Recapitulation of HIV-1 Neutralization Breadth in Plasma by the Combination of Two Broadly Neutralizing Antibodies from Different Lineages in the Same SHIV-Infected Rhesus Macaque.

Viral infection generally induces polyclonal neutralizing antibody responses. However, how many lineages of antibody responses can fully represent the neutralization activities in sera has not been well studied. Using the newly designed stable HIV-1 Env trimer as hook, we isolated two distinct broadly neutralizing antibodies (bnAbs) from Chinese rhesus macaques infected with SHIV1157ipd3N4 for 5 years. One lineage of neutralizing antibodies (JT15 and JT16) targeted the V2-apex in the Env trimers, similar to the J038 lineage bnAbs identified in our previous study. The other lineage neutralizing antibody (JT18) targeted the V3 crown region in the Env, which strongly competed with human 447-52D. Each lineage antibody neutralized a different set of viruses. Interestingly, when the two neutralizing antibodies from different lineages isolated from the same macaque were combined, the mixture had a neutralization breath very similar to that from the cognate sera. Our study demonstrated that a minimum of two different neutralizing antibodies can fully recapitulate the serum neutralization breadth. This observation can have important implications in AIDS vaccine design.

Differences in neutralizing antibody sensitivities and envelope characteristics indicate distinct antigenic properties of Nigerian HIV-1 subtype G and CRF02_AG.

The magnitude of the HIV-1 epidemic in Nigeria is second only to the subtype C epidemic in South Africa, yet the subtypes prevalent in Nigeria require further characterization. A panel of 50 subtype G and 18 CRF02_AG Nigerian HIV-1 pseudoviruses (PSV) was developed and envelope coreceptor usage, neutralization sensitivity and cross-clade reactivity were characterized. These PSV were neutralized by some antibodies targeting major neutralizing determinants, but potentially important differences were observed in specific sensitivities (eg. to sCD4, MPER and V2/V3 monoclonal antibodies), as well as in properties such as variable loop lengths, number of potential N-linked glycans and charge, demonstrating distinct antigenic characteristics of CRF02_AG and subtype G. There was preferential neutralization of the matched CRF/subtype when PSV from subtype G or CRF02_AG were tested using pooled plasma. These novel Nigerian PSV will be useful to study HIV-1 CRF- or subtype-specific humoral immune responses for subtype G and CRF02_AG.

Applying Flow Virometry to Study the HIV Envelope Glycoprotein and Differences Across HIV Model Systems.

The HIV envelope glycoprotein (Env) is a trimeric protein that facilitates viral binding and fusion with target cells. As the sole viral protein on the HIV surface, Env is important both for immune responses to HIV and in vaccine designs. Targeting Env in clinical applications is challenging due to its heavy glycosylation, high genetic variability, conformational camouflage, and its low abundance on virions. Thus, there is a critical need to better understand this protein. Flow virometry (FV) is a useful methodology for phenotyping the virion surface in a high-throughput, single virion manner. To demonstrate the utility of FV to characterize Env, we stained HIV virions with a panel of 85 monoclonal antibodies targeting different regions of Env. A broad range of antibodies yielded robust staining of Env, with V3 antibodies showing the highest quantitative staining. A subset of antibodies tested in parallel on viruses produced in CD4+ T cell lines, HEK293T cells, and primary cells showed that the cellular model of virus production can impact Env detection. Finally, in addition to being able to highlight Env heterogeneity on virions, we show FV can sensitively detect differences in Env conformation when soluble CD4 is added to virions before staining.

Intermediate open state of CD4-bound HIV-1 env heterotrimers in asia CRFs.

