Immunologic Efficiency And Mechanisms Of A Novel Integration-defective SIV Vaccine In A Chinese Rhesus Macaque Model

Acquired Immunodeficiency Syndrome(AIDS)is a major global epidemic disease caused by Human Immunodeficiency Virus(HIV).Highly Active Antiretroviral Therapy(HAART)is a widely-used method for the clinical treatment of AIDS.However,this method involves different drugs which can have some side effects to humans and cannot eliminate the latent viruses in infected patients.Therefore,developing an effective preventive or therapeutic vaccine against AIDS is imperative.However,over 30 years of research,it still failed to develop an effective HIV vaccine.Since the HIV genome is highly variable,it is very challenging to develop a vaccine which prevent the infection of various subtypes of HIV and suppress the replication of diverse viruses after infection.In addition,after HIV enters into host cells,viral RNA will be converted into c DNA by reverse transcriptase and then integrated into the chromosome of host cells.This means that once the virus invades the body,it cannot be completely removed.Therefore,due to the safety concern,live attenuated vaccines which are common and very effective methods used for traditional vaccine development cannot be directly applied to AIDS vaccine research.In some early studies,it was found that using Simian Immunodeficiency Virus(SIV)live attenuated vaccines tested in non-human primate(NHP)models showed effective immune protection.However,it was also found that these live attenuated SIV vaccines can also cause AIDS in infant and adult monkeys.Therefore,the adaptation of protective immunity induced by SIV live attenuated vaccines and improvement of its safety and immunogenicity can lead to development of successful AIDS vaccines.The integrase encoded by HIV genome is required for the integration of proviral genome into host cell genomes.Although such integration-defective HIV will not integrate into the host cell genome after infection,viral proteins still can be translated from some circular forms of proviral genome in the cytoplasm,and these extrachromosomal DNA(E-DNA)can exist in some types of cells which are not active in proliferation for a long time.The viral proteins translated from E-DNA in the cytoplasm can serve as antigens to stimulate the immune system for generation of virus-specific immune responses.This suggests that integration-defective HIV may be developed into a safer vaccine strategy.Studies have shown that integration-defective lentiviral vector(IDLV)vaccines can induce strong antigen-specific systematic and mucosal immune responses in the mouse and NHP models.However,the protective efficacy induced by this IDLV vaccine has been evaluated in the NHP model.Therefore,in this study,we used the SIVmac239 strain which can infect rhesus macaques to develop a safe integration-defective SIV(idSIV)vaccine.Multiple mutations at the sites required for integration were introduced into the SIVmac239 genome to ensure the safety at different levels;We preserved all SIVmac239 viral proteins as antigens and replaced the viral LTR promoter with the Cytomegalovirus immediate early(CMV-IE)promoter which can achieve efficient gene expression in eukaryotic cells to enhance the levels of immunogen proteins.After construction of the genome-modified idSIV genome using the molecular cloning method,we first studied the virological characteristics of idSIV.We found that idSIV could successfully assemble into infectious viral particles but its infectivity was weak compared to wild-type SIVmac239.To enhance the infectivity of idSIV,we used two different serotypes of Vesicular Stomatitis Virus glycoproteins(VSV-G)to generate VSV-G pseudotyped idSIV particles and found that the infectivity was more than 20 times higher than idSIV.No integration and continuous replication were detected in CEM×174 cells infected with idSIV or VSV-G pseudotyped viruses.The expression of early and late viral proteins from E-DNA could be detected in infected TZM-bl cells,suggesting that viral antigens could stimulate the immune system after infection.To induce both cellular and humoral immune responses,we immunized eight Chinese rhesus macaques using a prime-boost strategy(three times of idSIV DNA and four times of viral particles)and used another eight macaques received PBS as control.Fourteen weeks after the last immunization,animals of vaccine and control groups were intravenously challenged with a single dose of 50 TCID50 SIVmac239 and followed for 28 weeks.The viremia of vaccinated rhesus macaques was significantly reduced during the acute infection stage and considerably suppressed at the set point.T cell immune responses,binding antibodies,neutralizing antibodies and antibody-dependent cell-mediated cytotoxicity(ADCC)activity were detected in the vaccinated macaques.T cell immune responses were mainly induced by idSIV DNA and primarily targeted to Gag,while humoral immune responses were mainly induced by viral particles.Vaccine-induced neutralizing antibodies showed strong neutralization activity on a tier 1 heterologous virus SIVmac251 but weak activity on autologous SIVmac239.ADCC activity could be detected in four of the seven macaques.The protection efficacy of vaccine was further analyzed to explain its Immunologic Efficiency and mechanisms.By high-throughput sequencing virus populations in both vaccine and control macaques around the peak viremia and epitope analysis,we found six amino acid mutations in Nef,Gag and Env protein sequences only from the vaccine group animals.These mutations were likely to be selected under the immune pressure generated from the T cell immune responses and humoral immune responses induced by the idSIV vaccine.To further analyze the role of idSIV DNA and viral particles in reducing plasma viral loads in the acute infection stage,we immunized two groups of Chinese rhesus monkeys with idSIV DNA or virus particles alone in a small pilot study.Our results confirmed that the T cell immune responses and humoral immune responses were induced by idSIV DNA and viral particles respectively,and found that idSIV DNA immunization alone is critical to control viral replication during the acute infection stage.Finally,we explored the application possibility of idSIV DNA as a therapeutic vaccine,and found that it could also contribute to the control of viral replication during the chronic infection stage.In conclusion,in this study a novel integration-defective SIV vaccine was successfully generated and evaluated in Chinese rhesus macaques for its immunogenicity,protective efficacy and virologic characteristics under the vaccine-induced immune selection pressure.We found plausible explanations for protection mechanisms of the idSIV vaccine and the key signatures associated with the protection.We anticipate that this study will provide a theoretical and experimental basis for new AIDS vaccine design and immune protection mechanism research.