  that the c o m p o s i t i o n o f r e s u l t i n g p r o t e o l i p o s o m e s  These experiments i n d i c a t e d that i n i t i a l detergent concentrations need to be 15-fold  higher than the C M C for complete s o l u b i l i z a t i o n o f the p h o s p h o l i p i d . F o l l o w i n g detergent dialysis, c o m p l e t e v e s i c u l a t i o n o f the p h o s p h o l i p i d w a s observed.  W h e n the detergent/lipid  m o l a r ratio p r i o r to d i a l y s i s w a s 5:1, the m i n i m u m concentration required for o b t a i n i n g a clear s o l u t i o n , o n l y about 5 0 % o f the l i p i d w a s f o u n d to be i n v e s i c u l a r f o r m , whereas the remainder w a s i n n o n - v e s i c u l a r f o r m ( M i m m s et al.,  1981).  I n i t i a l detergent-to-lipid and  i n i t i a l p r o t e i n - t o - l i p i d ratio may affect the protein i n c o r p o r a t i o n efficiency. T h e present study compares p r o t e i n reconstitution into l i p o s o m e s generated f r o m a n o n - i o n i c or i o n i c detergent. T h e detergents used, o c t y l g l u c o p y r a n o s i d e ( O G P ) and s o d i u m cholate,  have  been  (Paternostre et al,  employed  i n either  1988, R i g a u d et al,  protein  i s o l a t i o n and/or  1988, and A n g r a n d et al,  reconstitution  1997). O G P is a n o n - i o n i c  detergent that has p r e v i o u s l y been used i n the reconstitution o f c y t o c h r o m e - P 4 5 0 proteins ( S c h w a r z et al.,  1984).  studies  membrane  O n e advantage to u s i n g this detergent is that it has a  relatively h i g h c r i t i c a l m i c e l l a r concentration (21 m M ) and c a n be r a p i d l y r e m o v e d d u r i n g the d i a l y s i s procedure ( G o u l d et al,  1981).  A s the present study illustrates, complete P o r  protein i n c o r p o r a t i o n into l i p o s o m e s c a n be a c h i e v e d b y O G P - m e d i a t e d reconstitution, as has been reported for other proteins ( R i g a u d et al,  1988 and A n g r a n d et al,  1997). A n a l y s e s o f  reconstituted proteoliposomes have s h o w n r e s i d u a l detergent levels to be w e l l b e l o w the C M C , levels that are non-membrane l y t i c ( A n g r a n d etal, 1997). S o d i u m cholate, an i o n i c detergent, has also been used i n the reconstitution o f membrane proteins into l i p o s o m e s ( M a d d e n et al,  1983 and R i g a u d et al,  1988).  Studies  94  report that p r o t e o l i p o s o m e s generated b y cholate-mediated reconstitution tend to be smaller i n diameter than v e s i c l e s generated f r o m O G P ( S c h w a r z et al,  1984).  H o w e v e r , it was  observed that P o r - c o n t a i n i n g v e s i c l e s generated f r o m cholate w e r e as large o r larger than vesicles prepared from O G P . I n addition, the rate o f r e m o v a l o f cholate w a s m u c h s l o w e r than that o f O G P .  Furthermore, cholate-mediated reconstitution resulted i n l o w e r p r o t e i n  i n c o r p o r a t i o n efficiencies than for O G P - m e d i a t e d reconstitution.  T h e differences  between  these t w o detergents may be attributed to t w o factors. F i r s t , the a n i o n i c charge o n the cholate m a y be i n v o l v e d i n an electrostatic interaction w i t h protein residues.  T h i s interaction c o u l d  have resulted i n some protein denaturation, thus p r e v e n t i n g c o m p l e t e i n c o r p o r a t i o n o f the g o n o c o c c a l membrane protein.  S e c o n d , the steroid-like structure, l o w e r C M C and negative  charge o n cholate m a y retard or substantially reduce the rate o f d i s s o c i a t i o n o f detergent m o l e c u l e s f r o m the l i p i d / p r o t e i n c o m p l e x e s , resulting i n s l o w e r detergent r e m o v a l .  Studies  suggest that the presence o f p o l a r h y d r o x y l g r o u p s o f cholate i n the h y d r o p h o b i c c o r e o f liposomes  and  its  lower  C M C may  proteoliposomes ( R i g a u d et al,  play  a  role  i n the  time  1988 and L i c h t e n b e r g , 1985).  required  to  generate  S l o w i n g the reconstitution  process m a y f a v o r protein denaturation and/or aggregation and hence reduce the e f f i c i e n c y o f protein  incorporation.  presented  i n this  paper  Analysis indicates  o f the octyl  detergent-mediated g l u c o s i d e to  be  the  reconstitution detergent  experiments  o f choice  p r o t e o l i p o s o m e reconstitution, i n particular, for the i n c o r p o r a t i o n o f bacterial  for  membrane  proteins into a l i p o s o m a l m a t r i x to be used to produce a subunit v a c c i n e . A s mentioned earlier, it has been suggested that the c o m p o s i t i o n and characteristics o f the reconstituted system w i l l be d e t e r m i n e d b y the relative i n i t i a l concentrations o f l i p i d , protein and detergent ( M i m m s et al,  1981). W i t h an initial detergent concentration b e l o w its  95  C M C , o n l y partial protein i n c o r p o r a t i o n w a s attainable ( R i g a u d et al,  1988).  Complete  protein i n c o r p o r a t i o n into l i p o s o m e s w a s a c h i e v e d w h e n i n i t i a l detergent levels w e r e above the C M C  Studies i n v o l v i n g the i n c o r p o r a t i o n o f the c y t o c h r o m e - P 4 5 0 e n z y m e system into  l i p o s o m e s u s i n g O G P have reported the e f f i c i e n c y o f protein i n c o r p o r a t i o n to be dependent o n the initial p r o t e i n - t o - l i p i d ratio ( S c h w a r z et al,  1984). T h i s is consistent w i t h the present  results w h i c h indicate that protein i n c o r p o r a t i o n is saturable for a g i v e n l i p i d concentration. A s observed, initial p r o t e i n - t o - l i p i d ratios above this saturation l i m i t result i n i n c o m p l e t e protein i n c o r p o r a t i o n w i t h consequent  denaturation  and aggregation  of  non-incorporated  protein. F o r a subunit v a c c i n e preparation to be most effective, it w o u l d be advantageous to have m a x i m u m presentation o f the antigenic determinants o n the surface o f the l i p o s o m e . addition, complete antigen i n c o r p o r a t i o n into l i p o s o m e s m a y reduce t o x i c i t y and  In  increase  i m m u n o g e n i c i t y o f the antigen, subsequently y i e l d i n g a v a c c i n e w i t h increased potency and efficacy. OGP-mediated  reconstitution  of  meningococcal  outer  membrane  proteins  was  observed to be efficient w i t h a large p r o p o r t i o n o f protein associated w i t h the p h o s p h o l i p i d as determined b y i s o p y c n i c density gradient centrifugation.  However, M O M P  incorporation  w a s i n c o m p l e t e at the same P / L ratio o f 0.01 w h e r e g o n o c o c c a l P o r protein w a s c o m p l e t e l y incorporated into l i p o s o m e s .  T h i s m a y have been due to heterogeneity  o f the  MOMP  sample, as it consisted o f a m i x t u r e o f proteins r a n g i n g i n m o l e c u l a r w e i g h t f r o m 2 5 - 1 0 0 k D a (data not shown).  A g g r e g a t i o n and denaturation  o f certain protein m o l e c u l e s m a y  have  prevented i n c o r p o r a t i o n o f the a p p r o x i m a t e l y 2 0 % p r o t e i n observed at the b o t t o m o f the gradient  Therefore, a single p r o t e i n w o u l d be preferred  formulation.  i n a potential subunit v a c c i n e  96  In a d d i t i o n to  O G P , another detergent m i x t u r e that w a s  employed  p r o t e o l i p o s o m e reconstitution w a s E m p i g e n B B w i t h s o d i u m cholate.  in  MOMP  E m p i g e n B B is a  r e l a t i v e l y m i l d , z w i t t e r i o n i c detergent that has been c o m m o n l y used i n p r o t e i n s o l u b i l i z a t i o n and p u r i f i c a t i o n studies ( L o w t h e r t et al,  1995 and M u k h l i s et al,  1986).  M a n y detergents  have been used i n protein i s o l a t i o n experiments; h o w e v e r , protein p u r i f i c a t i o n often results i n loss o f the antigenic determinants and thus the antigenicity o f the protein ( C h r i s t i e et 1988).  E m p i g e n B B - e x t r a c t e d antigen preparations  e x h i b i t an a b i l i t y to elicit  greater  antibody responses than preparations extracted w i t h v a r i o u s other detergents (Jennings et 1988 and Jennings and E r t u r k , 1990).  al,  al,  These studies indicate E m p i g e n B B to be m i l d e r and  less denaturing o n isolated proteins a l l o w i n g for the retention o f antigenic activity. B a s e d o n these properties,  meningococcal proteoliposomes  were  reconstituted  from a mixture o f  s o d i u m cholate and E m p i g e n B B to compare these v e s i c l e s to p r o t e o l i p o s o m e s f o r m e d f r o m O G P . T h e present study demonstrates, h o w e v e r , that no v e s i c u l a t i o n or protein i n c o r p o r a t i o n o c c u r r e d d u r i n g reconstitution f r o m a c h o l a t e / E m p i g e n B B m i x t u r e . dialysis, effective  r e m o v a l w a s . not  a c h i e v e d and  Despite prolonged  hence no protein reconstitution  possible. T h e C M C for E m p i g e n B B (1.2 m M ) (de l a M a z a et al,  was  1998) is l o w e r than those  o f O G P and s o d i u m cholate but this factor alone does riot appear to be sufficient to e x p l a i n w h y detergent r e m o v a l w a s not a c h i e v e d .  O n e p o s s i b i l i t y is that, w i t h i n m i x e d m i c e l l e s  c o n t a i n i n g p h o s p h o l i p i d , P o r , cholate and E m p i g e n B B , the effective C M C o f each detergent c o m p o u n d is s i g n i f i c a n t l y l o w e r than for the i n d i v i d u a l pure surfactant.  I n this regard, it  should be noted that the z w i t t e r i o n i c character o f E m p i g e n B B is s i m i l a r to some p h o s p h o l i p i d s ( A l l e n and H u m p h r i e s , 1975).  membrane  97  In  summary,  protein  incorporation  was  complete  proteoliposomes f r o m O G P at a p r o t e i n / l i p i d ratio o f 0.01.  when  reconstituting  However, incorporation was  i n c o m p l e t e at higher P / L ratios above 0.02, i n d i c a t i n g that protein i n c o r p o r a t i o n is saturable and l i m i t e d for a g i v e n l i p i d  concentration.  I n contrast,  it w a s observed that protein  i n c o r p o r a t i o n w a s inefficient and i n c o m p l e t e d u r i n g s o d i u m cholate-mediated  reconstitution  at a P / L o f 0.01. Interestingly, reconstitution studies u s i n g E m p i g e n B B / c h o l a t e detergent m i x t u r e s h o w e d that a m i x t u r e o f a z w i t t e r i o n i c and i o n i c detergent c o u l d not be r e m o v e d b y d i a l y s i s and thus no v e s i c u l a t i o n or protein i n c o r p o r a t i o n o c c u r r e d d u r i n g the reconstitution process.  These results i n d i c a t e d that O G P w o u l d be the i d e a l detergent to use i n subsequent  reconstitution experiments.  I n addition, reconstitution o f a single outer m e m b r a n e protein,  Por, resulted i n greater i n c o r p o r a t i o n e f f i c i e n c y c o m p a r e d to the reconstitution o f a m i x t u r e o f outer membrane proteins, M O M P .  98  CHAPTER 4 INFLUENCE OF LIPID COMPOSITION ON POR RECONSTITUTION AND CHARACTERIZATION OF THE RESULTING PROTEOLIPOSOMES  In the p r e v i o u s chapter, factors i n f l u e n c i n g protein i n c o r p o r a t i o n w e r e investigated. These factors ranged from detergent properties, reconstitution time, p r o t e i n - t o - l i p i d ratios and the nature o f the m e m b r a n e p r o t e i n sample, single protein or a m i x t u r e o f m e m b r a n e proteins. F r o m the observations and other considerations (see S e c t i o n 1.1.1), it is c o n c l u d e d that a single protein w o u l d be preferred i n a potential v a c c i n e f o r m u l a t i o n . Therefore, h a v i n g characterized i n c o r p o r a t i o n o f b o t h P o r and M O M P , subsequent studies focussed o n P o r proteoliposomes.  