The HIV-1 envelope glycoprotein (Env) plays crucial role in viral infection by facilitating viral attachment to host cells and inducing fusion of the virus with the host cell membrane. This fusion allows the HIV-1 viral genome to enter the target cell then triggering various stages of the viral life cycle. The native Env directly interacts with the main receptor CD4 and the co-receptor (CCR5 or CXCR4) in human cell membrane then induces membrane fusion. The elucidation of the structure of Env with CD4 and co-receptors in different HIV-1 subtypes is essential for the understanding of the mechanism of virus entry. Here we report the Cryo-EM structure of the CD4-bound HIV-1 heterotrimeric Env from Asia prevalent CRF07_BC CH119 strain. In this structure, the binding of three CD4 molecules with Env induced extensively conformational changes in gp120, resulting in the transformation of the Env from close state to intermediate open state. Additionally, the conformational shift of V1/V2 loops of the heterotrimeric Env allosterically expose the V3 loop and promoting the further interactions with co-receptor CCR5 or CXCR4. These findings not only illustrate the structural complexity and plasticity of HIV-1 Env but also give new insights how the biological trimeric Env initialize the immune recognition and membrane fusion.

Evaluating the antibody response elicited by diverse HIV envelope immunogens in the African green monkey (Vervet) model.

African Green (Vervet) monkeys have been extensively studied to understand the pathogenesis of infectious diseases. Using vervet monkeys as pre-clinical models may be an attractive option for low-resourced areas as they are found abundantly and their maintenance is more cost-effective than bigger primates such as rhesus macaques. We assessed the feasibility of using vervet monkeys as animal models to examine the immunogenicity of HIV envelope trimer immunogens in pre-clinical testing. Three groups of vervet monkeys were subcutaneously immunized with either the BG505 SOSIP.664 trimer, a novel subtype C SOSIP.664 trimer, CAP255, or a combination of BG505, CAP255 and CAP256.SU SOSIP.664 trimers. All groups of vervet monkeys developed robust binding antibodies by the second immunization with the peak antibody response occurring after the third immunization. Similar to binding, antibody dependent cellular phagocytosis was also observed in all the monkeys. While all animals developed potent, heterologous Tier 1 neutralizing antibody responses, autologous neutralization was limited with only half of the animals in each group developing responses to their vaccine-matched pseudovirus. These data suggest that the vervet monkey model may yield distinct antibody responses compared to other models. Further study is required to further determine the utility of this model in HIV immunization studies.

Contemporary HIV-1 consensus Env with AI-assisted redesigned hypervariable loops promote antibody binding.

An effective HIV-1 vaccine must elicit broadly neutralizing antibodies (bnAbs) against highly diverse Envelope glycoproteins (Env). Since Env with the longest hypervariable (HV) loops is more resistant to the cognate bnAbs than Env with shorter HV loops, we redesigned hypervariable loops for updated Env consensus sequences of subtypes B and C and CRF01_AE. Using modeling with AlphaFold2, we reduced the length of V1, V2, and V5 HV loops while maintaining the integrity of the Env structure and glycan shield, and modified the V4 HV loop. Spacers are designed to limit strain-specific targeting. All updated Env are infectious as pseudoviruses. Preliminary structural characterization suggests that the modified HV loops have a limited impact on Env's conformation. Binding assays show improved binding to modified subtype B and CRF01_AE Env but not to subtype C Env. Neutralization assays show increases in sensitivity to bnAbs, although not always consistently across clades. Strikingly, the HV loop modification renders the resistant CRF01_AE Env sensitive to 10-1074 despite the absence of a glycan at N332.

Vaccine induction of heterologous HIV-1-neutralizing antibody B cell lineages in humans.

A critical roadblock to HIV vaccine development is the inability to induce B cell lineages of broadly neutralizing antibodies (bnAbs) in humans. In people living with HIV-1, bnAbs take years to develop. The HVTN 133 clinical trial studied a peptide/liposome immunogen targeting B cell lineages of HIV-1 envelope (Env) membrane-proximal external region (MPER) bnAbs (NCT03934541). Here, we report MPER peptide-liposome induction of polyclonal HIV-1 B cell lineages of mature bnAbs and their precursors, the most potent of which neutralized 15% of global tier 2 HIV-1 strains and 35% of clade B strains with lineage initiation after the second immunization. Neutralization was enhanced by vaccine selection of improbable mutations that increased antibody binding to gp41 and lipids. This study demonstrates proof of concept for rapid vaccine induction of human B cell lineages with heterologous neutralizing activity and selection of antibody improbable mutations and outlines a path for successful HIV-1 vaccine development.

