Evolution of the envelope proteins E1 and E2 and of specific humoral immune response to these proteins in a group of patients infected by HCV in a single-source outbreak
Duisburg, Essen (2011), Vii, 145 Bl.
Dissertation / Fach: Biologie
Fakultät für Biologie
Medizinische Fakultät » Universitätsklinikum Essen » Institut für Virologie
Roggendorf, Michael (Doktorvater, Betreuerin)
The current study deals with several important aspects of HCV research, including evolution of the envelope proteins and dynamic of the humoral immune response during the long term virus persistence. The essential feature of the project was the use of a special group of patients - women infected with the same HCV strain (AD78) by a contaminated anti-D globulin in a single-source outbreak. This unique cohort of patients infected with the same virus strain allowed the minimization of factors, including route of transmission, size of inoculum, duration of infection, age and sex of infected individuals, playing a significant role in evolution of the virus during the chronic infection. Nevertheless, even under these dramatic reduction of factors affecting intrapatient viral evolution, the established parameters, such as a rate of selective pressure, as well as a pattern of mutations in the E1/E2 region, differed markedly between AD-patients. Analysis of the rate and character of nucleotide and amino acid substitutions in the E1/E2 region revealed no evidence for a strong positive selection, while a high frequency of reverse mutations to a consensus HCV 1b sequence was observed, indicating a critical role of functional/structural constrains in evolution of envelope proteins. The obtained data allowed to formulate an unequivocal conclusion that HCV envelope proteins evolution is a patient-specific phenomenon, and that purifying (negative) selection is the major force acting on HCV populations in chronic infection. Analysis of the character and localization of a.a. substitutions in envelope sequences of AD78 isolates obtained from AD-patients at different time points of chronic infection have shown that a significant part of mutations occurred in the HVRI, suggesting a possibilty of emergence of HCV variants escaping neutralization mediated by anti-HVRI antibodies. A significant number of mutations was also localized in known human mAbs targeted sites and, especially often, in CD-81-binding sites or, in other words, in the functionally relevant domains of the protein. These data indicated that in most AD-patients the whole ectodomain of E2 protein and not only the HVRI remains under evident selective pressure during the long term chronic infection. The other aim of the current study was the analysis of the dynamic of the HCV-specific humoral immune response in long term chronic patients. Application of the HCVpp system allowed detecting relatively high titres of cross-reacting and cross-neutralizing antibodies in sera of AD-patients; however, no evidence of immune escape during HCV persistence was registered. These experiments provided no evidence that humoral immune response lags behind the changes in envelope sequences of the virus circulating in the host at a given time point. In other words, our data indicated that the postulated mechanism of escape from antibody-mediated neutralization is not operative in all chronic patients. The results of the current study suggested, however, that the absence of escape from neutralization of virions transmitted via cell-free route does not mean that another mechanism of immune escape, namely, the escape from anti-HVRI antibodies, which very efficiently neutralize a virus transmitted by a cell-to-cell route, is not operative and does not contribute to HCV persistence. Finally, a new interesting structural feature of envelope sequences of some HCV strains belonging to different subtypes and genotypes - presence of additional 1 to 4 a.a. residues at the N-terminus of the E2 protein - was studied using a set of hymeric E1/E2 plasmids, in which the additional a.a. residues were swapped with a number of natural or artificial a.a. stretches. These experiments have demonstrated that the presence of additional a.a. track may result in a significant increase of pseudoparticles infectivity suggesting that the viruses bearing E2 proteins with modified N-terminus might also posess unique biological properties in vivo.