Tick-borne encephalitis virus (TBEV) is a serious viral infection that affects the central nervous system. It was estimated that between 1990 and 2009 a total of 169,937 cases of TBE were recorded in Europe. TBEV belongs to genus Flavivirus that comprises over 70 viruses, many of them are important human pathogens. Most members are either transmitted by mosquitoes e.g. West Nile virus (WNV), Dengue virus (DENV) or ticks e.g. TBEV. Due to the extensive antigenic cross-reactivity among flaviviruses serological diagnosis of TBE infections is commonly difficult in areas where more than one virus type circulates. Particularly, a situation now exists in Europe, where TBEV and WNV are endemic in many countries Thus, this thesis focused on the one hand on optimization of serological test systems using recombinant envelope protein domain III (rED3). It represents domain 3 of the major antigen, the surface protein E, which additionally has been shown to induce flavivirus specific neutralizing antibodies. Therefore rED3 was expressed and purified and its application as antigen in ELISA for TBEV diagnosis was verified.
On the other hand a memory B-cell assay was established to analyse antiviral immunity after TBEV-vaccination. Here rED3 was used as antigen to determine the frequency of rED3 specific antibody-secreting cells (ASCs). Vaccination is the most effective method of preventing TBE disease and is currently recommended for all those who live and work or travel to areas of TBE endemicity. An essential requirement of any vaccine is the induction of long-term protective immunity. Several vaccines have defined levels of serum antibody (as measured by ELISA, haemagglutination inhibition test, or neutralisation test) that serve as correlates or surrogates of protective immunity. But this does not take account of vaccine induced memory B cells. Although, not providing direct protection against infection, they represent an important second line of immune defence that is initiated only if pre existing antibody levels are too low to prevent infection or if the invading pathogen is able to circumvent the pre-existing antibody response. A thorough understanding of the frequencies of antigen-specific memory B cells and their relationship with the antibodies in serum serological memory is likely to be critical to give information about the long-term efficacy of vaccine as well as its correlates of protection.
This thesis focuses on the establishment of a recombinant protein based ELISA and of a memory B cell assay for analysis of specific antiviral immunity after vaccination with the following two objectives: (1) Expression and purification of recombinant envelope protein domain 3 (rED3) and verification of its application as antigen in ELISA for TBEV diagnosis; (2) Establishment of a memory B cells assay using rED3 for determination of frequency of rED3 specific antibody-secreting cells (ASCs) in individuals vaccinated against TBE.
In this study, TBEV- and WNV-specific antigen ED3 was expressed in E. coli as MBP fusion proteins with C-terminal histidine tag using pMAL-c2x vector. By purification with amylose affinity chromatography followed by nickel affinity chromatography, highly purified TBEV rED3 and WNV-rED3 were obtained. Nevertheless, using TBEV-rED3 protein in Western Blot unspecific reaction with serum antibodies of negative serum was detected and a differentiation between WNV infection and TBEV was impossible, probably due to the MBP moiety.
However, using the purified rED3 protein as antigen in ELISA, TBE virus-specific antibodies were detected specifically. Twenty-three serum samples predefined as TBEV positive were tested positive by rED3-based ELISA and commercial IgG ELISA. Five predefined negative serum samples were tested negative by rED3-based IgG ELISA as well as commercial IgG ELISA. But cross reactivity of WNV and DENV positive sera was detected in 15 of 18 sera by commercial ELISA. On the other hand, these samples were found negative in TBEV rED3-based ELISA. Thus, TBEV-rED3-based ELISA allows a differentiation of infections caused by TBE serogroup and mosquito-borne flaviviruses but not the inactivated virus based commercial ELISA. Interestingly, compared to neutralisation test the specificity of rED3-based ELISA obtained 100% with a sensitivity of 91.6%. In contrast, the commercial ELISA obtained 100 % sensitivity but a low specificity with only 42.8%.