In this  chapter, b i o p h y s i c a l and  g o n o c o c c a l P o r v a c c i n e are characterized. determine  the effect  antigenic  properties  o f a liposomal  T h e objective o f these experiments  was  o f l i p i d c o m p o s i t i o n o n P o r i n c o r p o r a t i o n into l i p o s o m e s and  to the  orientation o f P o r i n the l i p i d b i l a y e r s .  4.1 INTRODUCTION A s mentioned earlier, Neisseria gonorrhoeae is a m u c o s a l pathogen that c o l o n i z e s b y c o m p e t i n g w i t h l o c a l m i c r o f l o r a for adherence to the m u c o s a l e p i t h e l i a l cells and  the  o r g a n i s m has adapted several m e c h a n i s m s for f a c i l i t a t i n g i n f e c t i o n and a v o i d i n g the host i m m u n e response ( B r i t i g a n et al.,  1985).  I n d i v i d u a l s w i t h g o n o c o c c a l infections  produce  bactericidal, o p s o n i c antibodies that m a y p o s s i b l y protect host cells b y i n h i b i t i n g bacterial attachment at m u c o s a l surfaces serum k i l l i n g ( W a r d et al,  or b y p r o m o t i n g p h a g o c y t o s i s and  1978 and V i r j i ,  1981).  complement-mediated  These observations  have  prompted  99  research o f g o n o c o c c a l surface  structures  revealed protein I as a potential candidate.  as possible v a c c i n e target antigens P r o t e i n I ( P o r ) is a major,  and  have  channel-forming  protein that orients i n a " h a i r p i n " fashion w i t h b o t h ends inserted i n the p l a s m a membrane, w i t h the l o o p p o r t i o n e x t e n d i n g out f r o m the p e r i p l a s m i c surface ( G r e c o et al, B l a k e et al., 1981).  1980 and  Studies have s h o w n that protective, b a c t e r i c i d a l antibodies are directed  towards, and b i n d an epitope located w i t h i n , the surface-exposed l o o p r e g i o n ( V i r j i et 1986 and F l e t c h e r et al,  al.,  1986).  E a r l i e r studies reported i n c o r p o r a t i o n o f P o r into l i p i d b i l a y e r s and c o m p a r e d this l i p o s o m a l f o r m u l a t i o n to p r e v i o u s l y u s e d v a c c i n e s c o n t a i n i n g the P o r protein ( W e t z l e r et 1988 and W e t z l e r et al,  1992)  These studies demonstrated  al,  that c o m b i n i n g g o n o c o c c a l  protein I w i t h a l u m i n u m phosphate or F r e u n d ' s adjuvant increased the o v e r a l l l e v e l o f p o r i n reactive antibodies, as c o m p a r e d to P o r alone; h o w e v e r , there w a s a reduced titer o f bactericidal antibodies that w e r e reactive w i t h the g o n o c o c c a l c e l l surface.  The reduction in  bactericidal a n t i b o d y titers was l i k e l y due to inadequate presentation o f the protein l o o p r e g i o n as a result o f protein denaturation and aggregation p r o d u c e d b y the adjuvants.  In  contrast, protein I incorporated into l i p o s o m e s e l i c i t e d the highest titer o f surface-reactive, bactericidal antibodies i n the rabbit m o d e l . These observations suggest that l i p o s o m e s w o u l d be preferred adjuvants i n a g o n o c o c c a l subunit v a c c i n e . It has been s h o w n that l i p o s o m e s are r e m o v e d from c i r c u l a t i o n and a c c u m u l a t e i n organs o f the reticuloendothelial system, s u c h as the lungs, l i v e r , spleen and b o n e m a r r o w ( A l l e n and C h o n n , 1987), where they are taken up b y antigen presenting cells ( A P C ) . R e s e a r c h o n macrophages, w h i c h are a major subset A P C , has suggested that the c e l l surface charge m a y influence particulate phagocytosis ( M u t s a e r s and P a p a d i m i t r i o u , 1988).  Studies  100  conducted b y N a k a n i s h i et al. (1997) s h o w e d that p o s i t i v e l y c h a r g e d l i p o s o m e s w e r e taken up m o r e efficiently b y macrophages than neutral o r n e g a t i v e l y charged carriers.  I n addition,  researchers have suggested that antigenic a c t i v i t y m a y be dependent o n the p h y s i c a l state o f the p h o s p h o l i p i d m o i e t y ( G o m e z - G u t i e r r e z et al., 1994 and G o m e z - G u t i e r r e z et al,  1995).  These studies indicate that the polar head group, the electrostatic interactions b e t w e e n the antigen and p h o s p h o l i p i d , as w e l l as the fatty a c i d c o m p o s i t i o n o f the p h o s p h o l i p i d m a y influence the r e c o v e r y o f the antigenic a c t i v i t y o f p u r i f i e d antigens. T h e early studies c o n d u c t e d b y W e t z l e r and c o w o r k e r s (1988, 1992) e x a m i n e d l i p i d mixtures o f P O P C : P O P E ; therefore, studies w e r e c o n d u c t e d to determine the influence o f l i p i d c o m p o s i t i o n , P O P C alone or P O P C : P O P E , o n i n c o r p o r a t i o n and p r o t e i n orientation.  In  addition, P o r protein w a s reconstituted into l i p o s o m e s c o m p o s e d o f a n i o n i c or c a t i o n i c l i p i d s to determine the effect o f l i p i d charge o n i n c o r p o r a t i o n efficiency. P o r P r o t e o l i p o s o m e s w e r e also characterized i n terms o f v e s i c l e size and m o r p h o l o g y .  F u r t h e r m o r e , the issue o f  sterilization o f a l i p o s o m a l g o n o c o c c a l subunit v a c c i n e f o r m u l a t i o n w a s also addressed.  101 4.2 RESULTS  4.2.1 P o r P r o t e i n reconstitution determined b y i s o p y c n i c density gradient centrifugation P r e v i o u s studies b y W e t z l e r and c o w o r k e r s ( 1 9 8 8 , 1992) e x a m i n e d P o r incorporated i n l i p o s o m e s c o n s i s t i n g o f mixtures o f P O P C : P O P E , whereas reconstitution  experiments  described i n the p r e v i o u s chapter w e r e c o n d u c t e d w i t h P O P C alone. Therefore, P o r protein w a s reconstituted into l i p o s o m e s w i t h different l i p i d c o m p o s i t i o n , P O P C alone or a m i x t u r e o f P O P C P O P E , to determine the effect o f l i p i d c o m p o s i t i o n o n i n c o r p o r a t i o n efficiency. F o l l o w i n g reconstitution, p r o t e o l i p o s o m e s w e r e centrifugation to separate components densities.  a n a l y z e d b y i s o p y c n i c density  gradient  w i t h i n a m i x t u r e o n the basis o f their s p e c i f i c  I n F i g u r e s 4.1 and 4.2 are s h o w n the density gradient profiles obtained  proteoliposomes reconstituted w i t h either P O P C alone o r P O P C : P O P E .  for  It can be seen that  c o - m i g r a t i o n o f p h o s p h o l i p i d and protein o c c u r s to a p o s i t i o n a p p r o x i m a t e l y m i d w a y d o w n the gradient.  P r o t e i n i n c o r p o r a t i o n w a s o b s e r v e d to be 9 0 % , w h i c h was associated w i t h the  l i p i d band, i n d i c a t i n g efficient i n c o r p o r a t i o n d u r i n g reconstitution.  F u r t h e r m o r e , i n the case  o f systems prepared f r o m P O P C alone, the p r o t e i n / l i p i d b a n d extends over a r e l a t i v e l y n a r r o w density range i n d i c a t i n g that the v e s i c l e s are h i g h l y h o m o g e n o u s w i t h respect p r o t e i n - t o - l i p i d ratio.  In the case o f reconstituted  systems prepared w i t h  to  POPCPOPE,  h o w e v e r , t w o n a r r o w bands o f p r o t e o l i p o s o m e s are observed ( F i g u r e 4.2). These t w o bands appear to arise f r o m v e s i c l e p o p u l a t i o n s o f d i f f e r i n g p r o t e i n - t o - l i p i d ratio. Inspection o f the data presented i n F i g u r e 4.2 shows that the larger, denser v e s i c l e fraction exhibits a p r o t e i n t o - l i p i d ratio ( P / L ) o f 0.026, whereas the lighter fraction e x h i b i t s a P / L o f 0.012.  These  i n i t i a l experiments also established the i m p o r t a n c e o f r e m o v i n g any i n s o l u b l e or aggregated  102  0  1  2  3  4  5  6  7  8  9  10  11  12  13  Gradient Depth (ml)  Figure 4.1: I s o p y c n i c density gradient centrifugation p r o f i l e for P o r reconstituted i n P O P C l i p o s o m e s . T h e p r o t e o l i p o s o m e s a m p l e w a s centrifuged at 1 1 0 , 0 0 0 g a continuous F i c o l l gradient (0-10%). concentrations for each fraction.  S h o w n are the protein ( • )  av  at 4°C for 20 hours o n and p h o s p h o l i p i d ( O )  103  0  1  2  3  4  5  6  7  8  9  10 11 12 13  Gradient Depth (ml)  Figure 4.2: I s o p y c n i c density gradient centrifugation p r o f i l e for P o r reconstituted i n P O P C P O P E (1:1) l i p o s o m e s .  T h e p r o t e o l i p o s o m e sample was centrifuged at 1 1 0 , 0 0 0 g  4 ° C for 20 hours o n a continuous F i c o l l gradient (0-10%). p h o s p h o l i p i d ( O ) concentrations for each fraction.  S h o w n are the protein ( • )  av  at  and  104  Figure 4 . 3 : I s o p y c n i c density gradient centrifugation p r o f i l e for i n s o l u b l e P o r reconstituted i n P O P C P O P E (1:1) liposomes.  I n this sample, the P o r protein w a s not filtered p r i o r to  reconstitution. T h e p r o t e o l i p o s o m e sample w a s centrifuged at 1 1 0 , 0 0 0 g o n a continuous F i c o l l gradient ( 0 - 1 0 % ) . concentrations for each fraction.  av  at 4 ° C for 20 hours  S h o w n are the protein ( • ) and p h o s p h o l i p i d ( O )  105  P o r protein p r i o r to reconstitution. A s described i n the M e t h o d s section, some batches o f P o r protein contained s m a l l amounts o f i n s o l u b l e material. T h i s denatured or aggregated protein w a s not incorporated into l i p o s o m e s d u r i n g reconstitution and c o u l d be separated proteoliposomes b y i s o p y c n i c density gradient centrifugation. 4.3 w h i c h  s h o w s the  density gradient  from  the  T h i s is illustrated i n F i g u r e  p r o f i l e for reconstituted  systems  prepared  P O P C : P O P E u s i n g a P o r protein b a t c h that had not been subjected to filtration.  with Again  protein bands are seen associated w i t h the l i p o s o m e s , as i n F i g u r e 4.1 and 4.2, but i n addition, protein is found near the b o t t o m o f the gradient w i t h no associated l i p i d .  In all  subsequent studies, therefore, any i n s o l u b l e material w a s r e m o v e d f r o m the O G P - s o l u b i l i z e d P o r (as described under M e t h o d s ) p r i o r to reconstitution.  4.2.2 P o r orientation i n reconstituted p r o t e o l i p o s o m e s A s i n d i c a t e d earlier, i n the bacterial p l a s m a membrane, P o r is f o l d e d i n a " h a i r p i n " l o o p c o n f i g u r a t i o n w i t h the l o o p p o r t i o n e x p o s e d o n the p e r i p l a s m i c surface.  Ideally, P o r  should retain this orientation i n the reconstituted proteoliposomes, thereby ensuring that the major antigenic site o n the l o o p d o m a i n w a s exposed.  T o evaluate protein orientation,  reconstituted systems w e r e incubated w i t h t r y p s i n or a - c h y m o t r y p s i n .  C l e a v a g e sites for  these proteases are located i n the l o o p d o m a i n as discussed i n S e c t i o n 1.3.1. protease digestion, samples were a n a l y z e d b y S D S - P A G E .  Following  A s s h o w n i n F i g u r e 4.4, w h e n  O G P - s o l u b i l i z e d P o r is incubated w i t h a - c h y m o t r y p s i n (lane C ) , the 3 9 K protein is c l e a v e d to produce 2 3 K , 1 7 K and 1 4 . 5 K 2 9 K , 2 3 K , 2 1 K and  fragments.  1 4 . 5 K fragments.  I n contrast, t r y p s i n cleavage (lane D ) p r o d u c e d These results correlate w e l l w i t h the  digestion studies o f p u r i f i e d outer membranes reported p r e v i o u s l y ( B l a k e et al,  protease 1981).  In  106  F i g u r e 4 . 4 : T r y p s i n and a - c h y m o t r y p s i n cleavage o f d e t e r g e n t - s o l u b i l i z e d and reconstituted P o r protein.  In lane A are s h o w n m o l e c u l a r w e i g h t standards, p h o s p h o r y l a s e b ( 9 7 . 4 K ) ,  serum a l b u m i n ( 6 6 . 