Vaccine priming of rare HIV broadly neutralizing antibody precursors in nonhuman primates.

Germline-targeting immunogens hold promise for initiating the induction of broadly neutralizing antibodies (bnAbs) to HIV and other pathogens. However, antibody-antigen recognition is typically dominated by heavy chain complementarity determining region 3 (HCDR3) interactions, and vaccine priming of HCDR3-dominant bnAbs by germline-targeting immunogens has not been demonstrated in humans or outbred animals. In this work, immunization with N332-GT5, an HIV envelope trimer designed to target precursors of the HCDR3-dominant bnAb BG18, primed bnAb-precursor B cells in eight of eight rhesus macaques to substantial frequencies and with diverse lineages in germinal center and memory B cells. We confirmed bnAb-mimicking, HCDR3-dominant, trimer-binding interactions with cryo-electron microscopy. Our results demonstrate proof of principle for HCDR3-dominant bnAb-precursor priming in outbred animals and suggest that N332-GT5 holds promise for the induction of similar responses in humans.

Epidemic characteristics of local HIV-2 transmission across Hunan province, China.

OBJECTIVE: To elucidate the epidemiological features of HIV-2 in Hunan Province, China, utilizing sequence analysis. METHODS: Thirteen individuals diagnosed with HIV-2 infection in Hunan Province, China, from 2017 to 2023 were included in this study. Amplification of HIV-2 env and pol regions was conducted, followed by Sanger sequencing. Phylogenetic and molecular transmission network analyses were performed to delineate molecular features and transmission dynamics. RESULTS: All 14 individuals contracted HIV-2 through heterosexual intercourse, comprising 7 males and 7 females, with a median age of 58 years. Among them, three couples (HN001 and HN013, HN010 and HN011, HN008 and HN009) were identified, along with commercial sexual activity engagement reported for subject HN004. Notably, subjects HN001, HN003, HN008, and HN010 engaged in commercial sexual activities at the same location as subject HN004. Phylogenetic analysis of the pol gene revealed close proximity of sequences from all subjects to reference sequences from Gambia (Sub-type A). Employing a genetic distance threshold of 1.5 %, eight out of the 14 subjects formed a molecular transmission network, with HN002 and HN004 identified as central nodes. CONCLUSION: From 2017 to 2023, all HIV-2-infected individuals in Hunan Province, China, acquired the virus through identifiable routes, indicating transmission of similar HIV-2 strains among them.

CoPoP liposomes displaying stabilized clade C HIV-1 Env elicit tier 2 multiclade neutralization in rabbits.

One of the strategies towards an effective HIV-1 vaccine is to elicit broadly neutralizing antibody responses that target the high HIV-1 Env diversity. Here, we present an HIV-1 vaccine candidate that consists of cobalt porphyrin-phospholipid (CoPoP) liposomes decorated with repaired and stabilized clade C HIV-1 Env trimers in a prefusion conformation. These particles exhibit high HIV-1 Env trimer decoration, serum stability and bind broadly neutralizing antibodies. Three sequential immunizations of female rabbits with CoPoP liposomes displaying a different clade C HIV-1 gp140 trimer at each dosing generate high HIV-1 Env-specific antibody responses. Additionally, serum neutralization is detectable against 18 of 20 multiclade tier 2 HIV-1 strains. Furthermore, the peak antibody titers induced by CoPoP liposomes can be recalled by subsequent heterologous immunization with Ad26-encoded membrane-bound stabilized Env antigens. Hence, a CoPoP liposome-based HIV-1 vaccine that can generate cross-clade neutralizing antibody immunity could potentially be a component of an efficacious HIV-1 vaccine.

Further Characterization of the Antiviral Transmembrane Protein MARCH8.