In order to determine frequency of antigen-specific antibody secreting cells (ASCs) produced by individuals who received the vaccination against TBE, peripheral blood mononuclear cells (PBMCs) were isolated from blood samples. Subsequently, memory B cells were activated with R848 (Resiquimod) and human recombinant IL-2 (hrIL-2) for 72 hours in 37°C, 5% CO2, 90% humidity. After 72 hours of incubation, Enzyme-Linked Immunospot (ELISPOT) detected antigen-specific memory B cells. In order to evaluate specificity of TBEV-rED3 in ELISPOT, other antigens including WNV-rED3, Maltose binding protein (MBP) and Influenza Nucleoprotein (NP) were included. Study subjects could be separated into two groups: last vaccination before 5 years or longer than 10 years. TBEV-rED3 specific ASCs could be detected in 11 of 12 TBE vaccinated individuals with different vaccination history and even low serum anti-TBE antibodies levels. TBEV-rED3 specific ASCs were found with frequency of ranging 0.016-0.188 % per total IgG ASCs and lower than frequency of Influenza-NP specific ASCs (between 0.012-0.51%). But TBE-specific memory B cells could be maintained for more than 20 years of post-vaccination. There was a significant difference in number of ASCs between vaccinated and non-vaccinated group (p<0.05). Interestingly, there was no significant difference between TBE vaccinated groups between 1-5 years (mean of 2.5 years) and >10 years (mean of 16.5 years) since vaccination (p>0.05). These finding proved that memory B cells have been stable for years and are maintained up to 25 years since last vaccination.
A statistic analysis showed that there was no significant correlation between serum levels of anti-TBEV antibodies and the frequency of rED3 specific IgG ASCs (p>0.05, Spearman’s coefficient r = 0.36). A similar result was also indicated for influenza-vaccinated individuals (p>0.05, Spearman’s coefficient r = 0.27). These findings revealed that memory B cells and plasma cells maybe play an independently role in maintaining of immunological memory. Anyway, neutralizing antibodies have been found in all vaccines (a Geometric mean titre (GMT) of 96.04, 95% confident interval (CI): 52.76-174.8) and thus were maintained for a long time since last vaccination. Interestingly, the quantitative determination of specific IgG in TBE post-vaccination sera by rED3-based ELISA exhibits a good correlation with neutralizing antibody titres. The presence of specific antibodies in rED3-based ELISA is therefore highly predictive for the presence of neutralizing antibodies, and this correlation can probably be used in the future to establish guidelines for recommendations of booster vaccinations. Additionally, it became apparent that the number of previous booster vaccinations correlated strongly with the frequency of circulating memory B cells. As expected, individuals who received a booster increased both the specific antibody titre and frequency of antigen specific memory B cells. This suggests that immunological long-term memory induced by booster immunizations is better reflected by the circulating memory B cells than the amount of the antibody titre. Thus, memory B cells seem to be a more reliable parameter for the assessment of long-term immunity.
Taken together, a highly antigenic rED3 using the bacterial expression system was produced and it is a promising alternative to whole inactivated virus in ELISA. Notably, rED3 was a reliable antigen for detecting antigen-specific memory B cells in individuals who have been previously TBEV vaccinated. This study provides data on immunological memory for TBE vaccination and might be useful for reconsideration of recommendations for booster dose.
In conclusion, boosters of vaccination should be recommended for all individuals who live and work or travel to areas of TBE endemicity. As consequently, vaccine-induced protection is enhanced by both strong humoral and cell-mediated immune responses. If pace of pathogenesis is rapidly growing, pre-existing virus-specific antibodies represent the first line of defence against infection before the memory response is fully activated and implemented. They clearly function best together to efficiently protect against disease.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa.de:bsz:15-qucosa-151733 |
| Date | 15 September 2014 |
| Creators | Le Van, Tuan |
| Contributors | Institut für Virologie, Medizinische Fakultät, Prof. Dr. med. Uwe Gerd Liebert, Prof. Dr. med. Uwe Gerd Liebert |
| Publisher | Universitätsbibliothek Leipzig |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | doc-type:doctoralThesis |
| Format | application/pdf |
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