Structural dynamics reveal isolate-specific differences at neutralization epitopes on HIV Env.

The envelope glycoprotein (Env) is the sole target for neutralizing antibodies against HIV and the most rapidly evolving, variable part of the virus. High-resolution structures of Env trimers captured in the pre-fusion, closed conformation have revealed a high degree of structural similarity across diverse isolates. Biophysical data, however, indicate that Env is highly dynamic, and the level of dynamics and conformational sampling is believed to vary dramatically between HIV isolates. Dynamic differences likely influence neutralization sensitivity, receptor activation, and overall trimer stability. Here, using hydrogen/deuterium-exchange mass spectrometry (HDX-MS), we have mapped local dynamics across native-like Env SOSIP trimers from diverse isolates. We show that significant differences in epitope order are observed across most sites targeted by broadly neutralizing antibodies. We also observe isolate-dependent conformational switching that occurs over a broad range of timescales. Lastly, we report that hyper-stabilizing mutations that dampen dynamics in some isolates have little effect on others.

In vivo Serial Passaging of Human-Simian Immunodeficiency Virus Clones Identifies Characteristics for Persistent Viral Replication.

We previously reported that a human immunodeficiency virus type 1 with a simian immunodeficiency virus vif substitution (HSIV-vifNL4-3) could replicate in pigtailed macaques (PTMs), demonstrating that Vif is a species-specific tropism factor of primate lentiviruses. However, infections did not result in high-peak viremia or setpoint plasma viral loads, as observed during simian immunodeficiency virus (SIV) infection of PTMs. Here, we characterized variants isolated from one of the original infected animals with CD4 depletion after nearly 4years of infection to identify determinants of increased replication fitness. In our studies, we found that the HSIV-vif clones did not express the HIV-1 Vpr protein due to interference from the vpx open reading frame (ORF) in singly spliced vpr mRNA. To examine whether these viral genes contribute to persistent viral replication, we generated infectious HSIV-vif clones expressing either the HIV-1 Vpr or SIV Vpx protein. And then to determine viral fitness determinants of HSIV-vif, we conducted three rounds of serial in vivo passaging in PTMs, starting with an initial inoculum containing a mixture of CXCR4-tropic [Vpr-HSIV-vifNL4-3 isolated at 196 (C/196) and 200 (C/200) weeks post-infection from a PTM with depressed CD4 counts] and CCR5-tropic HSIV (Vpr+ HSIV-vif derivatives based NL-AD8 and Bru-Yu2 and a Vpx expressing HSIV-vifYu2). Interestingly, all infected PTMs showed peak plasma viremia close to or above 105 copies/ml and persistent viral replication for more than 20weeks. Infectious molecular clones (IMCs) recovered from the passage 3 PTM (HSIV-P3 IMCs) included mutations required for HIV-1 Vpr expression and those mutations encoded by the CXCR4-tropic HSIV-vifNL4-3 isolate C/196. The data indicate that the viruses selected during long-term infection acquired HIV-1 Vpr expression, suggesting the importance of Vpr for in vivo pathogenesis. Further passaging of HSIV-P3 IMCs in vivo may generate pathogenic variants with higher replication capacity, which will be a valuable resource as challenge virus in vaccine and cure studies.

Vaccinia virus-based vaccines confer protective immunity against SARS-CoV-2 virus in Syrian hamsters.

COVID-19 in humans is caused by Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that belongs to the beta family of coronaviruses. SARS-CoV-2 causes severe respiratory illness in 10-15% of infected individuals and mortality in 2-3%. Vaccines are urgently needed to prevent infection and to contain viral spread. Although several mRNA- and adenovirus-based vaccines are highly effective, their dependence on the "cold chain" transportation makes global vaccination a difficult task. In this context, a stable lyophilized vaccine may present certain advantages. Accordingly, establishing additional vaccine platforms remains vital to tackle SARS-CoV-2 and any future variants that may arise. Vaccinia virus (VACV) has been used to eradicate smallpox disease, and several attenuated viral strains with enhanced safety for human applications have been developed. We have generated two candidate SARS-CoV-2 vaccines based on two vaccinia viral strains, MVA and v-NY, that express full-length SARS-CoV-2 spike protein. Whereas MVA is growth-restricted in mammalian cells, the v-NY strain is replication-competent. We demonstrate that both candidate recombinant vaccines induce high titers of neutralizing antibodies in C57BL/6 mice vaccinated according to prime-boost regimens. Furthermore, our vaccination regimens generated TH1-biased immune responses in mice. Most importantly, prime-boost vaccination of a Syrian hamster infection model with MVA-S and v-NY-S protected the hamsters against SARS-CoV-2 infection, supporting that these two vaccines are promising candidates for future development. Finally, our vaccination regimens generated neutralizing antibodies that partially cross-neutralized SARS-CoV-2 variants of concern.

