| Human immunodeficiency virus(HIV-1),which have caused tens of thousands of infections and deaths all around the world, is severely threatening public health. Developing a safe and useful vaccine to fight against virus infection is always being an important part of study on HIV-1. However, several barriers stand in the way of designing such a HIV-1 vaccine, including the highly variability of virus genes, the ability of virus to escape from host immune system, the different distribution of virus subtypes in different geographical regions, and the genetic polymorphism of HLA molecules in humans. Although scientists worldwide have been making efforts to develop a vaccine against HIV-1 and have made some progression, the problem still remains to be solved. As a result, some new strategies need to be considered to promote research on HIV-1 vaccine design.In this study, we focused on developing an epitope-based HIV-1 candidate vaccine for Chinese population by taking the virus subtypes in mainland China and the Chinese dominant MHC alleles into account together. The optimal design of such a multi-epitope vaccine was performed using the selected epitopes which are highly conserved among viral subtypes prevalent in China and also matching Chinese dominant MHC restriction. Then, different types of mice were immunized to evaluate the vaccine immunogenicity, and the PBMCs samples from HIV-1 infected patients were further used to explore the applicability of the multi-epitope protein among Chinese population as a prophylactic and therapeutic vaccine. 1. Selection of HIV-1 epitopes restricted to Chinese dominant MHC alleles and design of a multi-epitope vaccine(1) Selection and identification of candidate epitopes. To obtain the ideal epitopes for vaccine design, the data from HIV DATABASES were summarized and analyzed. Candidate epitopes having the following characteristics were selected: 1) located in virus structural proteins Gag or Env or protein Pol; 2) highly conserved within virus subtypes prevalent in China(China B, CRF01_AE, CRF07_BC and CRF08_BC); 3) restricted to dominant Chinese MHC alleles. After analysis of the MHC restriction and sequence conservatism, 9 CTL epitopes in total were acquired, of which 6 located in Gag, 2 in Env and 1 in Pol. In addition, a Th-epitope-rich region which located in Env protein and consist of 36 amino acids were also selected for vaccine design.(2) Rational design, construction and identification of multi-epitope DNA vaccine. To obtain the optimal design of a multi-epitope vaccine, several multi-epitope constructs based on the candidate epitopes selected above were designed by connecting epitopes in different orders or with different linkers. Finally three DNA fragments, MEG1, MEG2 and MEG3, were chosen to construct the corresponding DNA vaccines. The structural characteristics of the three designs were analyzed in silico. The expression in cell lines and the immunogenicity in animal models of them were also evaluated in vitro and in vivo respectively. Results showed that p JW4303-MEG1 was similar to p JW4303-MEG2. Both of them expressed well and induced detectable immune responses. However, p JW4303-MEG3 presented a quite different protein structure when compared with the other two constructs, and gave negative results in expression detection and immunogenicity evaluation. Collectively, the linker between epitopes significantly affected the vaccine efficacy, and the flexible linker ‘GGGS’ worked better.(3) Evaluation of the immunogenicity and the immune protection efficacy of multi-epitope DNA vaccine p JW4303-MEG1. The female BALB/c mice were intramuscularly immunized to evaluate cellular immune response to single epitopes and memory immune response induced by DNA vaccine p JW4303-MEG1. Results showed that mice immunized with p JW4303-MEG1 could generate immune responses specific to all the H-2d restricted epitopes, and also developed immunologic memory which could last for nearly one year. Then the immunized mice were further challenged with recombinant virus r TTV-lucgag, and the luciferase activity in ovary issue was detected to show whether the immune response developed in mice could prevent virus replication or not. The virus load in immunized mice was slightly lower when compared with control. However, the difference was not significantly. 2. Evaluation of immunogenicity of the multi-epitope protein MEP1 in BALB/c mice.(1) Preparation and identification of a multi-epitope protein MEP1. To obtain the recombinant protein MEP1, the plasmid p QE30-MEG1.E was constructed by inserting E.coli codon-optimized gene MEG1.E into prokaryotic expression vector p QE30, and was identified with restriction enzyme analysis. The target protein MEP1 was mainly expressed in the form of inclusion body, then the crude purified inclusion body containing MEP1 was purified using High Affinity Ni-Charged Resin, and further identified utilizing SDS-PAGE and WB. Finally, the protein MEP1 with high putiry was successfully produced and could be used in the follow-up experiment.