2 K ) , o v a l b u m i n ( 4 5 K ) , c a r b o n i c anhydrase ( 3 I K ) , i n h i b i t o r ( 2 1 . 5 K ) and l y s o z y m e ( 1 4 . 5 K ) .  soy bean t r y p s i n  T h e r e m a i n i n g lanes represent the f o l l o w i n g : P o r  s o l u b i l i z e d i n O G P alone (lane B ) , P o r i n O G P treated w i t h a - c h y m o t r y p s i n (lane C ) , P o r i n O G P treated w i t h t r y p s i n (lane D ) , P o r reconstituted i n P O P C l i p o s o m e s treated w i t h a chymotrypsin  (lane  E ) , Por in P O P C  reconstituted i n P O P C P O P E  l i p o s o m e s treated  l i p o s o m e s treated  with  t r y p s i n (lane  F), Por  w i t h a - c h y m o t r y p s i n (lane G ) , P o r i n  P O P C P O P E l i p o s o m e s treated w i t h t r y p s i n (lane H ) , a - c h y m o t r y p s i n alone (lane I), and trypsin alone (lane J).  107  this earlier w o r k , a - c h y m o t r y p s i n w a s f o u n d to cleave P o r protein I ( 3 4 K ) into 2 3 K and 1 4 K fragments w h i l e t r y p s i n severed at t w o sites to p r o d u c e 3 fragments. the protein into 2 8 K and 1 0 K fragments.  Initially, t r y p s i n cleaves  T h e 2 8 K fragment is then further digested to a 2 I K  fragment and a s m a l l e r undetected fragment.  In the present study, h o w e v e r , i n c u b a t i o n w i t h  t r y p s i n also p r o d u c e d 2 3 K and 1 4 . 5 K fragments w h i c h appear to result f r o m residual ctc h y m o t r y p s i n activity.  A l p h a - c h y m o t r y p s i n c a n c l e a v e the 2 9 K fragment  t r y p s i n into a 2 3 K fragment and a s m a l l e r undetected fragment.  generated  by  W h e n P o r , reconstituted into  either P O P C or P O P C P O P E vesicles, is i n c u b a t e d w i t h t r y p s i n o r a - c h y m o t r y p s i n ( F i g u r e 4.4 lanes E - H ) cleavage patterns s i m i l a r to the O G P - s o l u b i l i z e d p r o t e i n are seen.  However,  for the reconstituted systems, a p o r t i o n o f u n c l e a v e d p r o t e i n c a n also be seen. T h i s proteaseresistant p o p u l a t i o n l i k e l y represents reconstituted P o r that is i n w a r d l y oriented, w i t h the l o o p d o m a i n f a c i n g the v e s i c l e interior, and hence u n a v a i l a b l e to external protease.  To  determine the relative proportions o f o u t w a r d l y and i n w a r d l y oriented P o r i n reconstituted proteoliposomes, gels w e r e scanned u s i n g a laser densitometer to determine the relative amounts o f each peptide band. F r o m the densitometer scan, fragments p r o d u c e d b y protease digestion accounted for an average o f 8 3 . 5 % o f the total p r o t e i n i n each o f the p r o t e i n digests ( T a b l e 4.1). These results suggest that o v e r 8 0 % o f reconstituted P o r w a s o u t w a r d l y oriented w i t h i n the l i p i d b i l a y e r w i t h the l o o p d o m a i n e x p o s e d o n the external surface o f the l i p o s o m e as has been described i n studies o n the bacterial c e l l m e m b r a n e ( B l a k e et al,  1981).  Studies  have s h o w n that l i p o s o m e s are i m p e r m e a b l e to e n z y m e s ( O b e r h o l z e r et al. 1999). Therefore, the 2 0 % o f the P o r protein that w a s not degraded b y the e n z y m e s m a y represent a p r o p o r t i o n o f P o r protein that was either i n w a r d l y oriented or sequestered to the inner l a m e l l a e o f m u l t i l a m e l l a r v e s i c l e s and thus not susceptible to protease cleavage.  108  P e a k A r e a s (°/o) Band  Lane B  Lane E  100  Lane F  Lane G  Lane H  15.2  13  19.1  18.6  -  19.5  -  13  41.4  25.3 12.9  43  25.7 14.9  18.8 24.5  -  14500  -  29.4  18.3 19.6  27.8  Total Fragments  0  84.7  87.1  80.9  81.4  Moi. Wt. 39000 29000 23000 21000 17000  -  -  -  %  Table 4.1: T r y p s i n and a - c h y m o t r y p s i n cleavage o f reconstituted P o r - p r o t e o l i p o s o m e s : D e n s i t o m e t r i c analysis o f S D S - P A G E g e l .  S h o w n are the areas under each b a n d as a  percentage o f the total for the c o r r e s p o n d i n g lane.  109  4.2.3 S i z e r e d u c t i o n o f reconstituted P o r p r o t e o l i p o s o m e s P h a r m a c e u t i c a l p r o d u c t i o n o f a l i p o s o m a l P o r v a c c i n e w o u l d be greatly facilitated i f the reconstituted p r o t e o l i p o s o m e s c o u l d be s t e r i l i z e d b y t e r m i n a l filtration.  Reconstitution  f r o m O G P , h o w e v e r , generates systems o f m e a n diameter greater than about 500 n m w h i c h therefore cannot be sterilized b y passage t h r o u g h a 0.2 m i c r o n filter. Therefore, experiments w e r e c o n d u c t e d to e x a m i n e w h e t h e r s m a l l e r v e s i c l e s c o u l d be generated b y e x t r u s i o n o f the proteoliposomes t h r o u g h p o l y c a r b o n a t e  filters  o f defined  size.  Proteoliposomes  were  sequentially extruded t h r o u g h 6 0 0 , 4 0 0 , 2 0 0 and 100 n m p o l y c a r b o n a t e filters and after each extrusion step, v e s i c l e size d i s t r i b u t i o n w a s e x a m i n e d b y Q E L S and p r o t e i n and p h o s p h o l i p i d r e c o v e r y also determined.  A s s h o w n i n F i g u r e 4.5, little or no protein o r p h o s p h o l i p i d loss  occurs o n e x t r u s i o n o f p r o t e o l i p o s o m e s reconstituted f r o m either P O P C or P O P C P O P E .  As  expected, however, the m e a n diameter o f the extruded systems is r e d u c e d as filter pore size is decreased ( T a b l e 4.2). P r o t e o l i p o s o m e s reconstituted f r o m P O P C g e n e r a l l y e x h i b i t s l i g h t l y smaller  mean  POPC:POPE.  diameters  following  extrusion  compared  to  systems  prepared  from  F o l l o w i n g e x t r u s i o n t h r o u g h 2 0 0 or 100 n m pore size filters, for example,  P O P C p r o t e o l i p o s o m e s have m e a n diameters o f about 134 and 88 n m , respectively, whereas POPCPOPE  proteoliposomes g i v e m e a n diameters o f about  196 and 104.  In addition,  vesicles p r o d u c e d b y e x t r u s i o n t h r o u g h 2 0 0 n m and 100 n m filters e x h i b i t r e l a t i v e l y n a r r o w size distributions as i n d i c a t e d b y the c a l c u l a t e d standard deviations ( T a b l e 4.2). It s h o u l d be anticipated that P o r p r o t e o l i p o s o m e s prepared from P O P C and extruded t h r o u g h either 100 n m or 2 0 0 n m filters w e r e then suitable for s t e r i l i z a t i o n b y t e r m i n a l  filtration.  I n the case o f  proteoliposomes prepared from P O P C P O P E , h o w e v e r , o n l y systems extruded t h r o u g h 100 nm  filters  w e r e s m a l l e n o u g h to be s t e r i l i z e d t h r o u g h a 0.2 m i c r o n filter.  The vesicle  110  Lipid  F i l t e r P o r e Size  M e a n Diameter  Composition  (nm)  (nm)  (nm)  POPC  Initial  494.3  286.0  600  251.1  118.1  400  210.1  70.4  200  133.8  40.0  100  88.5  25.7  Initial  975.7  655.3  600  592.9  357.7  400  347.1  161.8  200  195.6  64.0  100  104.0  33.8  POPE POPC  Standard D e v i a t i o n  Table 4 . 2 : Size r e d u c t i o n o f reconstituted P o r p r o t e o l i p o s o m e s b y extrusion.  Ill Figure  4.5:  Recovery  proteoliposomes.  of  protein  Reconstituted P O P C  and  phospholipid  following  ( A ) or P O P C P O P E  extruded t h r o u g h sequentially smaller pore size p o l y c a r b o n a t e M a t e r i a l s and M e t h o d s .  extrusion  of  ( B ) proteoliposomes filters  Por were  as described under  113  m o r p h o l o g i e s o f reconstituted and extruded p r o t e o l i p o s o m e s are discussed later i n the next section.  4.2.4 R e c o n s t i t u t i o n o f P o r into l i p o s o m e s c o m p o s e d o f charged l i p i d s P o r protein w a s reconstituted into l i p o s o m e s c o n t a i n i n g charged l i p i d s , a n i o n i c or cationic, to determine the effect o f l i p i d properties o n p r o t e o l i p o s o m e f o r m a t i o n and protein incorporation. T h e charged l i p i d s u t i l i z e d w e r e the n e g a t i v e l y and p o s i t i v e l y charged l i p i d s P O P S (or P O P G ) and D O D A C , respectively.  D i f f e r e n c e s i n i o n i c character o f these l i p i d  species m a y influence the P o r protein i n c o r p o r a t i o n e f f i c i e n c y into l i p o s o m e s . G o n o c o c c a l protein I (Por) w a s reconstituted into l i p o s o m e s c o n s i s t i n g o f P O P C and 5, 10 and 2 5 % (by w t . ) P O P S , P O P G o r D O D A C f r o m O G P (as described under M e t h o d s ) . A s discussed i n S e c t i o n 4.2.1, reconstituted neutral p r o t e o l i p o s o m e s w e r e found to have a m e a n v e s i c l e diameter o f about 500 n m ( T a b l e 4.2).  A s shown previously in Figure 4.1,  reconstitution o f P O P C l i p o s o m e s resulted i n the majority ( 8 5 % ) o f the P o r protein b e i n g associated w i t h the p h o s p h o l i p i d b a n d and c o - m i g r a t i n g i n the top h a l f o f the gradient.  A  peak fraction o f free protein (15 % ) w a s observed at the b o t t o m o f the gradient, i n d i c a t i n g that there is i n c o m p l e t e protein i n c o r p o r a t i o n into P O P C v e s i c l e s at this p r o t e i n - t o - l i p i d ratio ( P / L = 0 . 0 2 ) . In studies e m p l o y i n g the a n i o n i c l i p i d s , P O P S and P O P G , reconstituted  systems  w e r e found to have diameters o f about 500 n m and standard deviations greater than 150 n m , characteristics w h i c h w e r e s i m i l a r to that o f v e s i c l e s c o m p o s e d solely o f P O P C ( T a b l e 4.3). T h e reconstituted  proteoliposomes, c o n t a i n i n g P O P C alone or a m i x t u r e o f P O P C w i t h  a n i o n i c l i p i d , e x h i b i t e d large C h i values (greater than five), i n d i c a t i n g the v e s i c l e p o p u l a t i o n 2  had a m u l t i - m o d a l d i s t r i b u t i o n .  I n addition, a n i o n i c p r o t e o l i p o s o m e s  exhibited similar  114  Sample  Charged Lipid (%)  POPC POPC/POPG  0 5 10 25 5 10 25 5 10 25  POPC/POPS POPC/DODAC  Mean Vesicle Diameter (nm) 494.3 459 473 434 519 419 412 702 720 1180  Standard Deviation (nm) 286 195 193 149 259 200 209 301 354 675  Table 4.3: Q E L S size analysis and protein i n c o r p o r a t i o n e f f i c i e n c y o f P o r proteoliposomes reconstituted w i t h v a r y i n g l i p i d c o m p o s i t i o n .  115  gradient profiles to neutral l i p o s o m e s ; protein and l i p i d w e r e observed to co-migrate i n peak fractions spanning a n a r r o w density range w i t h free, u n i n c o r p o r a t e d protein at the gradient b o t t o m (Figures 4.6 and 4.7). F o r a m i x t u r e c o n t a i n i n g 5 % P O P G , reconstitution generated vesicles w i t h l o w e r protein i n c o r p o r a t i o n e f f i c i e n c y ( 7 5 % ) c o m p a r e d to that observed for neutral l i p o s o m e s . A n increase i n the p r o p o r t i o n o f P O P G (10 or 2 5 % ) resulted i n a decrease i n the amount o f P o r protein ( 6 5 % ) associated w i t h the l i p i d 4.6C).  Reconstitution o f 5%  and  10% P O P S  samples  fraction  ( F i g u r e s 4 . 6 B and  yielded proteoliposomes  a p p r o x i m a t e l y 9 0 % and 8 0 % protein i n c o r p o r a t i o n ( F i g u r e 4 . 7 A and 4 . 7 B ) , w h i c h  with was  s i m i l a r to that seen for P O P C p r o t e o l i p o s o m e s w h e n t a k i n g into account a 5 % v a r i a b i l i t y b e t w e e n reconstitution preparations.  A s s h o w n i n F i g u r e 4 . 7 C , an increase i n the P O P S  content to 2 5 % resulted i n a decrease ( 7 5 % ) i n the relative amount o f protein incorporated. These profiles indicate that the electrostatic interaction o f the charged l i p i d and charged domains o f the protein m a y be i n f l u e n c i n g protein structure,  subsequently  affecting  the  efficiency o f protein insertion into the b i l a y e r . P o r reconstitution i n v e s i c l e s c o n t a i n i n g p o s i t i v e l y charged l i p i d , D O D A C , resulted i n significantly larger p r o t e o l i p o s o m e s and these systems d i s p l a y e d v e r y different i s o p y c n i c density gradient profiles ( T a b l e 4.3 and F i g u r e 4.8) f r o m those seen w i t h neutral or a n i o n i c l i p i d mixtures.  