Study Of Mechanisms For Development Of Anti-HIV-1 Broadly Neutralizing Antibodies In SHIV-infected Chinese Rhesus Macaques

Acquired Immunodeficiency Syndrome(AIDS)is one of the infectious diseases that has become a huge threat on human public health.AIDS is caused by Human Immunodeficiency Virus(HIV)infection.The available treatment schemes can prolong the life of patients effectively,but they are not able to cure the disease because virus latent reservoir in HIV-1 infected patients cannot be completely cleared out.And not only that,the side effects on patients and drug resistance problems that these treatments brought needed to be solved.Therefore,the development of an effective HIV vaccine is an important intervention to control the epidemic of AIDS.Broadly neutralizing antibody(bn Ab)is a kind of humoral response gradually induced by long-term infection in HIV-1-infected individuals.Bn Abs possess the great neutralizing potency against majority HIV-1 circulating strains,which provide new directions for HIV treatment and prevention strategies.For this reason,whether it can induce bn Ab in human has also become an important criterion for HIV vaccines.However,only around20% of HIV-1-infected individuals could develop neutralizing antibodies with significant breadth.Bn Abs could only mature after 2-4-year evolution through the researches on evolutionary process of bn Abs in the human.As the only animal model which is suitable for the evaluation of protection efficacy of HIV-1 vaccine,Simian/human immunodeficiency virus(SHIV)infected Non-human primate(NHP)has been widely used,but it is not totally clear that whether it could develop bn Abs as human and how the bn Abs evolve in NHP.Therefore,establishing of a reasonable SHIV-NHP model to study the development and evolution mechanisms of bn Abs in NHP is very meaningful for further evaluation of HIV-1 vaccines.In the previous studies,the broadly neutralizing activities were rarely detected in SHIV-infected rhesus macaques,only a few macaques were found to have relatively broad neutralization activity but no monoclonal bn Ab was isolated.This is because SHIV-infected NHPs were generally not maintained more than 2-year post infection owing to the planned experiments had been accomplished,bn Abs did not have enough time to develop in rhesus macaques.So the characterization and evolution pathway of bn Ab in NHP has not been revealed in detail due to the lack of a suitable long-term infection model.In this study,we established long-term infection NHP model for over6 years infected with two SHIVs respectively.The broadly neutralizing activity was elicited in rhesus macaques by long-term SHIV infection and the circulating HIV-1strains with different sensitivities were used to analyze the neutralizing potency and epitopes of plasma samples.Then we isolated the monoclonal bn Abs from SHIVinfected macaques and evaluate their neutralizing potency,breadth and epitopes.We also investigated the bn Ab mature process and virus-antibody co-evolutionary mechanism by analyzing monoclonal antibodies(m Abs)lineage and the mutations on Env of SHIV.More importantly,the comparison between development of bn Abs in NHPs and humans will be critical for better use of the SHIV-NHP model on evaluation of efficacy of HIV-1 Env-based vaccines.Firstly,two infectious SHIVs(SHIV1157ipd3N4 and SHIVSF162P3)were chosen to challenge Chinese rhesus macaques by intravenous or intrarectal route and 31 macaques were infected by SHIV.It could be found that the viral loads reached peak during acute infection stage in macaques,and there was persistence viremia during the whole chronic infection stage.The viral loads of two macaques infected with SHIV1157ipd3N4(G1015R and G1020R)were monitored until 6 years after infection and became increasing in the late stage of infection,which was the antigen base of bn Ab development.Through the neutralizing activity analysis of the plasma from six SHIV-infected macaques with archived longitudinal samples,only limited cross-reactive neutralizing antibodies were detected at early stage and the neutralizing breadth significantly increased over time,an average of 65% tier 2 viruses could be neutralized by G1015 R and G1020 R after 6-year infection.Comparing the neutralizing breadth induced by different SHIV-infection,SHIV1157ipd3N4 was superior to SHIVSF162P3 and SHIVCHN19P4 for induction of broadly neutralization responses and the intrarectal challenge was the better way.The neutralizing epitope characterization showed the mutations resistant to known bn Abs in V2,V3 or CD4 bs regions could reduce neutralizing sensitivities by plasma from SHIV-infected macaques,indicated that like the bn Ab in human,the neutralizing activity elicited in G1015 R and G1020 R could target V2,V3 or CD4 bs regions.To further determine the specificities of bn Ab in NHP,the m Abs were isolated from macaques.Single epitope-specific memory B cells were sorted from week 350 PBMC of G1015 R by fluorescence-activated cell sorting(FACS)and 44 paired Ig G genes were amplified.The m Abs which could bind autologous antigen were selected by ELISA and the neutralizing potency of them were determined with purified Ig G.Twelve neutralizing m Abs were isolated and six of them could neutralize several tier 2viruses.J038 is the m Ab with the strongest neutralizing ability,could neutralize 54%of circulating global strains,which is the broadest antibody isolated from SHIVinfected NHP.Analysis of the gene family of these antibodies revealed that the six m Abs with cross-reactive neutralization were from the same lineage(VH4-2 * 01 F and VK1-20 * 01 F).Epitope mapping suggested the N156/N160 glycans and the amino acid residue of V2 loop were forming the recognition epitope of J038-lineage antibodies.J038 Fab-C1080 Env Cryo-EM structure revealed that the 18-aa-long HCDR3 of J038 formed extensive anti-parallel ?-strand hydrogen bonding interactions with the V2 loop instead of inserting into the trimer interface at the V2 apex.However,the J038 binding is heavily dependent on glycans,which occupied nearly half of the total binding surface.This is consistent with the characteristics of V2 specific bn Abs isolated from human.Then we investigated the evolution pathway of J038-lineage.The unmutated common ancestor(UCA)and Intermediate antibodies(IAs)were inferred by phylogenetic tree,the UCA and early IAs could not neutralize heterologous viruses,suggesting that the neutralizing ability of J038-lineage were gained from accumulating mutation during infection.Analysis of the binding interface of UCA and IAs at each stage with homologous modeling showed that the binding area of the N156 and N160 glycans in the V2 region has gradually increased during the evolution process of J038 lineage,which accounted for the maturation of broad neutralizing antibody.The gene sequences from various infection stage of G1015 R were examined and there were several mutations emerged in V2 region which could be recognized by J038-lineage.The I165 L,K171R and V172 A mutation rendered the virus more resistant to J038-lineage m Abs,suggesting viruses with these mutations had escaped from this lineage of bn Abs.Although the escape mutations detected in NHP which produced under the selection pressure of bn Abs are not exactly same as those detected in humans,the development of bn Abs in NHP also share many similarities with the evolution mechanisms of bn Ab in humans.In summary,the longest-term SHIV-NHP infection model was successfully established in this study,and broadly neutralizing activity was induced in NHP.We obtained the J038 lineage bn Ab,which was the broadest neutralizing antibody isolated from SHIV-infected rhesus macaques.J038 has short complementary determining loops and neutralizes 54% of global circulating HIV-1 strains.The research on specificities and evolution process of bn Ab developed in NHP suggested J038 lineage recognized the similar V2 region as those V2-tageted bn Abs isolated from humans,and continuous maturation enhances neutralization potency and breadth of J038 against diverse heterologous tier 2 viruses via expanding glycan contact areas.Not only that,viruses in G1015 R have accumulated escape mutations under the immune selected pressure of J038 lineage.Reasonable analysis and description of whether NHP can develop bn Ab similar as those in human and the bn Ab maturation mechanisms were conducted by this research.We hope that the knowledge learned from this study can provide some theoretical and experimental basis for the better application of SHIVNHP model to evaluate HIV-1 vaccine.