Construction Of SHIV/Chinese-origin Rhesus Macaque Models And Application In Immune Protective Effect Evaluation Of DNA-Tiantan Vaccinia Combined AIDS Vaccine

Human immunodeficiency virus(HIV) infection and AIDS continue to be a growing problem for the world's population.The need to develop a safe and efficacious vaccine against HIV is more pressing than ever.A pathogenic R5 simian-human immunodeficiency virus(SHIV) encoding genes of the predominant prevalent HIV-1 B'/C Recombinant(CRF07_BC) strain in China was highly desirable to study the role of HIV-1 envelopes in transmission and pathogenesis as well as to evaluate candidate AIDS vaccines in nonhuman primates. To this end,SHIV-XJ02170 and SHIV-CN97001 which also carrying CRF07_BC env, were adapted by serial passage in thirteen Chinese-origin Rhesus Macaques,separately. Up to now,there were no pathogenic viral strain obtained during in vivo passage by viral load and CD4/CD8 ratio analysis.But,it was revealed that the infectivity property of the two SHIVs enhanced during in vivo passage.The viral RNA load of third passage was highest during SHIV-CN97001 passage and two animals infected SHIV-XJ02170 had inclined setpoint viral load.To identify variation in the gpl20 region of SHIV-CN97001 and SHIV-XJ02170 during passage,the fragments of gpl20 gene were amplified by RT-PCR from the plasma of infected animals at the peak viral load time point and the gene distances (divergence,diversity) were calculated using DISTANCE.The analysis revealed that the genetic distances of SHIV-CN97001 in the third passage animals were the highest and the gene divergence from founder strain in M408 serially inclined during SHIV-XJ02170 in vivo adaption.It had a relationship between viral divergence from the founder strain and viral replication ability.In the two SHIV constructs,the nucleic acid sequence of the V3 region was highly conservative and all the RNA clones were predicted to be using CCR5 co-receptor on the basis of the critical amino acids within V3 loop.These results showed that there was no significant increase in the genetic distance during SHIV-CN97001 and SHIV-XJ02170 passage,and the two SHIV gp120 gene evolved toward ancestral states upon transmission to a new host.But,the SHIV-XJ02170 virus in one of second passage animals(408) maybe has involved the late infection phase according to gene distance analysis.This could partly explain why there has no pathogenic viral strain obtained during in vivo passage.To develop a SHIV-KB9/Chinese-origin rhesus macaques(Ch Rh) model for vaccine research and to compare the pathogenesis of SHIV-KB9 in Ch Rh with published reports from Indian-origin rhesus macaques(Ind Rh).Seven mamu-A*01 negative Ch Rh were inoculated intravenously with 1-1000 MID₅₀ of SHIV-KB9.The monkeys were monitored by measured viral load,CD4,CD8,SHIV-specific antibody and virus genetic variation.In Ch Rh,SHIV-KB9 displayed three identical disease progression patterns,compared to what observed in Ind Rh.However,the primary pattern was not same for the two subspecies.The level of plasma viremia differed in SHIV-KB9-infected Ch Rh exhibiting different outcomes in contrast to in Ind Rh. Generally,the viral load values and the maintenance of CD4⁺ T cells were associated with the humoral immune responses.Otherwise,the viral genetic distances (divergence,diversity) were larger in animals(M419,M425) in which the CD4⁺ T cells were profound depleted.Ch Rh model employing SHIV-KB9 displayed a relative slower progression to AIDS compared with Ind Rh,which may more accurately reflect the potential of candidate vaccines in humans.Interesting, SHIV-CN97001 and SHIV-KB9 infection in Chinese-origin Rhesus Macaques could induce heterologous and homologous immune protective response to SHIV-KB9 superinfection.So it is not suitable for SHIV-KB9 superinfection in Chinese monkeys systemicly infected with SHIVs.To contain the pandemic of HIV/AIDS in china,plasmids containing nef,gag, pol,gp140 and replication competent Tiantan vaccinia(rTV) containing gag,pol and gp140 from B'/C recombinant HIV-1 CN54,the predominant prevalent strain in China,were constructed as candidate AIDS vaccines.To evaluate the immunogenicity and protective effect against homologous SHIV-CN97001 and heterologous SHIV-KB9 challenge,12 Chinese-origin Rhesus Macaques were immunized and challenged,rTV-only vaccination was compared with a DNA prime/rTV boost strategy.Animals were followed prospectively for immune parameters and viral RNA loads.HIV-specific antibody titers and CTLs responses were determined with ELISA and ELISPOT based assay respectively.Neutralizing antibody titers were measured using a viral infectivity assay of TZM-bl cells.Compared to the DNA/rTV vaccine, the rTV-only vaccine raised less than 1/10 the number of vaccine-specific T cells,but comparative titers of binding antibody and neutralizing antibody.Otherwise,the candidate vaccines were able to induce strong memory responses in all the vaccinated animals nearly 9 months after the last immunization.After homologous SHIV-CN97001 challenge,the rTV-only-vaccinated animals had achieved better control of the infection.4 of 4 animals in rTV group were completely protected. However,only 1 of 3 animals in DNA prime/rTV boost group(M7) was complete protected,the other two animals were partially protected.After 10 months,we selected two animals which completely protected against SHIV-CN97001 to be rechallenged with heterologous SHIV-KB9,only one for each group.One monkey (M7) in DNA prime/ rTV boost group was also completely protected against SHIV-KB9 rechallenge,which was comfirmed by blood passage analysis,another one (M2) in rTV-only group was partially protected with declined peak and setpoint viral load.It was indicated that the rapid responses having neutralizing activity might contributed to the clearance of the homologous SHIV-CN97001 challenge virus and the IFN-γsecreting T cell immune response had correlation to protection from heterologous SHIV-KB9 rechallenge.DNA-replication competent Tiantan vaccinia combined vaccine could provide the potent,persistent immunity necessary to protect against homologous and heterologous SHIV.This study provides a foundation for evaluation of these candidate vaccines in human body.