It w a s observed that i n c r e a s i n g the relative amount o f D O D A C f r o m 5 - 2 5 %  resulted i n an increase i n v e s i c l e diameter f r o m a p p r o x i m a t e l y 700 n m to about l u M w i t h standard deviations r a n g i n g from about 3 0 0 to 700 n m .  T h e v e s i c l e populations w e r e also  observed to exhibit a m u l t i - m o d a l size d i s t r i b u t i o n as suggested b y a C h i Reconstituted  proteoliposomes  containing  5%  DODAC  exhibited  2  greater than complete  five.  protein  i n c o r p o r a t i o n as i n d i c a t e d b y c o - m i g r a t i o n o f the protein and l i p i d fractions and the absence  116  Figure 4 . 6 : P o r protein i n c o r p o r a t i o n into P O P C / P O P G l i p o s o m e s .  Isopycnic  gradient centrifugation profile for g o n o c o c c a l protein I i n c o r p o r a t e d into P O P C c o n t a i n i n g v a r y i n g amounts o f P O P G : centrifuged at 1 1 0 , 0 0 0 g  av  A ) 5%, B ) 10 %  and C ) 2 5 % .  liposomes  Samples  at 4 ° C for 2 4 hours o n a continuous F i c o l l gradient  P r o t e i n (circles). P h o s p h o l i p i d (triangles).  density were  (0-10%).  118  Figure 4.7: P o r protein i n c o r p o r a t i o n into P O P C / P O P S l i p o s o m e s .  Isopycnic  gradient centrifugation profile for g o n o c o c c a l protein I i n c o r p o r a t e d into P O P C c o n t a i n i n g v a r y i n g amounts o f P O P S : centrifuged at 1 1 0 , 0 0 0 g  av  A ) 5%, B ) 10 % and C ) 2 5 % .  Samples  at 4 ° C for 24 hours o n a continuous F i c o l l gradient  P r o t e i n (circles). P h o s p h o l i p i d (triangles).  density  liposomes were  (0-10%).  120  o f free protein at the b o t t o m o f the gradient ( F i g u r e 4.8 A ) .  Interestingly, as s h o w n i n figure  4 . 8 B , an increase i n the D O D A C content to 1 0 % resulted i n a shift o f the p r o t e i n / l i p i d peak d i s t r i b u t i o n towards the top o f the gradient.  A c o m p l e t e shift o f the p r o t e i n / l i p i d fraction to  the top o f the gradient w a s observed after reconstitution w i t h 2 5 % D O D A C , i n d i c a t i n g that proteoliposome  density  was  less  than  the  lowest  Ficoll  concentration  (Figure  4.8C).  R e c o n s t i t u t i o n w i t h c a t i o n i c l i p i d resulted i n c o m p l e t e protein i n c o r p o r a t i o n ; h o w e v e r , the properties o f the reconstituted systems w e r e altered relative to neutral or a n i o n i c v e s i c l e s , as demonstrated b y the shift o f the p r o t e i n / l i p i d b a n d t o w a r d s a l o w e r density.  These changes  w e r e associated w i t h reduced antibody b i n d i n g a c t i v i t y c o m p a r e d to neutral and a n i o n i c proteoliposomes, as discussed i n C h a p t e r 5.  4.2.5 V e s i c l e m o r p h o l o g i e s P r o t e o l i p o s o m e m o r p h o l o g i e s w e r e also e x a m i n e d u s i n g c r y o - e l e c t r o n m i c r o s c o p y ( C T E M ) . P o r proteoliposomes reconstituted f r o m O G P detergent are s h o w n i n F i g u r e s 4 . 9 A and B . Consistent w i t h Q E L S results, C T E M analysis s h o w s that v e s i c l e s are heterogeneous w i t h regard to l a m e l l a r i t y and m o r p h o l o g y . U n d e r l o w m a g n i f i c a t i o n ( F i g u r e 4 . 9 A ) , v e s i c l e s are observed as aggregated structures o f v a r y i n g m o r p h o l o g y w i t h large v e s i c l e s e n c l o s i n g m a n y smaller vesicles, h o w e v e r , these v e s i c l e s m a y have been s u p e r - i m p o s e d o n one another and thus they are p e r c e i v e d to be m u l t i l a m e l l a r . These smaller v e s i c l e s b e c o m e discernable under higher m a g n i f i c a t i o n ; h o w e v e r , P o r protein, due to its s m a l l size, is not v i s i b l e w i t h i n the l i p i d bilayers.  S i m i l a r v e s i c l e s c o n t a i n i n g a p o s i t i v e l y charged l i p i d , D O D A C , o r a  negatively charged l i p i d , P O P S , d i s p l a y s i m i l a r m o r p h o l o g i e s ( F i g u r e s 4 . 9 C - D and 4 . 9 E - F , respectively).  R e c o n s t i t u t e d systems w e r e also size-reduced b y e x t r u s i o n t h r o u g h t w o ,  121  Figure  4.8:  P o r protein i n c o r p o r a t i o n into P O P C / D O D A C  liposomes.  I s o p y c n i c density  gradient centrifugation profile for g o n o c o c c a l p r o t e i n I i n c o r p o r a t e d into P O P C c o n t a i n i n g v a r y i n g amounts o f D O D A C : A ) 5%, B ) 1 0 % and C ) 2 5 % . centrifuged at 1 1 0 , 0 0 0 g  av  at 4 ° C for 2 4 hours o n a continuous F i c o l l gradient  P r o t e i n (circles). P h o s p h o l i p i d (triangles).  liposomes  Samples  were  (0-10%).  Gradient Depth (ml)  123  Figure 4.9: C r y o - e l e c t r o n m i c r o g r a p h s o f reconstituted P o r p r o t e o l i p o s o m e  formulations  v i e w e d under different magnifications. A ) and B ) P O P C / P o r , C ) and D ) P O P C / D O D A C / P o r , E ) and F) P O P C / P O P S / P o r , G ) and H ) E x t r u d e d P O P C / P o r .  124  126  stacked polycarbonate  filters  o f 100 n m pore size (see M e t h o d s ) i n an attempt to generate  u n i l a m e l l a r v e s i c l e s o f a u n i f o r m size d i s t r i b u t i o n .  A s s h o w n i n F i g u r e s 4 . 9 G and H ,  extruded proteoliposomes are a p p r o x i m a t e l y 7 0 % u n i l a m e l l a r and spherical w i t h an average diameter o f about 100 nm. F u r t h e r m o r e , the extrusion procedure resulted i n m i n i m a l loss o f protein and l i p i d , as discussed i n S e c t i o n 4.2.3.  127  4.3 D I S C U S S I O N P o r is the major constituent o f the g o n o c o c c a l outer membrane, c o m p r i s i n g up to 6 0 % o f the protein content (Johnston and G o t s c h l i c h , 1974 and J o h n s t o n and G o t s c h l i c h , 1976). F u r t h e r m o r e , anti-Por antibodies have been s h o w n to confer protection against subsequent challenge b y g o n o c o c c a l o r g a n i s m s ( V i r j i et al, trials e m p l o y i n g g o n o c o c c a l m e m b r a n e protective effect ( A r m i n j o n et al,  blebs,  1987 and P l u m m e r et al, however,  have  failed  1987, T r a m o n t , 1989 and G u l a t i et al,  m a y have contributed to this l a c k o f success. m e m b r a n e constituents, notably Rmp  to  1989).  Vaccine  demonstrate  a  1991). T w o factors  F i r s t , c o n t a m i n a t i o n o f the blebs b y other  (protein III), m a y have triggered the p r o d u c t i o n o f  b l o c k i n g antibodies. R e c e n t studies, for e x a m p l e , have s h o w n that the presence o f anti-Rmp antibodies results i n an increased s u s c e p t i b i l i t y to g o n o c o c c a l i n f e c t i o n ( R i c e et al, P l u m m e r et al,  1986 and  1993). S e c o n d , P o r proteins m a y not have been adequately presented i n their  native tertiary structure, resulting i n o n l y l o w levels o f a n t i - P o r antibody b e i n g p r o d u c e d . A s described b e l o w , the l i p o s o m a l P o r v a c c i n e d e s c r i b e d herein a v o i d s b o t h o f these problems. T o prevent c o n t a m i n a t i o n b y Rmp, P o r protein w a s isolated from g o n o c o c c a l strains from w h i c h the rmp gene had been deleted ( W e t z l e r et al,  1989a and W e t z l e r et al,  1989b).  P o r i n s isolated f r o m these strains have been s h o w n to be f u n c t i o n a l l y and a n t i g e n i c a l l y identical to those isolated f r o m i s o g e n i c w i l d type parent strains.  P u r i f i e d P o r was then  reintroduced into a b i l a y e r e n v i r o n m e n t b y reconstitution w i t h p h o s p h o l i p i d s to generate w e l l - d e f i n e d proteoliposomes.  E f f i c i e n t and essentially c o m p l e t e P o r insertion c o u l d  be  demonstrated w h e n fully s o l u b i l i z e d protein w a s e m p l o y e d . It w a s noted, h o w e v e r , that any i n s o l u b l e protein i n i t i a l l y  present  d u r i n g reconstitution w a s  not  incorporated  into  the  resulting proteoliposomes.  P r o t e i n reconstitution w a s c o m p a r e d for systems c o n s i s t i n g o f a  128  m i x t u r e o f p h o s p h o l i p i d species ( P O P C : P O P E ) or a single l i p i d ( P O P C ) .  The lipid mixture  selected for this study consisted o f a b i l a y e r f o r m i n g species ( P O P C ) together w i t h a l i p i d ( P O P E ) w h i c h i n i s o l a t i o n c a n adopt the n o n - b i l a y e r , h e x a g o n a l H n phase ( E p a n d , 1985).  It  has p r e v i o u s l y been suggested that the h y d r o p h o b i c , b i l a y e r - s p a n n i n g d o m a i n o f i n t r i n s i c m e m b r a n e proteins may m o r e r e a d i l y be a c c o m o d a t e d i n b i l a y e r s c o m p o s e d o f m i x e d l i p i d species due to the a b i l i t y o f l i p i d s possessing different d y n a m i c m o l e c u l a r shapes to p a c k at the l i p i d - p r o t e i n interface w i t h o u t creating b i l a y e r defects ( N a v a r r o et al,  1984).  I n the  present study, h o w e v e r , no differences i n the e f f i c i e n c y o f protein reconstitution w e r e seen b e t w e e n P O P C v e s i c l e s and P O P C P O P E systems.  I n these reconstituted systems, P o r w a s  p r e d o m i n a n t l y inserted i n the same o u t w a r d l y oriented c o n f i g u r a t i o n found i n the native membrane.  F u r t h e r m o r e , the external h a i r p i n l o o p d o m a i n w a s r e a d i l y c l e a v e d b y t r y p s i n o r  a - c h y m o t r y p s i n to y i e l d characteristic peptide fragments.  A g a i n , this is consistent w i t h the  protein e x i s t i n g i n its native tertiary structure. T h e research presented i n this chapter characterized the effect o f l i p o s o m e charge o n protein i n c o r p o r a t i o n efficiency.  It w a s observed that P o r i n c o r p o r a t i o n into cationic  l i p o s o m e s w a s more efficient than for neutral or negatively c h a r g e d systems.  I n a d d i t i o n , the  i n c o r p o r a t i o n o f larger proportions o f the c a t i o n i c l i p i d , D O D A C , resulted i n changes i n density o f the p r o t e o l i p o s o m e s as seen b y the shift i n the p r o t e i n / l i p i d fractions t o w a r d the top o f the i s o p y c n i c density gradient ( F i g u r e 4.8). T h i s density change m a y have been due to the increase i n m e a n l i p i d cross-sectional area i n t r o d u c e d b y the c a r b o n d o u b l e bonds o f DODAC.  In addition, the absence o f phosphate groups and the l o w e r m o l e c u l a r w e i g h t o f  DODAC  compared  to  POPS  or  POPG  proteoliposomes t o w a r d a l o w e r density.  may  have  also  produced  the  shift  of  the  129  E a r l i e r studies have s h o w n that p r o t e o l i p o s o m e s generated b y reconstitution  from  O G P detergent w e r e largely u n i l a m e l l a r w i t h a m e a n diameter o f about 2 0 0 n m ( M i m m s et al,  1981). I n contrast, the c r y o - e l e c t r o n m i c r o g r a p h s presented i n this chapter i n d i c a t e that  reconstituted  systems  are  heterogeneous  in  their  size,  l a m e l l a r i t y and  morphology.  R e c o n s t i t u t e d proteoliposomes c o m p o s e d o f neutral o r charged l i p i d s w e r e o b s e r v e d to be o f v a r i a b l e l a m e l l a r i t y w i t h m e a n diameters greater than 500 n m . These systems c o u l d be s i z e reduced to vesicles o f about 100 n m m e a n diameter u s i n g an e x t r u s i o n procedure ( H o p e et al.,  1985 and M a y e r et al,  1986).  S m a l l e r v e s i c l e s w o u l d be favorable w i t h regard to  sterilization and safety for h u m a n use.  