The cellular transmembrane protein MARCH8 impedes the incorporation of various viral envelope glycoproteins, such as the HIV-1 envelope glycoprotein (Env) and vesicular stomatitis virus G-glycoprotein (VSV-G), into virions by downregulating them from the surface of virus-producing cells. This downregulation significantly reduces the efficiency of virus infection. In this study, we aimed to further characterize this host protein by investigating its species specificity and the domains responsible for its antiviral activity, as well as its ability to inhibit cell-to-cell HIV-1 infection. We found that the antiviral function of MARCH8 is well conserved in the rhesus macaque, mouse, and bovine versions. The RING-CH domains of these versions are functionally important for inhibiting HIV-1 Env and VSV-G-pseudovirus infection, whereas tyrosine motifs are crucial for the former only, consistent with findings in human MARCH8. Through analysis of chimeric proteins between MARCH8 and non-antiviral MARCH3, we determined that both the N-terminal and C-terminal cytoplasmic tails, as well as presumably the N-terminal transmembrane domain, of MARCH8 are critical for its antiviral activity. Notably, we found that MARCH8 is unable to block cell-to-cell HIV-1 infection, likely due to its insufficient downregulation of Env. These findings offer further insights into understanding the biology of this antiviral transmembrane protein.

Mutation-guided vaccine design: A process for developing boosting immunogens for HIV broadly neutralizing antibody induction.

A major goal of HIV-1 vaccine development is the induction of broadly neutralizing antibodies (bnAbs). Although success has been achieved in initiating bnAb B cell lineages, design of boosting immunogens that select for bnAb B cell receptors with improbable mutations required for bnAb affinity maturation remains difficult. Here, we demonstrate a process for designing boosting immunogens for a V3-glycan bnAb B cell lineage. The immunogens induced affinity-matured antibodies by selecting for functional improbable mutations in bnAb precursor knockin mice. Moreover, we show similar success in prime and boosting with nucleoside-modified mRNA-encoded HIV-1 envelope trimer immunogens, with improved selection by mRNA immunogens of improbable mutations required for bnAb binding to key envelope glycans. These results demonstrate the ability of both protein and mRNA prime-boost immunogens for selection of rare B cell lineage intermediates with neutralizing breadth after bnAb precursor expansion, a key proof of concept and milestone toward development of an HIV-1 vaccine.

Prevalence of resistance-associated viral variants to the HIV-specific broadly neutralising antibody 10-1074 in a UK bNAb-naïve population.

Broadly neutralising antibodies (bNAbs) targeting HIV show promise for both prevention of infection and treatment. Among these, 10-1074 has shown potential in neutralising a wide range of HIV strains. However, resistant viruses may limit the clinical efficacy of 10-1074. The prevalence of both de novo and emergent 10-1074 resistance will determine its use at a population level both to protect against HIV transmission and as an option for treatment. To help understand this further, we report the prevalence of pre-existing mutations associated with 10-1074 resistance in a bNAb-naive population of 157 individuals presenting to UK HIV centres with primary HIV infection, predominantly B clade, receiving antiretroviral treatment. Single genome analysis of HIV proviral envelope sequences showed that 29% of participants' viruses tested had at least one sequence with 10-1074 resistance-associated mutations. Mutations interfering with the glycan binding site at HIV Env position 332 accounted for 95% of all observed mutations. Subsequent analysis of a larger historic dataset of 2425 B-clade envelope sequences sampled from 1983 to 2019 revealed an increase of these mutations within the population over time. Clinical studies have shown that the presence of pre-existing bNAb mutations may predict diminished therapeutic effectiveness of 10-1074. Therefore, we emphasise the importance of screening for these mutations before initiating 10-1074 therapy, and to consider the implications of pre-existing resistance when designing prevention strategies.

Prolonged delivery of HIV-1 vaccine nanoparticles from hydrogels.

