Hiv-1 Ae Recombinant Envelope Protein Vaccine Construct And The Immunogen Transformation And Immunization Strategies

Human Immunodeficiency Virus Type I (HIV-1) is the etiologic agent for acquired immunodeficiency syndrome (AIDS) and there are 33.4 million people reported living with HIV/AIDS world widely (the prevalence rate is 6%o approximately), including around 700,000 cases from China. Once HIV-1 infection occurs, it is hardly to be eradicated by host immunity. The history of containing infectious diseases surmises that a safe and effective vaccine for HIV is the cost-effective way to control its pandemic. Epidemiological data showed that several subtypes and CRFs of HIV-1 circulate in China, CRF07_BC, CRF08_BC, B'(Thailand B) and CRF01_AE are the major forms. Currently, sexual contact has become the dominant risk factor of HIV infection in China. CRF01_AE usually spread with sexual transmitted population is on the rise consequently. Therefore, to develop a prophylactic vaccine against CRF_AE is desirable for controlling HIV-1 pandemic in China.Envelope protein of HIV-1 (Env) is a major immunogen for antibody based vaccine and has been tested in several vaccines in clinical trials. However, the natural form of Env is incapable of inducing broadly neutralizing antibodies because of its highly glycosylation. Several studies have shown that introducing particular modifications at the glycosylation sites of Env can improve its ability to elicit neutralizing antibodies. In the first section of this thesis, we constructed a DNA vaccine expressing human codon-optimized gp140 of CRF01_AE, a truncated Env without transmembrane and intracellular regions, and then further introduced N to Q mutations at the sites of N 157 and N 161 in the V1/V2 (m157/161) or of N 382 and N 388 in the V4 (m382/388) loops. Western Blot showed that all DNA vaccines can express gp140 effectively in a secreting form at a similar level and is detectable in both cell lysate and culture supernatant.To determine the immune response elicited by these DNA vaccines, we immunized mice four times at 2-week interval, and immune responses were examined in 2 weeks after the final injection. ELISPOT based on IFN-y secreting showed that the specific T cell responses elicited by gp140 (2432±586 SFCs/106 splenocytes), m157/161 (2682±893 SFCs/106 splenocytes), and m382/388 (2360±560 SFCs/106 splenocytes) did not reach any significant differences. This implies that each of the three DNA vaccines can elicit a robust specific T cell response, and the modification of selected glycans does not significantly affect specific T cell response. Then ELISA assay was performed to test the titer of binding antibody. The average antibody titer of gp140 group, m157/16, m382/388 is 4800,1600 and 1200, respectively. It's significantly decreased in the glycans deleted vaccine immunized groups (ml57/16 VS gp140 p<0.02, m382/388 VS gp140). This result implies that deglycosylation of the VI/V2 or V4 loops failed to enhance the levels of binding antibodies to its wild-type counterpart.We also tested the neutralizing activity of mice sera against three primary isolates of CRF07_BC (XBC6371, XBC0793, and XBC6431). Our data showed that sera of mice inoculated with gp140 did not have any neutralizing activities against XBC6371 and XBC6431, whereas several mice from glycosylation deleted groups showed a virus inhibition activity of 6%-16%. In contrast, for XBC0793, the mean inhibition of m157/161 group was 47% and m382/388 group was 49%, both are significantly higher than gp140 group (17.7%,p<0.05). It indicated m157/161 and m382/388 could enhance capacity of CRF₀1AE gp140 to elicit neutralizing antibodies against heterologous viruses, and the increased neutralizing activity was not associated with binding antibody activity. In conclusion, we successfully constructed a CRF01_AE Env vaccine that can effectively induce binding antibodies and T cell responses; the deglycosylation forms we tested here also provide prospects for designing vaccine aiming broad neutralizing antibody activities. Moreover, different subtypes of viruses should be further tested to confirm the spectrum of neutralization and the mechanism underlying the promotion of neutralizing activity should be further explored.As known, a proper vector can significantly improve a vaccine's efficiency. The clinically tested, safety proved replication-competent vaccinia virus will be one of the candidates for HIV-1 vaccine. Comparing with the strategy to use DNA or poxvirus alone, the DNA prime-poxvirus boost strategy is more widely used for its capability of inducing higher antigen specific immune responses without increasing of immunity against vector. In the second section of this thesis, we constructed recombinant replication-competent vaccinia vaccines expressing HIV-1 CRF01_AE Env, and the DNA prime/recombinant vaccinia boost strategy was used in a Balb/c mice model to test immunogenicity of this immunization strategy. Consistent with previous studies, g high level of B cell responses was elicited by secreting formed gp140, in contrast the membrane anchored gp145 with transmembrane region was capable of inducing high level T cell responses. As a result, we presumed that inoculated one animal with the two differently truncated Env forms as prime/boost immunogens may efficiently elicit both arms of the adaptive immunity. Therefore, in the second section of this thesis, we tested our hypothesis to select out the best combination. Firstly, we tested binding antibody titers elicited by vaccines using ELISA assay. Data showed that both groups priming with gp140 raised high-level of binding antibody titers with a geometric-mean of 128 00 in gp140 prime/gp140 boost group and 19 401 in gp140 prime/gp145 boost group respectively. On the contrary, in gp145 primed groups, only low antibody titers were elicited with a geometric-mean of 566 in gp145 prime/ gp145 boost group and 606 in gp145 prime/gp140 boost group respectively. Our data confirmed the fact that gp140 elicits better antibody responses than gp145, and we observed that the priming immunogen determines the final antibody level. We quantified vaccine activated specific T cell responses using IFN-γbased ELISPOT assay. Our data showed, T cell response elicited by gp145 prime/gp145 boost ((3424±650) SFCs/106 splenocytes) is higher than that elicited by gp140 prime/gp140 boost ((1918±442) SFCs/106 splenocytes). Furthermore, in heterologous prime/boost groups, gp145 also primed or boosted slightly higher T cell response than gp140. These data indicated that gp145 induced higher T cell response either as priming immunogen or as boosting immunogen than gp140 in DNA prime/recombinant vaccinia virus boost regimen. Taken together, we draw a conclusion that gp140 DNA prime/gp145 vaccinia virus boost strategy will be the best one for inducing both high-level of binding antibody and T cell responses among all regimens we tested.For further defining of the specific T cell responses, we mapped T cell epitopes by using HIV-1 Env derived peptides in Balb/c (H-2d) mice. Several epitopes were defined in peptides as following:No.35 (NSNNTTNGPNKIGNI), No.16 (ETEVHNVWATHACVP), No.136 (QQQSNLLRAIEAQQH) and No.106 (GQAMYAPPISGRINC). The No.35 peptide is a dominant T cell epitope, T cell responses induced by this epitope accounts for 61.3% of total T cell responses, and by the five epitopes accounts totally for 81.7% of the total T cell response. After blasting in Los Alamos database and comparing with other studies, we identified that the No.35 peptideis a newly discovered H-2d restricted T cell epitope. These results would be reference for analyzing immune responses elicited in Balb/c mice and other with H-2d genetic backgrounds. The limitation of our immunization strategy is that it failed to elicit broad T cell responses against its counterparts derived from different clades. The vaccines and immunization strategy should also be further explored in non-human private models.