C o n v e n t i o n a l heat s t e r i l i z a t i o n o r i r r a d i a t i o n o f the  v a c c i n e f o r m u l a t i o n w o u l d not be suitable s t e r i l i z a t i o n methods because they w o u l d l i k e l y cause denaturation o f the protein and loss o f the antigenic determinants.  I n order to retain the  i m m u n o g e n i c characteristics o f the v a c c i n e , the preparation w o u l d have to be sterilized under non-denaturing c o n d i t i o n s . procedure  O n e m e t h o d that w o u l d be suitable is t e r m i n a l filtration.  i n v o l v e s passing the  reconstituted  sample t h r o u g h  a 0.2  This  m i c r o n filter  that  effectively r e m o v e s any m i c r o b i a l o r g a n i s m s present i n the sample. C l e a r l y this requires that the proteoliposomes be less than 2 0 0 n m i n diameter. Therefore, the e x t r u s i o n procedure w i l l allow  vaccine  preparation  using  Good  c o n d i t i o n s that do not require full complexity  and  cost  of  vaccine  Manufacturing Practices  aseptic  ( G M P ) under  processing, w h i c h w i l l  manufacture.  In  addition,  the  clean  greatly reduce size-reduction  the of  proteoliposomes to L U V c o u l d lead to greater P o r protein e x p o s e d o n the external l i p o s o m e surface than for M L V , w h i c h m a y have some P o r protein sequestered i n internal l a m e l l a e . Therefore, L U V w o u l d l i k e l y have m o r e efficient antigen presentation and thus lead to an i m p r o v e d i m m u n e response.  130  CHAPTER 5 THE ANTIGENIC CHARACTERIZATON OF POR PROTEOLIPOSOMES  T h e previous chapter characterized P o r protein orientation i n the l i p i d b i l a y e r and the effect o f l i p i d c o m p o s i t i o n o n protein i n c o r p o r a t i o n e f f i c i e n c y , v e s i c l e size a n d m o r p h o l o g y . In the present chapter, P o r proteoliposomes, c o m p o s e d o f neutral l i p i d alone o r i n m i x t u r e s w i t h a n i o n i c o r cationic l i p i d , are characterized t o determine the effects o f l i p i d c o m p o s i t i o n o n in vitro antibody b i n d i n g a n d in vivo i m m u n o g e n i c i t y .  5.1 INTRODUCTION L i p o s o m e s , administered intravenously, are r e m o v e d f r o m c i r c u l a t i o n and a c c u m u l a t e i n organs o f the r e t i c u l o e n d o t h e l i a l system ( R E S ) , such as the lungs, liver, spleen a n d bone m a r r o w ( A l l e n and C h o n n , 1987). Preferential clearance o f l i p o s o m e s to tissues o f the R E S m a y be an advantageous aspect o f u s i n g l i p o s o m e s as carriers o f v a c c i n e s , because this m a y enable targeting and uptake o f l i p o s o m e s b y macrophages, w h i c h are major participants i n antigen p r o c e s s i n g ( U n a n u e , 1984). R e s e a r c h w i t h macrophages has suggested that the c e l l surface charge m a y influence particulate p h a g o c y t o s i s ( M u t s a e r s and P a p a d i m i t r i o u , 1988). Studies c o n d u c t e d b y N a k a n i s h i et al. (1997) s h o w e d that p o s i t i v e l y charged l i p o s o m e s w e r e taken u p m o r e efficiently b y macrophages and w e r e m o r e potent inducers o f h u m o r a l a n d c e l l - m e d i a t e d i m m u n i t y than neutral o r n e g a t i v e l y c h a r g e d carriers. R e s e a r c h c o n d u c t e d b y W e t z l e r et al. (1988 a n d 1992) has suggested that a l i p o s o m a l P o r f o r m u l a t i o n w o u l d be effective at i n d u c i n g h u m o r a l i m m u n i t y . T h e i r results s h o w e d that P o r proteoliposomes e x h i b i t h i g h in vitro a n t i b o d y b i n d i n g a c t i v i t y a n d thus g o o d surface  131  presentation o f the epitope for antibody r e c o g n i t i o n c o m p a r e d t o a l u m o r F r e u n d ' s adjuvant preparations c o n t a i n i n g P o r protein. T h e results presented i n C h a p t e r 4 demonstrated that g o n o c o c c a l protein EB ( P o r ) c a n be e f f i c i e n t l y i n c o r p o r a t e d into l i p o s o m e s i n a n orientation s i m i l a r to that seen i n the native bacterial membrane.  H o w e v e r , i n d e v e l o p i n g effective  v a c c i n e s for particular diseases, several factors must be taken into account.  Initial p r o t e c t i o n  to i n f e c t i o n appears to be conferred t h r o u g h a h u m o r a l i m m u n e response antibodies against the pathogen ( A h m e d a n d G r a y , 1996). such as Mycobacterium al,  1997).  tuberculosis,  V a c c i n e s , therefore,  mediated b y  S o m e i n t r a c e l l u l a r pathogens,  appear to i n d u c e c e l l - m e d i a t e d i m m u n i t y ( S k i n n e r et  should be designed t o induce a b r o a d i m m u n e  i n v o l v i n g a h u m o r a l i m m u n i t y against  the i n i t i a l exposure  response  a n d c e l l u l a r responses for  protection against intracellular infection. T h e studies o u t l i n e d i n the present chapter e x a m i n e the i n f l u e n c e o f p h o s p h o l i p i d c o m p o s i t i o n o n the in vitro and in vivo antigenic properties o f l i p o s o m a l g o n o c o c c a l subunit vaccine  formulations.  P o r proteoliposomes  described  i n the  previous  chapter  are  characterized w i t h regard to their antibody b i n d i n g a c t i v i t y a n d i m m u n o g e n i c i t y i n a m u r i n e i m m u n i z a t i o n m o d e l . I n a d d i t i o n , the m o u s e i m m u n e response is characterized t o determine the effect o f the route o f i n o c u l a t i o n o n i m m u n o g e n i c i t y . elicited are a n a l y z e d for i m m u n o g l o b u l i n serotypes response i n d u c e d , h u m o r a l o r c e l l - m e d i a t e d .  F u r t h e r m o r e , the antibody titers  t o determine  the type o f i m m u n e  132  5.2 RESULTS  5.2.1 A n t i b o d y b i n d i n g to P o r p r o t e o l i p o s o m e s determined u s i n g an E L I S A assay T h e b i n d i n g affinity o f p r o t e o l i p o s o m e s for a n t i - P o r antibody w a s e x a m i n e d for reconstituted systems prepared w i t h either P O P C o r P O P C P O P E (1:1) u s i n g t w o different batches o f p u r i f i e d P o r protein ( G M P and M S 1 1 ) . reconstituted  into  proteoliposomes  procedure to that described here.  composed  In an earlier study, P o r had  of P O P C P O P E  (1:4)  using  a  been  different  F o r c o m p a r i s o n , therefore, s i m i l a r p r o t e o l i p o s o m e s w e r e  prepared and their antibody b i n d i n g a c t i v i t y determined.  It is important to note that neither  w h o l e cells f r o m g o n o c o c c a l strain M S l l j e A r / w p n o r any o f the five l i p o s o m a l preparations demonstrated  any b i n d i n g a c t i v i t y w i t h antibodies directed at epitopes that appear to be  b u r i e d w i t h i n the e n v i r o n m e n t o f the g o n o c o c c a l outer membranes ( M . S . B l a k e , u n p u b l i s h e d results). antibodies  U s i n g an i n h i b i t i o n E L I S A assay, b i n d i n g o f three surface reactive, m o n o c l o n a l ( M A b ) and  a polyclonal  formulations w a s e x a m i n e d .  rabbit  serum  against  each  o f the  proteoliposome  A s s h o w n i n F i g u r e 5.1, each o f the P o r p r o t e o l i p o s o m e  preparations s h o w e d i n h i b i t o r y a c t i v i t y i n the E L I S A assay u s i n g the M A b s , 1 F 1 1 , 3 H 1 and 6F9.  These results suggest that P o r protein is oriented i n the l i p o s o m a l m e m b r a n e i n a  c o n f i r m a t i o n that is s i m i l a r to that seen i n the native bacterial membrane.  I n F i g u r e 5.1 A ,  P o r proteoliposomes prepared f r o m P O P C w i t h M S I 1 P o r and P O P C P O P E (1:1) w i t h G M P P o r both e x h i b i t e d 5 0 % i n h i b i t i o n at a p p r o x i m a t e l y 32 p g and 35 p g P o r , respectively, whereas p r o t e o l i p o s o m e s prepared w i t h P O P C and G M P P o r and P O P C P O P E (1:1) w i t h M S I 1 P o r demonstrated 5 0 % i n h i b i t i o n at 4 0 p g and 52 p g , respectively. V e s i c l e s prepared f r o m P O P C : P O P E (1:4), a c c o r d i n g to W e t z l e r and c o w o r k e r s (1992), s h o w e d the l o w e s t  133  F i g u r e 5.1: In vitro P o r antibody b i n d i n g a c t i v i t y . I n h i b i t i o n E L I S A assay s h o w i n g a n t i b o d y b i n d i n g activities o f five P o r - l i p o s o m e preparations w i t h m o n o c l o n a l antibodies  (MAbs)  generated against v a r i o u s g o n o c o c c a l strains: 1F11 M A b s . ( A ) , 3 H 1 M A b s ( B ) and 6 F 9 M a b s ( C ) . T h e p r o t e o l i p o s o m e formulations consisted o f p r o t e o l i p o s o m e s prepared from G M P P o r protein w i t h P O P C ( • ) and P O P C P O P E (1:1) ( A ) ; p r o t e o l i p o s o m e s prepared from M S 11 P o r reconstituted w i t h P O P C ( • ) and P O P C P O P E (1:1) ( T ) and p r o t e o l i p o s o m e s prepared from M S I 1 P o r w i t h P O P C P O P E (1:4) ( O ) .  135  potency w i t h 5 0 % i n h i b i t i o n at 55 u g P o r protein.  I n figure 5. I B , P O P C l i p o s o m e s w i t h  M S 11 P o r and P O P C w i t h G M P P o r demonstrated s i m i l a r potencies w i t h 5 0 % i n h i b i t i o n at 35 u g P o r protein, w h i c h w e r e greater than the p o t e n c y o f P O P C P O P E (1:1) systems w i t h G M P P o r w h i c h e x h i b i t e d 5 0 % i n h i b i t i o n w i t h 41 u g P o r .  Proteoliposomes composed o f  P O P C P O P E at a ratio o f 1:1 or 1:4 w i t h M S 11 P o r required 52 u g o f P o r to p r o d u c e 5 0 % inhibition. 5.1C).  S i m i l a r b i n d i n g activities w e r e o b s e r v e d w i t h m o n o c l o n a l a n t i b o d y 6 F 9 ( F i g u r e  O v e r a l l , the highest i n h i b i t i o n w a s seen for P O P C v e s i c l e s c o n t a i n i n g M S 11 P o r .  T h i s c o m p a r i s o n also h e l d w h e n the assay w a s c o n d u c t e d u s i n g a p o l y c l o n a l rabbit ( F i g u r e 5.2).  A s s h o w n i n F i g u r e 5.2, P O P C / M S 1 1 P o r p r o t e o l i p o s o m e s  serum  exhibit  50%  i n h i b i t i o n w i t h a p p r o x i m a t e l y 12 ng P o r , whereas greater than 25 u g P o r is required for 5 0 % i n h i b i t i o n w i t h each o f the other four f o r m u l a t i o n s . P O P C / M S 1 1 P o r proteoliposomes (1:4) w i t h M S 11 P o r .  A n a l y s i s o f the data indicates that  have 3 - f o l d greater b i n d i n g a c t i v i t y than  POPCPOPE  In w h o l e c e l l b i n d i n g assays, it w a s seen that a p p r o x i m a t e l y  10  9  g o n o c o c c i o f the strain M S I IJBAATW/? e x h i b i t e d 3 0 % b i n d i n g , whereas 18 u g o f P O P C / M S 1 1 gave the same 3 0 % b i n d i n g a c t i v i t y ( M . S . B l a k e , u n p u b l i s h e d results).  Therefore, u s i n g the  P O P C / M S 1 1 P o r f o r m u l a t i o n as the standard, a theoretical estimate o f the n u m b e r o f P o r proteins per b a c t e r i u m c a n be d e r i v e d . U s i n g the equation: Por proteins/Bacterium = ( D / M ) x A + C D = A m o u n t o f P o r protein i n m g M = M o l e c u l a r weight o f P o r A = A v a g a d r o ' s N u m b e r 6.02 x l O C = N u m b e r o f gonococcal cells  2 3  136  10  100  Porin-Liposome (pg protein)  Figure 5.2: In vitro P o r antibody b i n d i n g a c t i v i t y w i t h rabbit anti-sera.  Inhibition E L I S A  assay s h o w i n g a n t i b o d y b i n d i n g activities o f five l i p o s o m e preparations w i t h a p o l y c l o n a l rabbit  serum,  antiserum  2-859.  The  proteoliposome  formulations  consisted  proteoliposomes prepared f r o m G M P P o r protein w i t h P O P C ( • )  and P O P C P O P E  (A);  with  proteoliposomes  prepared  from  M S 11  Por  reconstituted  POPC  P O P C P O P E (1:1) ( T ) and p r o t e o l i p o s o m e s prepared from M S 11 P o r w i t h (1:4) ( O ) .  (•)  of (1:1) and  POPCPOPE  137  B a s e d o n a protein m o l e c u l a r w e i g h t o f 3 6 K , the c a l c u l a t i o n w o u l d suggest that each g o n o c o c c u s contains 3 x 1 0 m o l e c u l e s o f P o r protein o r 1 0 porins per b a c t e r i u m , w h i c h is 5  5  g o o d agreement w i t h other estimates ( N i k a i d o , 1993).  5.2.2 In vitro antibody b i n d i n g a c t i v i t y o f charged P o r p r o t e o l i p o s o m e s P r o t e o l i p o s o m e s c o m p o s e d o f P O P C alone o r a m i x t u r e o f P O P C and 1 0 % charged l i p i d , P O P S o r D O D A C , w e r e prepared a n d a n a l y z e d f o r their b i n d i n g affinity against an a n t i - P o r m o n o c l o n a l a n t i b o d y u s i n g a n i n h i b i t i o n E L I S A assay.  Subsequent t o the b i n d i n g  studies described above, it w a s observed that i m p r o v e d assay d e s i g n a l l o w e d m u c h greater sensitivity a n d detection o f n a n o g r a m quantities o f P o r protein.  I n F i g u r e 5.3, P O P C P o r  p r o t e o l i p o s o m e s e x h i b i t e d 3 8 % i n h i b i t i o n w i t h 2.4 n g P o r protein, whereas p r o t e o l i p o s o m e s c o n t a i n i n g P O P S o r D O D A C demonstrated 3 5 % and 1 7 % i n h i b i t o r y a c t i v i t y , respectively. A t 4.5 n g P o r protein, 5 0 % i n h i b i t i o n w a s observed f o r P O P C proteoliposomes.  POPS-  c o n t a i n i n g l i p o s o m e s s h o w e d 4 2 % i n h i b i t i o n w i t h 4.5 n g P o r protein, whereas o n l y 2 4 % inhibition was exhibited by D O D A C - c o n t a i n i n g liposomes. Control liposomes were found to have n e g l i g i b l e antibody b i n d i n g a c t i v i t y .  T h e results suggest that the m e m b r a n e  changes  i n t r o d u c e d b y D O D A C m a y prevent P o r protein from a d o p t i n g the correct b i l a y e r orientation required f o r epitope presentation and a n t i b o d y b i n d i n g .  5.2.3 In vivo i m m u n e responses to p r o t e o l i p o s o m e s T o determine  the antigenic properties  o f P o r proteoliposomes,  eight m i c e  were  i n o c u l a t e d w i t h either P o r i n c o r p o r a t e d into l i p o s o m e s , c o m p o s e d o f P O P C alone o r i n mixtures o f P O P C w i t h 1 0 % P O P S o r D O D A C ,  o r free (non-reconstituted)  P o r protein.  138  Figure 5.3:  Effect o f charged l i p o s o m e s o n antibody b i n d i n g activity.  Inhibition E L I S A  s h o w i n g b i n d i n g activities o f l i p o s o m e preparations w i t h a n t i - P o r m o n o c l o n a l antibody. liposomes  formulations  were:  control  POPC  (O),  control  POPC/POPS  (A),  The  control  P O P C / D O D A C ( V ) , P O P C / P o r ( • ) , P O P C / P O P S / P o r ( A ) and P O P C / D O D A C / P o r ( T )  139  A d m i n i s t r a t i o n s w e r e at 1 u g P o r protein g i v e n either intraperitoneally (i.p.) or i n t r a d e r m a l l y ( i d . ) at w e e k l y intervals (3 injections). m e a s u r i n g the a n t i b o d y titer.  T h e r e s u l t i n g i m m u n e response w a s a n a l y z e d b y  It w a s anticipated that the first i n o c u l a t i o n w o u l d i n d u c e a  p r i m a r y a n t i b o d y response and the f o r m a t i o n o f m e m o r y cells.  U p o n booster injections, the  antigen w o u l d trigger a faster and m o r e intense secondary a n t i b o d y response.  I n F i g u r e 5.4,  it c a n be seen that antibody titers elicited b y the first P o r p r o t e o l i p o s o m e i n o c u l a t i o n w e r e not significantly higher than titers observed for c o n t r o l l i p o s o m e s .  T h e first intraperitoneal  booster injection at 3 w e e k s i n d u c e d an average 5 0 - f o l d (1.7 l o g unit) increase i n a n t i b o d y titer, whereas a 100-fold (2 l o g unit) increase w a s o b s e r v e d for the p r o t e o l i p o s o m e s after the first  i d . booster i n o c u l a t i o n .  response  that o c c u r s after the  T h e m a r k e d increase i n titers signal the strong first  inoculation.  T h e second booster  secondary  injection o f P o r  proteoliposomes intraperitoneally or i n t r a d e r m a l l y at 7 w e e k s p r o d u c e d average increases i n antibody titers o f 18-fold and 14-fold, r e s p e c t i v e l y ( F i g u r e 5.4). F o r the i.p. route, the free P o r preparation e l i c i t e d an a n t i b o d y titer o f 4 . 6 2 l o g units after 9 weeks, w h i c h w a s slightly higher than the titer (4.24 l o g units) i n d u c e d b y D O D A C c o n t a i n i n g P o r proteoliposomes, but w a s not statistically significant (P>0.05) ( F i g u r e 5 . 4 A ) . H o w e v e r , the titer elicited b y free P o r c o n t r o l w a s s i g n i f i c a n t l y higher (P<0.05) than the titers, 3.83 and 3.90 l o g units, i n d u c e d b y P O P C and P O P S - c o n t a i n i n g P o r proteoliposomes. There w a s no significant difference b e t w e e n the a n t i b o d y titers generated b y the neutral and a n i o n i c proteoliposomes. neutral  F o r the i d . route o f i n o c u l a t i o n , free P o r protein, c a t i o n i c and  proteoliposome formulations  elicited  titers  o f 4 . 8 1 , 4.66  and  4.58  l o g units,  respectively, but w e r e not significantly different (P>0.05) ( F i g u r e 5 . 4 B ) . I n contrast, a n i o n i c proteoliposomes i n d u c e d a s i g n i f i c a n t l y l o w e r a n t i b o d y titer o f 4.13 l o g units (P<0.05).  140  Figure 5.4: A n t i - P o r I g G titers o f m i c e i m m u n i z e d w i t h P o r p r o t e i n preparations. Intraperitoneal and B ) Intradermal i m m u n i z a t i o n .  A)  H B S c o n t r o l ( O ) , c o n t r o l P O P C (A),  control P O P C / P O P S ( • ) , control P O P C / D O D A C ( V ) , Free P o r control ( • ) , P O P C / P o r ( • ) , POPC/POPS/Por (•), POPC/DODAC/Por (•).  A n t i b o d y titer was determined b y t a k i n g the  l o g o f the r e c i p r o c a l d i l u t i o n that gave an absorbance o f 0.2. E a c h data point represents the m e a n titer o f eight animals per t i m e point.  142  Control  l i p o s o m e s d i d not generate an appreciable  a n t i b o d y titer b y  either  route  of  inoculation. T h e effect o f the route o f a d m i n i s t r a t i o n w a s e x a m i n e d for the four P o r preparations. A t 9 w e e k s , neutral and c a t i o n i c p r o t e o l i p o s o m e s i n d u c e d s i g n i f i c a n t l y h i g h e r a n t i b o d y titers w h e n administered i n t r a d e r m a l l y (P<0.05).  T h e route o f i n o c u l a t i o n d i d not p r o d u c e any  significant difference i n titers i n d u c e d b y free P o r protein (P>0.05).  S i m i l a r l y , there w a s no  difference between a n t i b o d y responses for a n i o n i c p r o t e o l i p o s o m e s e l i c i t e d b y either i.p. or i d . injection.  5.2.4 A n t i b o d y i s o t y p i n g o f i m m u n e sera In a d d i t i o n to d e t e r m i n i n g the a n t i b o d y titers p r o d u c e d b y the proteoliposomes, the i m m u n e serum w a s characterized for a n t i b o d y subclasses present; m o r e specifically,  the  relative amounts o f I g G l or I g G 2 a w e r e determined. I n the case o f m i c e i n o c u l a t e d intraperitoneally w i t h free o r cationic P o r p r o t e o l i p o s o m e s , sera obtained at nine w e e k s d i d not exhibit a p r e d o m i n a n c e o f I g G l o r I g G 2 a ( F i g u r e 5 . 5 A ) .  H o w e v e r , sera from m i c e  i n o c u l a t e d w i t h neutral ( P O P C ) p r o t e o l i p o s o m e s c o n t a i n e d a s i g n i f i c a n t l y higher fraction o f I g G l antibodies than I g G 2 a (P<0.05).  Conversely, anionic P o r proteoliposomes induced an  a p p r o x i m a t e l y t w o - f o l d greater amount o f I g G 2 a antibodies c o m p a r e d to I g G l  (P<0.05).  U p o n i d . i n o c u l a t i o n , e x a m i n a t i o n o f the i m m u n e sera indicates a shift t o w a r d s higher I g G 2 a / I g G l ratios from 1.7:1 to 2.2:1 ( F i g u r e 5 . 5 B ) . It can be seen that sera i n d u c e d b y free P o r and c a t i o n i c P o r p r o t e o l i p o s o m e s consisted o f a t w o - f o l d higher l e v e l o f I g G 2 a c o m p a r e d to I g G l ; however, the relative difference w a s not statistically significant (P>0.05).  Similarly,  neutral and a n i o n i c p r o t e o l i p o s o m e i m m u n e sera c o n t a i n e d a s i g n i f i c a n t l y h i g h e r (P<0.05)  143  Figure 5.5: Effect o f i m m u n i z a t i o n route o n I g G isotypes o f a n t i - P o r antibodies. A ) Intraperitoneal and B ) Intradermal i m m u n i z a t i o n . A b s o r b a n c e at 4 9 0 g i v e s a measure o f the relative concentrations o f the I g G l and I g G 2 a antibody subclasses. D a t a represent the m e a n ± S E M o f five animals.  145  p r o p o r t i o n o f I g G 2 a antibodies c o m p a r e d t o I g G l .  T h e ratio o f the t w o antibody subclasses  m a y be i n d i c a t i v e o f the type o f i m m u n e response that is b e i n g i n d u c e d . A h u m o r a l response is associated w i t h elevated levels o f I g G l ,  whereas  a predominant  I g G 2 a response  is  i n d i c a t i v e o f a c e l l - m e d i a t e d i m m u n e response ( M o s m a n n and C o f f m a n , 1989).  5.2.5 H i s t o l o g y It w a s  observed  that animals r e c e i v i n g the  cationic proteoliposomes  exhibited  i n f l a m m a t i o n at the site o f intradermal i n j e c t i o n that w a s absent for the other p r o t e o l i p o s o m e and e m p t y l i p o s o m e formulations.  H i s t o l o g y o f the  skin  s h o w e d that empty,  control  l i p o s o m e s d i d not i n d u c e l o c a l i n f l a m m a t i o n or the i n f i l t r a t i o n o f l e u k o c y t e s to the i n j e c t i o n site and that the h i s t o l o g i c appearance o f the s k i n w a s s i m i l a r to that o f n o r m a l , n a i v e tissue (Beyaert etal., 1991). In contrast, h o w e v e r , cationic p r o t e o l i p o s o m e i m m u n i z a t i o n p r o d u c e d an intense l o c a l i m m u n e response, characterized b y the large i n f i l t r a t i o n o f neutrophils, mast cells and, to a lesser degree, e o s i n o p h i l s i n response to the D O D A C , i n association w i t h P o r protein antigen ( F i g u r e 5.6).  N e u t r o p h i l s w e r e observed as cells w i t h m u l t i - l o b e d dark,  violet-stained n u c l e i surrounded b y a lighter, p i n k - s t a i n e d c y t o p l a s m .  M a s t cells w e r e also  seen at the site o f injection, w h i c h w e r e characterized b y deep, v i o l e t staining o f the c y t o p l a s m and the nucleus, such that the nucleus c o u l d not be d i s t i n g u i s h e d cytoplasm.  from  the  O t h e r subsets o f l y m p h o c y t e s that w e r e observed w e r e m o n o c y t e s (histocytes)  and e o s i n o p h i l s . E o s i n o p h i l s w e r e identified b y the presence o f a b i - l o b e d nucleus, whereas m o n o c y t e s were characterized b y an indented, horseshoe-shaped  nucleus.  These  results  suggest that cationic l i p i d , D O D A C , m a y be t o x i c to the cells and thus c a u s i n g the l o c a l i n f l a m m a t i o n seen at the injection site.  146  Figure 5.6: P h o t o g r a p h magnification  o f s k i n cross-section o f the intradermal injection site v i e w e d at 4 0 x  A ) P O P C l i p o s o m e and B ) P C / D O D A C / P o r l i p o s o m e i m m u n i z a t i o n .  147  5.3  DISCUSSION  Liposomal vaccine preparations have been shown to elicit humoral (Therien et 1990) and cellular immune responses (Garcon and Six, 1991).  al,  Research using chemical  markers has demonstrated that mammalian cell membranes exhibit a net negative charge (Danon et al., 1972). Work on murine peritoneal macrophages demonstrated that cationic ferritin was internalized whereas native or anionic ferritin was not, suggesting that the negative charge on the cell surface influences particulate phagocytosis (Mutsaers and Papadimitriou, 1988).  