| The human immunodeficiency virus(HIV),which has seriously threatened global human health since its discovery,has been a hot topic in scientific research.Acquired immunodeficiency syndrome(AIDS),caused by HIV infection,is a major global health threat.According to UNAIDS in 2021,there were approximately 38 million people living with HIV globally.Although combination antiretroviral therapy(cART)can control chronic HIV infection,researchers believe that vaccines are the only route to completely control the virus.However,there are no vaccines to prevent HIV infection or curb this devastating pandemic.Therefore,there is an urgent need to develop an HIV vaccine that can induce broadly neutralizing antibodies(bnAbs).The enormous diversity of the envelope(Env)glycoprotein of HIV-1 and the high mutation rates in the HIV genome,especially in env genes have been tremendous challenges for the development of successful HIV-1 vaccines.Immunogens that can form components of a neutralizing antibody-based HIV vaccine include(a)germlinetargeting,(b)lineage-based,(c)CDRH3 feature-targeting,and(d)minimal epitope immunogens.Stable and native-like Env trimeric immunogens may induce bnAbs.There are two methods to guide the sequence design of vaccine antigens:using specific geographic isolates,or constructing a consensus sequence that is most closely related to the current circulating strains.Studies on the antibody-virus co-evolution process in HIV-1 infected individuals who developed broad neutralizing activities have demonstrated that primary infection is initiated by one or two transmitted/founder viruses(T/F viruses)in approximately 80%of heterosexually transmitted HIV-infected individuals.After initial infection,the T/F virus replicates substantially and generates escape mutations.At the primary infection stage,the initial nAb responses to the T/F viruses occur approximately three months after transmission and are strain-specific.The emergence of these nAbs drives the viruses to escape autologous neutralization,whereas immune escape mutants drive the maturation of bnAbs.Therefore,mimicking the natural infection process using Env trimers from HIV-1 infected individuals in whom broad-spectrum neutralizing antibodies have been isolated,may be an effective vaccine strategy to induce bnAbs.In our previous study,two bnAb lineages,CH103 and CH235,which neutralized approximately 55%and 90%of HIV-1 isolates,respectively,were isolated from an HIV-1-infected individual,CH505.Ab-virus co-evolution analysis showed that genetic diversification of the T/F virus led to the development of neutralization breadth and potency of both CH103 and CH235 bnAb lineages,strongly indicating that increased genetic diversity during viral evolution plays a key role in driving bnAb maturation.In this study,we attempted to evaluate the sequential immunisation strategy to induce bnAb lineage B cells,mimic the natural infection process of an HIV-1 infected individual,and replicate the mature process of bnAbs.We analysed 14 Env consensus sequences derived from the HIV-1 infector,CH505,and constructed a consensus sequence phylogenetic tree.Based on the Ab-virus co-evolution process,we inferred the key viruses that may drive this process.Consensus Env proteins from key viruses were expressed as immunogens for sequential immunisation.Two trimeric optimisation strategies were used to obtain trimeric immunogens.The first method consists in optimising the viral sequence,which is termed uncleaved prefusion-optimised(UFO).The other method uses the trimeric motif MTQ.HEK 293-6E cells were used to express trimeric immunogens that were used to sequentially immunise guinea pigs to reproduce mature bnAbs.Main results of this study include:First,we obtained the T/F viral sequence and 14 Env consensus sequences from weeks 4 to 160 after individual CH505 infection.Viral Env mutations occur less frequently during the initial stages of infection.During the maturation of bnAbs,viruses continually mutate under the selective pressure of Abs.The Env consensus sequences were divergent,particularly in the V1,V4,V5,and loop D regions,resulting in several key viruses that may drive Ab maturation.These key viruses have been termed Vir-w4,Vir-w22,Vir-w30,Vir-w53,and Vir-w78.A natural variant,Vir-w4.26,with a single amino acid mutation in the Env region,was also identified at week 4 post-infection.This variant showed higher binding affinity to Env and was more sensitive to autologous neutralization.These consensus sequences of key viruses Env and the unique sequence of Vir-w4.26 Env were all optimised based on the UFO structure.A clade C T/F virus BG505 UFO Env were also used as a native-like trimeric immunogen.Eukaryotic expression plasmids of the optimised sequences were constructed and trimeric Env proteins were expressed.Proteins were first purified by binding to Lentil Lectin columns followed by size exclusion chromatography.Purified trimeric proteins were used for sequential immunisations.Guinea pigs were primed with T/F or w4.26 proteins and boosted with proteins w22,w30,w53,and w78 with a three-weeks interval.Analysis of the broad spectrum of guinea pig sera showed the following:(1)sera in the sequential immunisation groups showed broader neutralizing activity than that of BG505 alone;both sequential immunisation groups induced 31.3%and 26.7%higher potency of neutralizing responses than the BG505 group.(2)Different primary immunogens used in the sequential immunisation groups induced different degrees of neutralizing activity.The T/F group neutralized seven pseudoviruses and the w4.26 group neutralized eight pseudoviruses.Neutralization specificity analysis showed that both sequential immunisation groups developed serum antibodies targeting the gp120/gp41 interface of Env,indicating a different evolutionary process between the vaccine and infection.Longitudinal serum neutralization activity showed that the breadth and potency of serum neutralization activity increased over time.After the third immunisation,sera in the sequential immunisation groups could only neutralize three pseudoviruses,whereas more than seven pseudoviruses were neutralized by the sera after the fifth immunisation.These results indicated that guinea pigs produced improved neutralization activity through sequential immunisation.We also used the MTQ motif to replace the gp41 region and constructed the trimeric proteins PM-T/F,w4.26,w22,w30,w53,and w78.Guinea pigs were also sequentially immunised with these immunogens.Compared with PM immune groups,guinea pig sera in the UFO immune groups had a broader spectrum.To our knowledge,this is the first study to mimic the natural infection process using a sequential consensus immunisation strategy.Thus,a broad neutralizing activity was induced in the guinea pig sera.Sequential immunised groups were primed with different proteins,resulting in immunogens with different abilities to induce bnAbs.Two different trimer optimisation methods were used in this study,yielding different immunogenicities and serum-neutralizing potencies.The sequential immunisation strategy induced a broader and more potent neutralizing activity.Taken together,sequential consensus immunisation with trimeric immunogens broadly induced serum-neutralizing activity in guinea pigs.Sequential immunisation significantly increased the neutralizing breadth and potency of guinea pig sera.By mimicking natural infections,a sequential immunisation strategy may become an effective mode for HIV vaccine development. |
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