Immunization is a straightforward concept but remains for some pathogens like HIV-1 a challenge. Thus, new approaches towards increasing the efficacy of vaccines are required to turn the tide. There is increasing evidence that antigen exposure over several days to weeks induces a much stronger and more sustained immune response compared to traditional bolus injection, which usually leads to antigen elimination from the body within a couple of days. Therefore, we developed a poly(ethylene) glycol (PEG) hydrogel platform to investigate the principal feasibility of a sustained release of antigens to mimic natural infection kinetics. Eight-and four-armed PEG macromonomers of different MWs (10, 20, and 40 kDa) were end-group functionalized to allow for hydrogel formation via covalent cross-linking. An HIV-1 envelope (Env) antigen in its trimeric (Envtri) or monomeric (Envmono) form was applied. The soluble Env antigen was compared to a formulation of Env attached to silica nanoparticles (Env-SiNPs). The latter are known to have a higher immunogenicity compared to their soluble counterparts. Hydrogels were tunable regarding the rheological behavior allowing for different degradation times and release timeframes of Env-SiNPs over two to up to 50 days. Affinity measurements of the VCR01 antibody which specifically recognizes the CD4 binding site of Env, revealed that neither the integrity nor the functionality of Envmono-SiNPs (Kd = 2.1 ± 0.9 nM) and Envtri-SiNPs (Kd = 1.5 ± 1.3 nM), respectively, were impaired after release from the hydrogel (Kd before release: 2.1 ± 0.1 and 7.8 ± 5.3 nM, respectively). Finally, soluble Env and Env-SiNPs which are two physico-chemically distinct compounds, were co-delivered and shown to be sequentially released from one hydrogel which could be beneficial in terms of heterologous immunization or single dose vaccination. In summary, this study presents a tunable, versatile applicable, and effective delivery platform that could improve vaccination effectiveness also for other infectious diseases than HIV-1.

Amino acid substitution of the membrane-proximal external region alter neutralization sensitivity in a chronic HIV-1 clade B infected patient.

The membrane-proximal external region (MPER) represents a highly conserved region of the Human Immunodeficiency Virus (HIV) envelope glycoprotein (env) targeted by several broadly neutralizing antibodies (bnAbs). In this study, we employed single genome amplification to amplify 34 full-length env sequences from the 2005 plasma sample of CBJC504, a chronic HIV-1 clade B infected individual. We identified three amino acid changes (N671S, D674N, and K677R) in the MPER. A longitudinal analysis revealed that the proportion of env sequences with MPER mutations increased from 26.5 % in 2005 to 56.0 % in 2009, and the sequences with the same mutation clustered together. Nine functional pseudoviruses were generated from the 34 env sequences to examine the effect of these mutations on neutralizing activity. Pseudoviruses carrying N674 or R677 mutations demonstrate increased sensitivity to autologous plasma and monoclonal antibodies 2F5, 4E10, and 10E8. Reverse mutations were performed in env including N674, R677, D659, and S671/N677 mutations, to validate the impact of the mutations on neutralizing sensitivity. Neutralization assays indicated that the N671S mutation increased neutralization sensitivity to 2F5 and 10E8. The amino acid R at position 677 increased viral resistance to 10E8, whereas N enhanced viral resistance to 4E10 and 10E8. It has been proposed that critical amino acids in the extra-MPER and the number of potential N-like glycosylation sites (PNGSs) in the V1 loop may have an impact on neutralizing activity. Understanding the mutations and evolution of MPER in chronically infected patients with HIV-1 is crucial for the design and development of vaccines that trigger bnAbs against MPER.

Alternative substitutions of N332 in HIV-1AD8 gp120 differentially affect envelope glycoprotein function and viral sensitivity to broadly neutralizing antibodies targeting the V3-glycan.