Immunization by exposure to live virus (SIVmne/HIV-2287) during antiretroviral drug prophylaxis may reduce risk of subsequent viral challenge.

RATIONALE/STUDY DESIGN: A major challenge in the development of HIV vaccines is finding immunogens that elicit protection against a broad range of viral strains. Immunity to a narrow range of viral strains may protect infants of HIV-infected women or partners discordant for HIV. We hypothesized that immunization to the relevant viral variants could be achieved by exposure to infectious virus during prophylaxis with antiretroviral drugs. To explore this approach in an animal model, macaques were exposed to live virus (SIVmne or HIV-2287) during prophylaxis with parenteral tenofovir and humoral and cellular immune responses were quantified. Subsequently, experimental animals were challenged with homologous virus to evaluate protection from infection, and if infection occurred, the course of disease was compared to control animals. Experimental animals uninfected with SIVmne were challenged with heterologous HIV-2287 to assess resistance to retroviral infection. METHODOLOGY/PRINCIPAL FINDINGS: Juvenile female Macaca nemestrina (N = 8) were given ten weekly intravaginal exposures with either moderately (SIVmne) or highly (HIV-2287) pathogenic virus during tenofovir prophylaxis. Tenofovir protected all 8 experimental animals from infection, while all untreated control animals became infected. Specific non-neutralizing antibodies were elicited in blood and vaginal secretions of experimental animals, but no ELISPOT responses were detected. Six weeks following the cessation of tenofovir, intravaginal challenge with homologous virus infected 2/4 (50%) of the SIVmne-immunized animals and 4/4 (100%) of the HIV-2287-immunized animals. The two SIVmne-infected and 3 (75%) HIV-2287-infected had attenuated disease, suggesting partial protection. CONCLUSIONS/SIGNIFICANCE: Repeated exposure to SIVmne or HIV-2287, during antiretroviral prophylaxis that blocked infection, induced binding antibodies in the blood and mucosa, but not neutralizing antibodies or specific cellular immune responses. Studies to determine whether antibodies are similarly induced in breastfeeding infants and sexual partners discordant for HIV infection and receiving pre-exposure antiretroviral prophylaxis are warranted, including whether these antibodies appear to confer partial or complete protection from infection.

Gene Transfer in Adeno-Associated Virus Seropositive Rhesus Macaques Following Rapamycin Treatment and Subcutaneous Delivery of AAV6, but Not Retargeted AAV6 Vectors.

Adeno-associated virus (AAV) vectors such as AAV6, which shows tropism for primary human CD4+ T cells in vitro, are being explored for delivery of anti-HIV therapeutic modalities in vivo. However, pre-existing immunity and sequestration in nontarget organs can significantly hinder their performance. To overcome these challenges, we investigated whether immunosuppression would allow gene delivery by AAV6 or targeted AAV6 derivatives in seropositive rhesus macaques. Animals were immune suppressed with rapamycin before intravenous (IV) or subcutaneous (SC) delivery of AAV, and we monitored vector biodistribution, gene transfer, and safety. Macaques received phosphate-buffered saline, AAV6 alone, or an equal dose of AAV6 and an AAV6-55.2 vector retargeted to CD4 through a direct ankyrin repeat protein (DARPin). AAV6 and AAV6-55.2 vector genomes were found in peripheral blood mononuclear cells and most organs up to 28 days postadministration, with the highest levels seen in liver, spleen, lymph nodes (LNs), and muscle, suggesting that retargeting did not prevent vector sequestration. Despite vector genome detection, gene expression from AAV6-55.2 was not detected in any tissue. SC injection of AAV6 facilitated efficient gene expression in muscle adjacent to the injection site, plus low-level gene expression in spleen, LNs, and liver, whereas gene expression following IV injection of AAV6 was predominantly seen in the spleen. AAV vectors were well tolerated, although elevated liver enzymes were detected in three of four AAV-treated animals 14 days after rapamycin withdrawal. One SC-injected animal had muscle inflammation proximal to the injection site, plus detectable T cell responses against transgene and AAV6 capsid at study finish. Overall, our data suggest that rapamycin treatment may offer a possible strategy to express anti-HIV therapeutics such as broadly neutralizing antibodies from muscle. This study provides important safety and efficacy data that will aid study design for future anti-HIV gene therapies.

Characterization of HIV-1 Nucleoside-Modified mRNA Vaccines in Rabbits and Rhesus Macaques.