(2) Evaluation of immunologic characteristics of protein MEP1 in female BALB/c mice. To evaluate the immunogenicity and immune protection efficacy of MEP1, the female BALB/c mice were immunized three times with alum adsorbed MEP1. Then, humoral and cellular immune responses were measured. ELISA results showed that protein MEP1 worked well in inducing high titer of specific Ig G. ELISPOT assay provided the evidences for the ability of MEP1, when combining with aluminium adjuvant, in stimulating strong Th1/Th2 balanced cellular immune responses including single peptide specific immune response. The protein could also protect mice from being infected with recombinant virus r TTV-lucgag. Further more, immune responses induced by the multi-epitope protein emerged very quickly and could be detected just 10 days after immunization.(3) Analysis of the influence of mice gender on immune responses induced by multi-epitope protein MEP1. To figure out whether gender is a key factor or not which would affect the results of MEP1 immunization, the immune responses in MEP1 immunized male BALB/c mice were measured. Results showed that the epitope-based protein MEP1 could also effectively induce humoral immune response and cellular immune response and provide protection against r TTV-lucgag infection in male mice. When compared with the female ones, the male mice developed slightly lower antibodies and Th2-biased cellular immune response, while the level of Th1-biased cellular immune responses was equal in different genders of mice. These results suggested that the recombinant protein MEP1 could elicit protective immune responses regardless of gender difference. 3. Evaluation of the relationship between MHC restriction and immune response induced by multi-epitope protein MEP1.(1) Detection of the immune responses induced by MEP1 in human MHC transgenic mice. To find out the relationship between immune response induced by MEP1 and MHC restriction, two different types of MHC transgenic mice, A2/DR1 transgenic mice and A11/DR1 transgenic mice, together with the genetic background mice, C57BL/6 mice, were immunized with protein plus aluminium adjuvant for three times. Results of ELISPOT assay and flow cytometry illustrated that difference in immune responses did exist between groups. The magnitude and breadth of the single peptide specific responses were greater in MHC transgenic mice than those in C57BL/6 mice. Further analysis by flow cytometry using A2/DR1 transgenic mice showed that the recombinant protein might affect the cellular immune response by regulating the amounts of immune cells and the expression of MHC-II molecules on the surfaces of APCs.(2) Evaluation of the response of PBMCs from HIV-1 infected patients to MEP1 stimulation in vitro. In this part, PBMCs from HIV-1 infected patients were treated with protein MEP1 to further evaluate the relationship between response to protein stimulation and MHC restriction as well as to evaluate the application of MEP1 within Chinese population. ELISPOT assay results showed that the cells from HIV-1 infected patients responded to the protein activation and secreted IFN-γ and IL-2 while cells form healthy donors kept silence. More importantly, MEP1 was capable to activate immune cells from patients with different HLA alleles and at different clinical status. This data supported that protein MEP1 might be useful to induce specific immune response within HIV-1 infected patients in China. 4. ConclusionIn this study, 9 HIV-1 CTL epitopes and a Th-epitope-rich region in Env protein were firstly selected. All of the epitopes are highly conserved within HIV-1 subtypes prevalent in china and restricted to dominant Chinese MHC alleles. Then, experiments on optimal design of multi-epitope vaccines based on the candidate epitopes were carried out and gave the results showing that linker between epitopes is one of the key factors of vaccine design. Our study developed a multi-epitope protein vaccine MEP1. The protein showed great immunogenicity and protective efficacy in animal models and could also induce PBMCs from HIV-1 infected patients to secrete cytokines. The immune responses induced by MEP1 closely matched the MHC restriction and the applicability of MEP1 among Chinese population is good. Our study contributes not only to design an effective vaccine against HIV-1, but also to build a protocol for epitope-based vaccine design for other pathogens. |
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