The envelope glycoprotein (Env) trimer on the surface of human immunodeficiency virus type I (HIV-1) mediates viral entry into host CD4+ T cells and is the sole target of neutralizing antibodies. Broadly neutralizing antibodies (bnAbs) that target gp120 V3-glycan of HIV-1 Env trimer are potent and block the entry of diverse HIV-1 strains. Most V3-glycan bnAbs interact, to a different extent, with a glycan attached to N332, but Asn at this position is not absolutely conserved or required for HIV-1 entry based on the prevalence of N332 in different circulating HIV-1 strains from diverse clades. Here, we studied the effects of amino acid changes at position 332 of HIV-1AD8 Envs on HIV-1 sensitivity to antibodies, cold exposure, and soluble CD4. We further investigated how these changes affect Env function and HIV-1 infectivity in vitro. Our results suggest robust tolerability of HIV-1AD8 Env N332 to changes, with specific changes that resulted in extended exposure of gp120 V3 loop, which is typically concealed in most primary HIV-1 isolates. Viral evolution leading to Asn at position 332 of HIVAD8 Envs is supported by the selection advantage of high levels of cell-cell fusion, transmission, and infectivity with high levels of cell surface expression and slightly higher gp120 shedding than most N332 variants. Thus, tolerance of HIV-1AD8 Envs to different amino acids at position 332 provides increased flexibility to respond to changing conditions/environments and evade the immune system. Modeling studies of the distance between N332 glycan and specific bnAbs were in agreement with N332 glycan dependency on bnAb neutralization. Overall, our studies provide insights into the contribution of specific amino acids at position 332 to Env antigenicity, stability on ice, and conformational states. IMPORTANCE: Glycan attached to amino acid asparagine at position 332 of HIV-1 envelope glycoproteins is a main target of a subset of broadly neutralizing antibodies that block HIV-1 infection. Here, we defined the contribution of different amino acids at this position to Env antigenicity, stability on ice, and conformational states.

HIResist: a database of HIV-1 resistance to broadly neutralizing antibodies.

MOTIVATION: Changing the course of the human immunodeficiency virus type I (HIV-1) pandemic is a high public health priority with approximately 39 million people currently living with HIV-1 (PLWH) and about 1.5 million new infections annually worldwide. Broadly neutralizing antibodies (bnAbs) typically target highly conserved sites on the HIV-1 envelope glycoproteins (Envs), which mediate viral entry, and block the infection of diverse HIV-1 strains. But different mechanisms of HIV-1 resistance to bnAbs prevent robust application of bnAbs for therapeutic and preventive interventions. RESULTS: Here we report the development of a new database that provides data and computational tools to aid the discovery of resistant features and may assist in analysis of HIV-1 resistance to bnAbs. Bioinformatic tools allow identification of specific patterns in Env sequences of resistant strains and development of strategies to elucidate the mechanisms of HIV-1 escape; comparison of resistant and sensitive HIV-1 strains for each bnAb; identification of resistance and sensitivity signatures associated with specific bnAbs or groups of bnAbs; and visualization of antibody pairs on cross-sensitivity plots. The database has been designed with a particular focus on user-friendly and interactive interface. Our database is a valuable resource for the scientific community and provides opportunities to investigate patterns of HIV-1 resistance and to develop new approaches aimed to overcome HIV-1 resistance to bnAbs. AVAILABILITY AND IMPLEMENTATION: HIResist is freely available at https://hiresist.ahc.umn.edu/.

Stoichiometry for entry and binding properties of the Env protein of R5 T cell-tropic HIV-1 and its evolutionary variant of macrophage-tropic HIV-1.

Human immunodeficiency virus type 1 typically requires a high density of CD4 for efficient entry as a mechanism to target CD4+ T cells (T-tropic), with CCR5 being used most often as the coreceptor. When target T cells are limiting, the virus can evolve to infect cells with a low density of CD4 such as macrophages (M-tropic). The entry phenotype is known to be encoded in the viral Env protein on the surface of the virus particle. Using data showing a dose response for infectivity based on CD4 surface density, we built a model consistent with T-tropic viruses requiring multiple CD4 molecules to mediate infection, whereas M-tropic viruses can infect cells using a single CD4 receptor molecule interaction. We also found that T-tropic viruses bound to the surface of cells with a low density of CD4 are released more slowly than M-tropic viruses which we modeled to be due to multiple interactions of the T-tropic virus with multiple CD4 molecules to allow the initial stable binding. Finally, we found that some M-tropic Env proteins, as the gp120 subunit, possess an enhanced affinity for CD4 compared with their T-tropic pair, indicating that the evolution of macrophage tropism can be reflected both in the closed Env trimer conformation on the virion surface and, in some cases, also in the open confirmation of gp120 Env. Collectively, these studies reveal differences in the stoichiometry of interaction of T-tropic and M-tropic viruses with CD4 and start to identify the basis of binding differences at the biochemical level. IMPORTANCE: Human immunodeficiency virus type 1 normally targets CD4+ T cells for viral replication. When T cells are limiting, the virus can evolve to infect myeloid cells. The evolutionary step involves a change from requiring a high surface density of CD4 for entry to being able to infect cells with a low density of CD4, as is found on myeloid lineage cells such as macrophage and microglia. Viruses able to infect macrophages efficiently are most often found in the CNS late in the disease course, and such viruses may contribute to neurocognitive impairment. Here, we examine the CD4 binding properties of the viral Env protein to explore these two different entry phenotypes.