Extrapolation o f these results suggests that the surface charge on  liposomes may effect phagocytosis by macrophages, and thus influence the type o f immune response.  In addition, experiments with rat peritoneal macrophages  have shown that  liposome-cell association can be enhanced with an increase in positive surface (Schwendener et al., 1984).  charge  Studies conducted by Nakanishi et al. (1997) showed that  positively charged liposomes were taken up more efficiently by macrophages and were more potent inducers o f humoral and cell-mediated immunity than neutral or negatively charged carriers. The  antibody-binding activities of various  Por  liposome  formulations  were  determined  using both anti-Por monoclonal antibodies with known reactivity against  gonococcal  organisms  and  an  immunized rabbit  serum.  The results  showed  that  proteoliposomes prepared from P O P C with M S 1 1 Por exhibited high antibody binding activity with several monoclonal anti-Por antibodies, as well as with rabbit immune serum. A l l the Por proteoliposome formulations prepared, using the methodology described herein, exhibited consistently higher levels o f antibody binding compared to systems prepared from P O P C P O P E (1:4), as described previously (Wetzler et al, 1992) (Figure 5.1 and 5.2).  148  E L I S A assays w e r e used t o determine the effect o f l i p o s o m e charge o n in vitro antibody b i n d i n g . D e s p i t e the higher protein i n c o r p o r a t i o n e f f i c i e n c y that w a s observed f o r D O D A C - c o n t a i n i n g p r o t e o l i p o s o m e s described i n C h a p t e r 4, E L I S A assays i n d i c a t e d that neutral and negatively charged systems h a d s i m i l a r a n t i b o d y b i n d i n g activities a n d profiles. N e u t r a l ( P O P C ) P o r p r o t e o l i p o s o m e s h a d an o v e r a l l 5 0 % greater antibody b i n d i n g a c t i v i t y than c a t i o n i c P o r p r o t e o l i p o s o m e s  ( F i g u r e 5.3).  These results  suggest that  membrane  changes p r o d u c e d b y D O D A C and/or charge interaction w i t h n e g a t i v e l y charged residues i n the P o r protein reduced the surface presentation o f the epitope f o r antibody r e c o g n i t i o n . T h e antibody b i n d i n g data indicate that neutral P o r p r o t e o l i p o s o m e s have the greatest epitope presentation and thus a greater p r o p o r t i o n o f P o r protein i n its native c o n f o r m a t i o n . In vivo i m m u n i z a t i o n studies i n a m o u s e m o d e l i n d i c a t e d that free protein a n d l i p o s o m a l P o r formulations a l l i n d u c e d s i m i l a r antibody titers that were greater than the control empty l i p o s o m e formulations. I m m u n i z a t i o n v i a the intraperitoneal route resulted i n free P o r protein e l i c i t i n g the highest antibody titer f o l l o w e d b y c a t i o n i c l i p o s o m e s .  The  neutral and negatively charged p r o t e o l i p o s o m e s w e r e the next efficacious o f the preparations tested (Figure 5.4). F r e e P o r , cationic a n d neutral p r o t e o l i p o s o m e s e l i c i t e d s i m i l a r a n t i b o d y profiles w h e n a d m i n i s t e r e d v i a the intradermal route. A l t h o u g h p u r i f i e d P o r protein has been s h o w n to be i m m u n o g e n i c i n o u r study as w e l l as i n p r e v i o u s studies, a h u m o r a l response alone is not sufficient i n p r o v i d i n g p r o t e c t i o n against the g o n o c o c c a l o r g a n i s m .  W e t z l e r et  al. (1988) s h o w e d P o r inserted into l i p o s o m e s i n its native orientation e l i c i t e d antibodies that agglutinated intact organisms a n d had a h i g h e r b a c t e r i c i d a l a n d o p s o n i c a c t i v i t y than a l u m generated  sera.  I n addition, w h o l e c e l l a b s o r p t i o n studies a n d synthetic peptide  ELISA  assays i n d i c a t e d that p r o t e o l i p o s o m e antisera contained a higher percentage o f antibodies  149  against  the  surface-exposed  phagocytosis.  epitopes  of Por  protein  required  for  opsonization  and  W e t z l e r et al. (1992) further demonstrated p r o t e o l i p o s o m e - i n d u c e d a n t i - P o r  I g G had a greater functional r e a c t i v i t y than free P o r o r alum-generated sera against  surface  exposed epitopes o f protein I i n an intact g o n o c o c c a l o r g a n i s m . A l t h o u g h p o s i t i v e l y charged p r o t e o l i p o s o m e s w e r e effective i n e l i c i t i n g a h u m o r a l immune  response,  it w a s  observed  that  animals  r e c e i v i n g this  formulation exhibited  i n f l a m m a t i o n at the site o f intradermal i n j e c t i o n that w a s absent for the other p r o t e o l i p o s o m e and e m p t y  l i p o s o m e formulations.  H i s t o l o g y o f the  s k i n revealed that there w a s  an  infiltration o f neutrophils, e o s i n o p h i l s and m o n o c y t e s i n d i c a t i n g that D O D A C , i n association w i t h antigen, causes an intense l o c a l i m m u n e response.  Studies have s h o w n that cationic  l i p o s o m e s are h i g h l y t o x i c t o w a r d p h a g o c y t i c cells, such as macrophages, but not t o w a r d n o n - p h a g o c y t i c T l y m p h o c y t e s ( F i l i o n and P h i l l i p s , 1997).  M i c e r e c e i v i n g empty D O D A C  proteoliposomes d i d not exhibit any s y m p t o m s o f t o x i c i t y n o r w a s there any i n f l a m m a t i o n at the site o f injection.  These results m i g h t suggest that empty D O D A C l i p o s o m e s do not  trigger an i m m u n e response; h o w e v e r , the presence o f a f o r e i g n antigen, P o r , m a y initiate phagocytosis and a subsequent c y t o k i n e response r e s u l t i n g i n the m a r k e d i n f i l t r a t i o n o f macrophages  at the injection site.  U p o n contact or uptake o f the cationic l i p o s o m e s ,  macrophages may process and present the antigen o n the M H C m o l e c u l e to elicit an i m m u n e response; h o w e v e r , the presence o f c a t i o n i c l i p i d m a y cause some c e l l t o x i c i t y . toxicity may  also trigger the  Macrophage  i n d u c t i o n Of c y t o t o x i c T l y m p h o c y t e s and thus  enhancing the i m m u n e response.  A s i n the case o f a l u m , i n f l a m m a t i o n at the site o f  i n o c u l a t i o n suggests that cationic p r o t e o l i p o s o m e s w o u l d be undesirable as a vaccine.  further  candidate  150  A l t h o u g h a h u m o r a l i m m u n i t y ( F A ) m a y afford protection against an extracellular pathogen,  c e l l - m e d i a t e d i m m u n i t y ( C M I ) w o u l d be required to combat  challenge.  an intracellular  I n order for a C M I response to o c c u r , the antigen has t o be  endogenously  processed into the c y t o s o l and presented b y the major h i s t o c o m p a t i b i l i t y c o m p l e x ( M H C ) class I m o l e c u l e s o n antigen presenting cells, s u c h as macrophages ( B r a c i a l e et al, 1987 and D a l M o n t e and S z o k a , 1989). taken u p and degraded  P r e v i o u s studies have suggested that l i p o s o m a l antigen is  i n l y s o s o m e s and then r e c y c l e d to endosomes and  subsequently  presented to T - c e l l s i n association w i t h M H C - c l a s s II m o l e c u l e s ( H a r d i n g et al, 1991). T h e i r research suggests that l i p o s o m a l i m m u n i z a t i o n favors the i n d u c t i o n o f H I o v e r C M I . H o w e v e r , other research has s h o w n that l i p o s o m a l antigen does enter the c y t o p l a s m and is processed i n the t r a n s - G o l g i o f m u r i n e macrophages, whereas free s o l u b l e antigen is l i k e l y degraded i n endosomes and is unable to reach the t r a n s - G o l g i ( R a o et al, 1997). results  may  explain w h y  l i p o s o m a l antigens  have  been  able  to  induce  These  cytotoxic  T  l y m p h o c y t e s ( C T L ) , whereas free soluble antigen d i d not ( R e d d y et al, 1992). M o r e o v e r , the type o f i m m u n e response has been antibody subclass.  associated  with a  particular  T y p i c a l l y , T h l type responses are characterized b y C T L s and h i g h levels  o f I g G 2 a i n d u c t i o n , whereas higher I g G l levels associated w i t h v e r y little o r no i n d u c t i o n o f C T L s are i n d i c a t i v e o f T h 2 type responses ( M o s m a n n and C o f f m a n , 1989).  Studies o f D N A  v a c c i n e s have demonstrated the balance b e t w e e n the T h l - and T h 2 - t y p e response ( C h o w et al, 1998).  M i c e i m m u n i z e d w i t h a hepatitis B v i r u s ( H B V ) D N A v a c c i n e and a T h l  c y t o k i n e gene w e r e s h o w n to have enhanced T h l c e l l s and C T L s w i t h c o n c o m i t a n t increase i n I g G 2 a antibodies. these observations.  A m a r k e d r e d u c t i o n o f T h 2 cells and I g G l antibodies a c c o m p a n i e d C o n v e r s e l y , c o - i n j e c t i o n o f H B V D N A v a c c i n e and a T h 2 c y t o k i n e gene  151  produced  an increase  differentiation demonstrated  and  i n T h 2 c e l l s and  reduction  in  IgG2a  IgGl  levels w i t h a suppression  production.  Others  researchers  of T h l  cell  have  also  the balance between T h l and T h 2 type responses ( K o s t e n s e et al,  1998).  S o m e investigators have also suggested that the route o f a d m i n i s t r a t i o n o f a v a c c i n e m a y influence the i m m u n o g l o b u l i n subclass and c y t o k i n e response (Pertmer et al, and S u n g , 1998).  1996 and L e e  T h e s k i n has a p o p u l a t i o n o f h i g h l y efficient A P C , such as  dendritic cells and L a n g e r h a n s ' c e l l s ( A n j e u r e et al,  1999).  dermal  Dendritic cells ( D C ) have  essential function i n the d e v e l o p m e n t o f the i m m u n e response against m i c r o b i a l pathogens, as w e l l as tumors. A n t i g e n s t i m u l a t i o n o f D C leads to the a c t i v a t i o n and m o b i l i z a t i o n o f D C , w h i c h c a n migrate and carry antigen f r o m the s k i n to the T h cells located i n the l y m p h nodes. D N A v a c c i n a t i o n studies have i n d i c a t e d that cutaneous D N A i m m u n i z a t i o n c a n activate and m o b i l i z e D C , s t i m u l a t i n g t h e m to p r o d u c e I L - 1 2 , a T h l - t y p e c y t o k i n e , and thus t r i g g e r i n g a T h l - p r e d o m i n a n t i m m u n e response (Jakob  etal,  1998 and J a k o b  etal,  1999).  T h e results o f the i m m u n e s e r o t y p i n g s h o w e d that u p o n intraperitoneal injection, there was either no predominant  a n t i b o d y subclass  or a tendency  t o w a r d higher  antibody levels, as seen for the neutral p r o t e o l i p o s o m e s ( F i g u r e 5.5). i m m u n i z a t i o n , a shift t o w a r d higher I g G 2 a / I g G l ratios w a s observed  After  IgGl  intradermal  for each  o f the  p r o t e o l i p o s o m e formulations, i n d i c a t i n g that a T h l type response m a y be prevalent.  These  results might suggest that the intradermal route o f v a c c i n a t i o n m a y be effective for targeting D C to induce a more predominant  T h l type response and hence the i n d u c t i o n o f C T L  response for protection against intracellular infection. H o w e v e r , detailed studies o f c y t o k i n e responses and C T L assays are required to further characterize the c e l l - m e d i a t e d i m m u n e response.  152  CHAPTER 6 SUMMARY  T h i s thesis describes the d e v e l o p m e n t a n d characterization o f l i p o s o m a l g o n o c o c c a l subunit  v a c c i n e formulations.  g o n o c o c c a l outer membrane  Experiments  examined  the i n c o r p o r a t i o n o f the  protein ( P o r ) into l i p o s o m e s a n d characterized the  i n f l u e n c i n g protein reconstitution.  