Hepatitis B Virus Core Protein As A Carrier For New Vaccines

The Hepatitis B virus core protein (HBc) is an excellent vaccine carrier for a number of reasons. It is able to self-assemble into particles in all viable prokaryotic and eukaryotic expression systems. It is highly immunogenic, is both a T cell-independent and a T cell-dependent antigen, exhibits a lack of genetic restriction, allows foreign insertion and is able to transfer these attributes to fused foreign sequences. It can display the foreign sequence on its outer surface, and ensure a high density of introduced sequence per particles. In addition, HBc particles are remarkably tolerant to modifications such as substitutions, additions, and deletions. It can be produced efficiently in E.coli. Chimeric HBc derivatives are easy to purify. So the major aim of this research is to utilize such advantages to establish the method of HBc used as a vaccine carrier with an epitope of anthrax protective antigen and an antigen of mycobacterium tuberculosis; to investigate the mechanism of HBc increasing the humoral immunity and cellular immunity; and to construct a versatile new vaccine platform for both humoral and cellular immune responses.A group of DNA fragments encoding different parts of the C-terminally truncated HBc (HBc-N144) were fused by overlap extension PCR. The synthesized HBc-N144 gene fragment was cloned into the BamHⅠand HindⅢdigested vector pET21a (+) to create the plasmid pET21a-HBc-N144. Based on the pET21a-HBc-N144, we inserted the DNA fragment encoding 2β2–2β3 loop of PA domain 2 into the major immunodominant region of the HBc DNA sequences by fusion PCR to generate the plasmid pET21a-HBc-N144-PA-loop2, which inserted 24 aa (302–325) of PA between aa 78 and 79 of HBc protein. 2β2–2β3 loop of PA domain 2 was identified to induce lethal factor (LF) / edema factor (EF) into the cytosol, the binding of protective antigen (PA) to the anthrax toxin acceptor, and it is believed to contain a neutralizing epitope of PA"SFFD".Then, the recombinant proteins were detected by Western blotting using a monoclonal antibody (mAb) against the polyhistidine tag in the C-terminal region of the fusion protein or a mAb against 2β2–2β3 loop of PA domain 2. As expected, both HBc-N144 and HBc-N144-PA-loop2 protein bands were detectable when anti-His tag antibody were used as primary antibody, while only HBc-N144-PA-loop2 was detectable when 5E12, a mAb binding to 2β2–2β3 loop of PA domain 2 was used. The negative staining electron microscopy was used to observe if our recombinant proteins can form particles. The results showed that both the HBc-N144 and HBc-N144-PA-loop 2 were able to form particles with the size around 30nm. It is confirmed that the chimeric HBc protein harboring 24 amino acids of PA 2β2–2β3 loop still keeps the same physical characteristic as the original HBc particles. To determine the immunogenicity of chimeric HBc particles, we immunized mice with HBc-N144 and HBc-N144-PA-loop2, respectively. The results of serological tests from the mice models showed that the chimeric HBc particles were able to induce PA-epitope specific antibodies in mice. Some researches have shown that the guinea pig was a better animal model to evaluate the efficiency of anthrax vaccine compared to mouse. So we used guinea pigs to comprehensively evaluate the immunogenicity of HBc-N144-PA-loop2 with rPA and HBc-N144 as controls. We found the chimeric HBc particles were able to induce both PA-epitope specific antibodies and anthrax toxin neutralizing antibodies in guinea pigs, and in the experiment of subcutaneously live Bacillus anthracis spores challenge, we found the HBc-N144-PA-loop2 could at least partially protect the immunized guinea pigs against the 40LD50 virulent anthrax spores challenge. However, previously, in the phage immunization, mice immunized with an amplified phage clone containing such"SFFD"sequence were only showing anti-PA antibodies. Besides, we also oberseved some adjuvant effects of HBc, chimeric HBc particles were able to induce PA-epitope specific antibodies and neutralizing antibodies regardless of whether alum adjuvant was used or not in guinea pigs, the HBc-N144-PA-loop2 without adjuvant can provide 57% protection of guinea pigs. However, compared to recombinant PA group, the anti-PA antibodies titer, neutralizing antibodies titer and the protection of HBc-N144-PA-loop2 were all needed to be increased. Since PA is an 83kDa protein presumably containing many toxin-neutralizing epitopes, while HBc-N144-PA-loop2 only contains one of them, the results is still promising for the vaccine development especially for the no adjuvant approach.To improve the protective effect of the HBc-N144-PA-loop2 as an epitope vaccine, we can optimize the epiotpes'arrangement, modify the molecular structutre of HBc, and induce other anthrax antigen, or change some potent new adjuvant etc. To enhance the ability of HBc to helpful increase the foreign sequence specific immunity response, we need to modify the molecular structutre of HBc, especially deletion some amino acids of the major immunodominant region of HBc. In addition, E.coli expressed HBc protein assembles to give two different sizes of shell, composed of 180 or 240 subunits arranged with T= 3 or T = 4 icosahedral symmetry, respectively, and with subunits paired to produce dimeric spikes protruding from the surface of the shell. Zlotnick etc found that the T = 4/T =3 ratio of HBc particles appears to increase as the C-terminus extending from 144 to 149. Previously, we proved the chimeric HBc particles HBc-N144-PA-loop2 could provide protective immunity, so based on pET21a-HBc-N144-PA-loop2, by inverse