Microsecond dynamics control the HIV-1 Envelope conformation.

The HIV-1 Envelope (Env) glycoprotein facilitates host cell fusion through a complex series of receptor-induced structural changes. Although remarkable progress has been made in understanding the structures of various Env conformations, microsecond timescale dynamics have not been studied experimentally. Here, we used time-resolved, temperature-jump small-angle x-ray scattering to monitor structural rearrangements in an HIV-1 Env SOSIP ectodomain construct with microsecond precision. In two distinct Env variants, we detected a transition that correlated with known Env structure rearrangements with a time constant in the hundreds of microseconds range. A previously unknown structural transition was also observed, which occurred with a time constant below 10 µs, and involved an order-to-disorder transition in the trimer apex. Using this information, we engineered an Env SOSIP construct that locks the trimer in the prefusion closed state by connecting adjacent protomers via disulfides. Our findings show that the microsecond timescale structural dynamics play an essential role in controlling the Env conformation with impacts on vaccine design.

A VRC13-like bNAb response is associated with complex escape pathways in HIV-1 envelope.

The rational design of HIV-1 immunogens to trigger the development of broadly neutralizing antibodies (bNAbs) requires understanding the viral evolutionary pathways influencing this process. An acute HIV-1-infected individual exhibiting >50% plasma neutralization breadth developed neutralizing antibody specificities against the CD4-binding site (CD4bs) and V1V2 regions of Env gp120. Comparison of pseudoviruses derived from early and late autologous env sequences demonstrated the development of >2 log resistance to VRC13 but not to other CD4bs-specific bNAbs. Mapping studies indicated that the V3 and CD4-binding loops of Env gp120 contributed significantly to developing resistance to the autologous neutralizing response and that the CD4-binding loop (CD4BL) specifically was responsible for the developing resistance to VRC13. Tracking viral evolution during the development of this cross-neutralizing CD4bs response identified amino acid substitutions arising at only 4 of 11 known VRC13 contact sites (K282, T283, K421, and V471). However, each of these mutations was external to the V3 and CD4BL regions conferring resistance to VRC13 and was transient in nature. Rather, complete resistance to VRC13 was achieved through the cooperative expression of a cluster of single amino acid changes within and immediately adjacent to the CD4BL, including a T359I substitution, exchange of a potential N-linked glycosylation (PNLG) site to residue S362 from N363, and a P369L substitution. Collectively, our data characterize complex HIV-1 env evolution in an individual developing resistance to a VRC13-like neutralizing antibody response and identify novel VRC13-associated escape mutations that may be important to inducing VRC13-like bNAbs for lineage-based immunogens.IMPORTANCEThe pursuit of eliciting broadly neutralizing antibodies (bNAbs) through vaccination and their use as therapeutics remains a significant focus in the effort to eradicate HIV-1. Key to our understanding of this approach is a more extensive understanding of bNAb contact sites and susceptible escape mutations in HIV-1 envelope (env). We identified a broad neutralizer exhibiting VRC13-like responses, a non-germline restricted class of CD4-binding site antibody distinct from the well-studied VRC01-class. Through longitudinal envelope sequencing and Env-pseudotyped neutralization assays, we characterized a complex escape pathway requiring the cooperative evolution of four amino acid changes to confer complete resistance to VRC13. This suggests that VRC13-class bNAbs may be refractory to rapid escape and attractive for therapeutic applications. Furthermore, the identification of longitudinal viral changes concomitant with the development of neutralization breadth may help identify the viral intermediates needed for the maturation of VRC13-like responses and the design of lineage-based immunogens.