| At present time,the treatments of HIV-1 mainly target the diverse steps of its life cycle,aiming to block its replicating in vivo.However,the therapies have nothing to do with the free viral particles or the integrated latent virus.Although the broadly applied highly active antiretroviral therapy could suppress viral replication in vivo and even block the transmission among individuals,the viral reservoirs are hardly impacted.The viral load would rapidly rebound when the administration of HAART was suspended.Clearing the free viral particles,limiting the size of latent reservoir and maintaining the durable control of HIV-1 to reach the aim of functional cure or even cure has become the focus of the therapeutic researches on HIV-1.Recent years,a swarm of broadly neutralizing antibodies with extraordinary potency has been isolated and characterized from the "elite" HIV-1 controllers,passive transferring of those neutralizing antibodies displayed dose dependent efficacy of immune-prophylaxis and protection in humanized mice and rhesus monkey models.It is promising and of great significance to transduce durably and stably antibody expression in HIV-1 infectors.Our lab has recently isolated and characterized an authorized monoclonal antibody termed Y498,in this study,we constructed several sera type 8 recombinant adenovirus-associated viruses(AAV8)to mediate diverse HIV-1 neutralizing antibodies expressing.The CD4 binding site directed antibody Y498 and VRC01,the V3 glycan region directed 10-1074,the membrane proximal external region(MPER)of gp41 directed 10E8,and both the CD4 binding site and CCR5 domain directed bi-specific antibody 4Dm2m were enrolled to construct recombinant antibody expressing AAV vectors.We aimed to evaluate the transgene expressing ability of these vectors in BALB/c mice and investigate the immunotherapeutic potency of the strategy in vivo.At the same time,we engineered the M428L/N434S mutant counterparts on Fc domain of those antibodies by site directed mutation to boost their binding affinity with the neo-natal Fc receptors(FcRns)in non-human primates and individuals,which would promote the half-life cycle of antibodies in vivo and thus help to maintain more durable circulation of the AAV vectors directed antibodies and hopefully to accumulate to a higher level,aiming to acquire better antiviral potency in vivo.In this study,we selected antibodies with more potent neutralizing potency and could be well expressed recombinant AAV-VRC01,AAV-10-1074 and AAV-10E8 vectors in BALB/c mice,and the M428L/N434S sites mutated vectors of AAV-VRC01-LS and AAV-10-1074-LS were applied in rhesus monkeys.Three doses of 1.0×1011,3.3×1010,and 1.1×1010vg were respectively inoculated in different groups of mice by intramuscularly injection.We found the expression of HIV-1 neutralizing antibodies lasted for 56 weeks in BALB/c mice sera and the vectored antibody concentrations were in positive correlation with the AAV injection doses.The high dose group of AAV-VRC01 reached peak concentration of 257.8?g/ml at 6th week post inoculation,the medium and low injection group got peak concentration of 219.5?g/ml and 139.8?g/ml at 12th and 20th week respectively.The AAV-10-1074 directed more potent antibody expressing in mice,and maintained higher antibody concentrations in plasma for a durable period than that in AAV-VRC01 injection groups.The high dose group of AAV-10-1074 reached peak concentration of 1478?g/ml at 24th week,the medium and low injection groups got their peak concentration of 1390.5?g/ml and 133.8?g/ml at 10th and 14th week respectively.We noticed that the antibody concentrations in vivo gradually got attenuated post the peak concentration and several mice in the low injection group of AAV-VRC01 constantly directed undetectable antibody expression.The pseudo-viral based in vitro neutralizing test demonstrated that the AAV-VRC01 and AAV-10-1074 injected mice sera own antibody concentration related neutralizing potency against HIV-1 tier 2 isolate JRFL,suggesting their affirmative antiviral potency in vivo.In rhesus monkeys with established SHIV-SF162P3 infection,the recombinant AAV-VRC01-LS and AAV-1074-LS also directed durable antibody expression,and the accumulated antibody in vivo displayed the potency to suppress the rebound SHIV viremia after the administration of HIV-1 fusion inhibitor LP-19 was suspended,among which three of five rhesus monkeys in AAV-VRC01-LS injection group and one in AAV-10-1074-LS injection group were depressed below detection limit.Besides,we detected potent humoral immune response against the AAV8 vectors in rhesus monkeys,which might severely impair the potential therapeutic efficacy of the AAV vectored immunotherapy.Taken together,in this study work,we constructed functional recombinant AAV8 vectors which directed durable and stable HIV-1 neutralizing antibody expression in experimental animals.And the vectored antibodies in rhesus monkeys provide effective therapeutic efficacy to persistently inhibit viral replication and decrease the plasma viral load.However,the host immunologic rejection against the exogenous AAV vectors needs to be prevented.All in all,our study supported the HIV-1 neutralizing antibody based and AAV vector mediated immunotherapy could be developed as an alternative tool for HIV-1 therapy and it might provide a novel perspective to reach the aim of HIV-1 functional cure. |
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