PCR, we deleted the 79-81 amino acids of HBc and added 145-149 amino acids of original HBc to the C-terminal of HBc-N144-PA-loop2, respectively. Besides, the extra sequence from plasmid pET21a and His tag contained by HBc-N144-PA-loop2 were removed, so we got a H144L2, the deletion mutant H144DL2 and the extendion mutant H149L2. By negative staining electron microscopy we still observed the particles formed by mutated chimeric HBc protein. By the differences of the immunity response in mouse model brougt by the H144L2, H144DL2 and H149L2, we evaluated the modification effect of HBc. Both anti-HBc antibodies and anti-PA antibodies could be detected in the serum from these mutated chimeric HBc proteins. H144DL2 could increase the anti-PA antibodies, and significantly reduced the anti-HBc antibodes comparing to HBc-N144 and the H144L2 group. Besides, the H144DL2 induced the second highest neutralizing antibodies of all, only less than rPA group, and the immunized serum from this group all could be detected the neutralizing antibodies, and the antibodies titer was resemble. In addition, H144DL2 could completely protect the immunized mice against the lethal dose anthrax toxin challenge.The H149L2 couldn't increase the anti-PA antibodies, and the anti-HBc antitodies increased a little, but it could significant impulse the neutralizing antibodies(comparing to HBc-N144 and the H144L2 group), all immunized serum from this group could be detected neutralizing antibodies, too. In spite of the lower antibodies titer than H144DL2 group, the H149L2 prolonged the death time of mice which be injected the lethal dose toxin. The results of adjuvant effect shown that H144L2 without adjuvant could induce the highest anti-PA antibodies comparing to H144L2 with aluminum hydroxide and H144L2 with Freund's adjuvant group, respectively, but the ability to induce neutralizing antibodies still needed to be increased. Though it couldn't completed protect immunized mice against the anthrax toxin challenge, H144L2 without adjuvant also could prolong the death time of mice, and could provide 50% protection. This suggested that the H144DL2 without adjuvant maybe a better HBc/anthrax epitope vaccine formation. Besides, we optimized the purification methods for recombinant proteins prepation, and avoiding the interference of LPS.On the other hand, we also utilized the antigen of mycobacterium tuberculosis, ESAT-6 to investigate the mechanism of HBc increasing the cellular immunity. We used the plasmid PAS22-ESAT-6 and the pET21a-H144L2 as templates, and the enzyme digest site MscⅠin the HBc DNA sequence was used to bring the full length of ESAT-6 gene (288bp) into the HBc. We also used western blotting to detect the binding of HBc/ TB subunits vaccine HE6 to the anti-HBc antibodies and anti-ESAT-6 antibodies. We proved the HE6 particles by negative staining electron microscopy. The protective immune responses against M. tuberculosis are primarily mediated by the cellular immune system, and the CD4+ Th1 cellular immunity response is the key point to the effect of TB vaccine. To determine the immunogenicity of HBc/ TB subunits vaccine HE6, we immunized mice with HE6. The results of serological tests from the mice models showed that the HBc/ TB subunits vaccine HE6 with or without aluminum hydroxide were able to induce ESAT-6 specific IgG2a and IgG1, and the antibodies titer were significant higher than rESAT-6 with aluminum hydroxide group.The rESAT-6 with aluminum hydroxide group induced the lowest IgG2a, it was about 2.81×102, this suggested that the rESAT-6 with aluminum hydroxide couldn't stimulate Th1 immunity response, effectively. Though, the IgG2a antibodies titer caused by HE6 with or without aluminum hydroxide groups were exceed 103, the ratio of IgG2a/ IgG1 wasn't significant, and it is suggested that the molecular structure of HE6 was needed to be optimized. The ELISPOT results showed that the HE6 with or without adjuvant group generated significantly increased number of IFN-γ-secreting T cells responding to specific stimulator rESAT-6 in comparison to single use rESAT-6 with aluminum hydroxide group or blank group. It demonstrated that ESAT-6 within the HBc can effectively induced specific IFN-γsecreting T cells. To further investigating the contributions of CD4+ and CD8+ T cells to the IFN-γ-production, intracellular cytokine staining assays were performed.The results showed that the increased frequencies of IFN-γ-secreting CD4+ and CD8+ T cell in HE6 without adjuvant group were significant higher than the single using rESAT-6 with aluminum hydroxide group and the blank group. The increased frequencies of IFN-γ-secreting CD4+ and CD8+ T cell in HE6 with adjuvant group were significant higher than the blank group. This result was consistent with the ELISPOT data. It suggested the HBc as a vaccine carrier could effectively increase the ESAT-6 specific cellular response, although the vaccine was needed to be optimized. The HBc is a promising vaccine carrier to increase the cellular immune response.In this reseach, we gained a better structure of HBc vaccine carrier (H144D), and optimized the purification methods, by the epitope of anthrax protective antigen. We proved that the HBc as a vaccine carrier could increase the anthrax epiotpe specific humoral immune response in mouse and anthrax epiotpe specific protective humoral immune response in guinea pig model. And by the antigen of mycobacterium tuberculosis, ESAT-6, we proved that the HBc as a vaccine carrier could increase the ESAT-6 specific cellular immune response. As a versatile vaccine platform for both humoral and cellular immune responses, HBc has a promising applied perspective.