UBC Theses and Dissertations
Comparative analysis of rubella specific antibody responses in congenitally and postnatally rubella infected humans : a model for selective tolerance Mauracher, Christoph Andreas
Rubella virus (RV) is an enveloped, positive sense, single stranded RNA virus in the family Togaviridae. The virus consists of a host derived lipid bilayer membrane, two trans-membrane proteins E1 and E2, and the C protein which together with viral RNA forms the icosahedral nucleocapsid. RV causes natural infections in humans only, where it can lead to a variety of pathological conditions. The most severe outcome, Congenital Rubella Syndrome (CRS), occurs when the fetus is infected in the first trimester of gestation. RV can be isolated from CRS patients at birth and is believed to establish a persistent infection. Although congenital RV infection remains of clinical importance it also provides a rare opportunity for studying the effects of intrauterine viral infection on the human fetus. This thesis examines the hypothesis that intrauterine infection with RV can establish immunological tolerance which is reflected in the serological response to RV. The study on RV-specific tolerance may help in the understanding by which mechanism self-recognition is established in the human. The induction of immunological tolerance may also be an important mechanism in viral persistence and play a role in chronic inflammatory disorders such as Rubella Associated Arthritis (RAA). A comprehensive study of the antibody mediated immunity of healthy individuals responding to postnatal infection with RV is compared to that of CRS patients. Assays were developed to measure antibody quantity, affinity and kinetics of the antibody response to whole virus as well as that to the individual RV structural proteins, E1, E2 and C. The viral proteins were purified from whole RV preparation by differential centrifugation followed by preparative SDS-PAGE under non-reducing conditions. Separated proteins were analyzed for their structural integrity by assaying their biological activity. RV protein-specific ELISAs were developed and used for antibody quantitation and in IgG affinity assays using the chaotropic elution technique. Biological activity of sera was assayed by HAl assay. The specificity of antibodies to linear and topographic epitopes was investigated by using reducing and non-reducing Western blotting. Observations made during the development of protein separation protocols have led to the description of an uncoating mechanism for RV. While establishing ELJSA protocols, improved buffers were developed by including heat denatured blocking proteins and studies on a novel anti-RV IgG ELISA, which will give a more accurate assessment of protective immunity than ELISA technology used to date, were initiated. Results indicate that CRS patients can produce similar amounts of IgG than control patients if measured by whole RV ELISA. Group differences were detected at the level of the protein specific responses. CRS patients exhibited significantly lower levels and had reduced affinities of E1-specific antibodies. The most consistent feature of this patient population was their inability to produce IgG to linear epitopes of the E1 protein. The potential role of the tolerization of anti-E1 Th cells due to the exposure of the immature fetal immune system to RV is discussed. A model is proposed which can accommodate the fmdings on serological and lymphoproliferative immune responses of CRS patients and which supports the hypothesis that congenital RV infection in the early gestational period leads to viral antigen tolerization. Tolerance to the immunodominant E1 protein may lead to a sufficient depression in responder T and B cells in order to allow chronic, low grade RV replication.
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