I n particular, the effects  major factors  o f p r o t e i n - l i p i d ratio,  lipid  species, l i p o s o m e charge, duration o f d i a l y s i s a n d detergent properties w e r e e x a m i n e d . F o r comparison,  a m e n i n g o c o c c a l protein  preparation,  proteins, w a s reconstituted into l i p o s o m e s .  representing  a mixture  o f different  T h e b i o p h y s i c a l properties o f reconstituted P o r  proteoliposomes w e r e characterized i n terms o f protein orientation w i t h i n the l i p i d b i l a y e r , as w e l l as v e s i c l e size and m o r p h o l o g y .  F u r t h e r m o r e , studies characterized the in vitro and in  vivo antigenicity o f P o r proteoliposomes. G o n o c o c c a l membrane protein, P o r , w a s reconstituted into l i p o s o m e s u s i n g detergent dialysis.  T h e rate o f detergent r e m o v a l w a s observed to be m o r e rapid f o r n o n - i o n i c , o c t y l  g l u c o p y r a n o s i d e ( O G P ) than for the i o n i c detergent s o d i u m cholate.  R e s i d u a l levels o f  detergents after extended d i a l y s i s w e r e b e l o w the c r i t i c a l m i c e l l e concentration ( C M C ) , at concentrations  that are u n l i k e l y t o affect  proteoliposome  stability.  Cholate-mediated  reconstitution resulted i n i n c o m p l e t e P o r protein i n c o r p o r a t i o n into l i p o s o m e s , whereas a l l o f the protein w a s reconstituted into the l i p i d b i l a y e r d u r i n g O G P - m e d i a t e d reconstitution at p r o t e i n - l i p i d ratios o f 0.01:1.  T h i s difference m a y result f r o m the s l o w e r rate o f r e m o v a l o f  s o d i u m cholate d u r i n g d i a l y s i s a n d hence the greater o p p o r t u n i t y f o r protein and/or denaturation p r i o r to b i l a y e r formation.  aggregation  T h e results o f this research demonstrate that  153  detergent properties influence p r o t e i n i n c o r p o r a t i o n and thus the c h o i c e o f detergent for production  o f proteoliposomes  must  be  taken  into  account  when  characterizing  the  reconstitution process. Studies were p e r f o r m e d to e x a m i n e the effects o f p r o t e i n - l i p i d ratios o n protein i n c o r p o r a t i o n efficiency.  It w a s o b s e r v e d that for a g i v e n l i p i d c o n c e n t r a t i o n there is a f i x e d  amount o f protein that can be i n c o r p o r a t e d into the l i p i d b i l a y e r . R e c o n s t i t u t i o n experiments w i t h p r o t e i n concentrations above this l e v e l resulted i n increased amounts o f u n i n c o r p o r a t e d protein as observed f o l l o w i n g  i s o p y c n i c density gradient centrifugation.  To maximize  protein insertion into the b i l a y e r , the d e v e l o p m e n t o f an effective subunit v a c c i n e requires determination o f the factors for o p t i m a l i n c o r p o r a t i o n and surface presentation o f the protein antigen.  E f f i c i e n t use o f p u r i f i e d p r o t e i n antigens w o u l d reduce the amount o f antigen  required and hence decrease v a c c i n e costs. A n o t h e r aspect o f protein reconstitution e x a m i n e d i n this research related to v e s i c l e size and m o r p h o l o g y .  U s i n g b o t h quasi-elastic light scattering ( Q E L S ) and c r y o - e l e c t r o n  microscopy  reconstituted  (CTEM),  proteoliposomes  were  found  to  be  heterogeneous  structures v a r y i n g i n l a m e l l a r i t y and shape w i t h m e a n diameters i n i n excess o f 300 n m . potential  vaccine  for  human  use  would  have  to  be  sterilized  during  A  preparation.  C o n v e n t i o n a l s t e r i l i z a t i o n methods, such as steam s t e r i l i z a t i o n or i o n i z i n g radiation, w o u l d l i k e l y be unsuitable due to protein and l i p i d degradation.  Therefore, t e r m i n a l filtration o f  proteoliposomes t h r o u g h 0.2 u m filters w a s e x a m i n e d as a potential s t e r i l i z a t i o n procedure. H o w e v e r , reconstituted samples w e r e i n i t i a l l y t o o large to be s t e r i l i z e d b y  filtration  and  therefore it w a s necessary to size-reduce the p r o t e o l i p o s o m e s p r i o r to t e r m i n a l filtration. A g a i n , the techniques o f Q E L S and C T E M w e r e used to c o n f i r m that p r o t e o l i p o s o m e s c o u l d  154  be size-reduced to about 100 n m i n diameter u s i n g an e x t r u s i o n p r o c e d u r e w i t h m i n i m a l loss o f protein and p h o s p h o l i p i d . These size-reduced systems w o u l d then be suitable for t e r m i n a l filtration  t h r o u g h 0.2 p m filters.  F u r t h e r m o r e , C T E M revealed that a p p r o x i m a t e l y 7 0 % o f  the size-reduced p r o t e o l i p o s o m e s w e r e u n i l a m e l l a r . T h e research presented i n this thesis e x a m i n e d the effects o f l i p i d species and charge o n protein i n c o r p o r a t i o n and P o r antigenicity.  R e c o n s t i t u t i o n studies w i t h b i l a y e r - ( P O P C )  and n o n - b i l a y e r - f o r m i n g ( P O P E ) l i p i d s s h o w e d that there w e r e n o differences i n p r o t e i n i n c o r p o r a t i o n efficiencies, o r the orientation o f P o r w i t h i n the l i p i d b i l a y e r s , for different lipid  formulations.  In addition, in  vitro  antibody b i n d i n g experiments  i n d i c a t e d that  proteoliposomes c o n s i s t i n g o f neutral b i l a y e r f o r m i n g l i p i d ( P O P C ) c o m p o s i t i o n s e x h i b i t e d greater antibody b i n d i n g a c t i v i t y and cross-reactivity w i t h v a r i o u s strain s p e c i f i c a n t i - P o r antibodies than reconstituted systems c o n t a i n i n g P O P C P O P E mixtures. POPCPOPE  Por  proteoliposomes  e x h i b i t e d greater  antibody  B o t h P O P C and  b i n d i n g a c t i v i t y than  a  reconstituted system prepared as described i n earlier p u b l i c a t i o n s f r o m a different research group.  M o r e o v e r , the P o r formulations d e v e l o p e d i n the present research e x h i b i t e d h i g h  reactivity w i t h rabbit i m m u n e serum, further i n d i c a t i n g that there is a h i g h degree o f surface exposure o f the antibody r e c o g n i t i o n site. P o r protein w a s also reconstituted into l i p o s o m e s c o n t a i n i n g a n i o n i c o r cationic l i p i d s to characterize the  effect  o f charge  o n protein i n s e r t i o n e f f i c i e n c y and the  properties o f the resulting p r o t e o l i p o s o m e s .  antigenic  F o r P o r proteoliposomes containing 5 w e i g h t %  a n i o n i c l i p i d ( P O P S or P O P G ) , c o m p a r a b l e protein i n c o r p o r a t i o n efficiencies w e r e c o m p a r e d to neutral proteoliposomes.  seen  Increasing the w e i g h t % c o m p o s i t i o n o f a n i o n i c l i p i d  resulted i n a decrease i n i n c o r p o r a t i o n e f f i c i e n c y . I n contrast, there w a s essentially c o m p l e t e  155  protein i n c o r p o r a t i o n w h e n P o r w a s reconstituted into cationic l i p o s o m e s c o n t a i n i n g u p to 25 weight% D O D A C .  Subsequent  in vitro a n t i b o d y b i n d i n g studies revealed that  neutral  proteoliposomes e x h i b i t e d greater b i n d i n g a c t i v i t y than n e g a t i v e l y o r p o s i t i v e l y c h a r g e d systems.  T h i s m a y indicate that neutral p r o t e o l i p o s o m e s have a greater surface exposure o f  the epitope for antibody r e c o g n i t i o n a n d b i n d i n g o r that antibody b i n d i n g is i n h i b i t e d b y electrostatic effects. Studies w e r e then c o n d u c t e d  i n m i c e t o evaluate the i m m u n e response  to P o r  p r o t e o l i p o s o m e formulations. These studies c o m p a r e d a n t i b o d y titers o v e r three i n o c u l a t i o n s b y either intraperitoneal  o r intradermal  injection o f proteoliposomes  o f differing lipid  c o m p o s i t i o n a n d free P o r protein. T h e results o f in vivo i m m u n i z a t i o n o f m i c e s h o w that free protein, neutral a n d c a t i o n i c p r o t e o l i p o s o m e s e l i c i t e d s i m i l a r antibody titers that w e r e greater than titers i n d u c e d b y a n i o n i c p r o t e o l i p o s o m e s b y either route o f administration. A l t h o u g h these results indicate that each o f the p r o t e o l i p o s o m e f o r m u l a t i o n s a n d free P o r protein are effective at i n d u c i n g a n antibody titer, a h i g h titer alone m a y not be sufficient for protection against infection. C o r r e l a t i o n o f the in vitro a n d in vivo results indicate that efficient epitope presentation and h i g h a n t i b o d y titers w o u l d l i k e l y i n d u c e protective i m m u n i t y . T h e fact that free protein induces a h i g h antibody titer m a y not be sufficient f o r e l i c i t i n g protective i m m u n i t y , because antibodies generated native P o r protein c o n f o r m a t i o n .  against denatured protein m a y not r e c o g n i z e the  Therefore, an effective a n t i b o d y titer must c o n t a i n a h i g h  concentration o f antibodies directed against the correct surface epitope i n order to trigger o p s o n i z a t i o n a n d p h a g o c y t o s i s o f the g o n o c o c c a l pathogen.  T h e a n t i b o d y b i n d i n g assays  indicate P O P C p r o t e o l i p o s o m e s to be the most effective f o r m u l a t i o n i n terms o f epitope presentation a n d i n d u c t i o n o f effective a n t i b o d y titers.  156 Immune  sera w e r e characterized w i t h regard to the  specific a n t i b o d y  serotypes  generated b y i m m u n i z a t i o n w i t h P o r p r o t e o l i p o s o m e s o f different l i p i d c o m p o s i t i o n and free P o r protein. T h e ratios o f I g G l and I g G 2 a serotypes m a y indicate whether a h u m o r a l o r c e l l mediated i m m u n e response is predominant. elicited  higher  I g G 2 a titers  and  an  Intradermal i n o c u l a t i o n w i t h p r o t e o l i p o s o m e s  increase  i n the  ratio  of  IgG2a/IgGl,  whereas  intraperitoneal i m m u n i z a t i o n i n d u c e d greater I g G l titers than I g G 2 a . These f i n d i n g s suggest that p r o t e o l i p o s o m e i m m u n i z a t i o n v i a the intradermal route enables antigen d e l i v e r y to the cytoplasm  and  subsequent  antigen  processing  h i s t o c o m p a t i b i l i t y c o m p l e x class I m o l e c u l e .  and  presentation  to  the  major  Therefore, p r o t e o l i p o s o m e s m a y be potential  inducers o f c e l l - m e d i a t e d i m m u n i t y and associated c y t o t o x i c T l y m p h o c y t e s ( C T L s ) that are required for c o m b a t i n g intracellular infections. T h e research presented i n this thesis outlines the d e v e l o p m e n t and characterization o f a l i p o s o m a l g o n o c o c c a l subunit v a c c i n e . T h e experiments described herein demonstrate the conditions for effective and o p t i m a l i n c o r p o r a t i o n o f a m e m b r a n e p r o t e i n antigen. A n t i g e n i c characterization o f these p r o t e o l i p o s o m e s suggests that this system m a y have potential as a l i p o s o m a l subunit v a c c i n e against Neisseria gonorrhoeae.  T h e p r o m i s i n g results reported i n  this thesis w i l l h o p e f u l l y l e a d to further characterization o f this preparation i n terms o f protection against g o n o c o c c a l challenge, i n d u c t i o n o f serum l y s i s and k i l l i n g o f intact organisms, as w e l l as i n d u c t i o n o f C T L s for c o n f e r r i n g protection against i n t r a c e l l u l a r infection, e s p e c i a l l y at m u c o s a l sites.  U l t i m a t e l y , it is h o p e d that such a candidate subunit  v a c c i n e w o u l d be used c l i n i c a l l y i n the p r e v e n t i o n or c o n t r o l o f g o n o c o c c a l infection.  157  REFERENCES A h m e d , R . and G r a y , D . (1996) I m m u n o l o g i c a l understanding their relation. 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