Despite the enormous effort in the development of effective vaccines against HIV-1, no vaccine candidate has elicited broadly neutralizing antibodies in humans. Thus, generation of more effective anti-HIV vaccines is critically needed. Here we characterize the immune responses induced by nucleoside-modified and purified mRNA-lipid nanoparticle (mRNA-LNP) vaccines encoding the clade C transmitted/founder HIV-1 envelope (Env) 1086C. Intradermal vaccination with nucleoside-modified 1086C Env mRNA-LNPs elicited high levels of gp120-specific antibodies in rabbits and rhesus macaques. Antibodies generated in rabbits neutralized a tier 1 virus, but no tier 2 neutralization activity could be measured. Importantly, three of six non-human primates developed antibodies that neutralized the autologous tier 2 strain. Despite stable anti-gp120 immunoglobulin G (IgG) levels, tier 2 neutralization titers started to drop 4 weeks after booster immunizations. Serum from both immunized rabbits and non-human primates demonstrated antibody-dependent cellular cytotoxicity activity. Collectively, these results are supportive of continued development of nucleoside-modified and purified mRNA-LNP vaccines for HIV. Optimization of Env immunogens and vaccination protocols are needed to increase antibody neutralization breadth and durability.

Evidence for persistence of the SHIV reservoir early after MHC haploidentical hematopoietic stem cell transplantation.

Allogeneic transplantation (allo-HCT) has led to the cure of HIV in one individual, raising the question of whether transplantation can eradicate the HIV reservoir. To test this, we here present a model of allo-HCT in SHIV-infected, cART-suppressed nonhuman primates. We infect rhesus macaques with SHIV-1157ipd3N4, suppress them with cART, then transplant them using MHC-haploidentical allogeneic donors during continuous cART. Transplant results in ~100% myeloid donor chimerism, and up to 100% T-cell chimerism. Between 9 and 47 days post-transplant, terminal analysis shows that while cell-associated SHIV DNA levels are reduced in the blood and in lymphoid organs post-transplant, the SHIV reservoir persists in multiple organs, including the brain. Sorting of donor-vs.-recipient cells reveals that this reservoir resides in recipient cells. Moreover, tetramer analysis indicates a lack of virus-specific donor immunity post-transplant during continuous cART. These results suggest that early post-transplant, allo-HCT is insufficient for recipient reservoir eradication despite high-level donor chimerism and GVHD.

Intestinal damage precedes mucosal immune dysfunction in SIV infection.

HIV and pathogenic SIV infection are characterized by mucosal dysfunction including epithelial barrier damage, loss of Th17 cells, neutrophil infiltration, and microbial translocation with accompanying inflammation. However, it is unclear how and when these contributing factors occur relative to one another. In order to determine whether any of these features initiates the cycle of damage, we longitudinally evaluated the kinetics of mucosal and systemic T-cell activation, microbial translocation, and Th17 cell and neutrophil frequencies following intrarectal SIV infection of rhesus macaques. We additionally assessed the colon proteome to elucidate molecular pathways altered early after infection. We demonstrate increased T-cell activation (HLA-DR+) beginning 3-14 days post-SIV challenge, reduced peripheral zonulin 3-14 days post-SIV, and evidence of microbial translocation 14 days post-SIV. The onset of mucosal dysfunction preceded peripheral and mucosal Th17 depletion, which occurred 14-28 days post-SIV, and gut neutrophil accumulation was not observed. Proteins involved in epithelial structure were downregulated 3 days post-SIV followed by an upregulation of immune proteins 14 days post-SIV. These data demonstrate that immune perturbations such as Th17 loss and neutrophil infiltration occur after alterations to epithelial structural protein pathways, suggesting that epithelial damage occurs prior to widespread immune dysfunction.

Nucleoside-modified mRNA vaccines induce potent T follicular helper and germinal center B cell responses.

T follicular helper (Tfh) cells are required to develop germinal center (GC) responses and drive immunoglobulin class switch, affinity maturation, and long-term B cell memory. In this study, we characterize a recently developed vaccine platform, nucleoside-modified, purified mRNA encapsulated in lipid nanoparticles (mRNA-LNPs), that induces high levels of Tfh and GC B cells. Intradermal vaccination with nucleoside-modified mRNA-LNPs encoding various viral surface antigens elicited polyfunctional, antigen-specific, CD4+ T cell responses and potent neutralizing antibody responses in mice and nonhuman primates. Importantly, the strong antigen-specific Tfh cell response and high numbers of GC B cells and plasma cells were associated with long-lived and high-affinity neutralizing antibodies and durable protection. Comparative studies demonstrated that nucleoside-modified mRNA-LNP vaccines outperformed adjuvanted protein and inactivated virus vaccines and pathogen infection. The incorporation of noninflammatory, modified nucleosides in the mRNA is required for the production of large amounts of antigen and for robust immune responses.

Differential impact of transplantation on peripheral and tissue-associated viral reservoirs: Implications for HIV gene therapy.

Autologous transplantation and engraftment of HIV-resistant cells in sufficient numbers should recapitulate the functional cure of the Berlin Patient, with applicability to a greater number of infected individuals and with a superior safety profile. A robust preclinical model of suppressed HIV infection is critical in order to test such gene therapy-based cure strategies, both alone and in combination with other cure strategies. Here, we present a nonhuman primate (NHP) model of latent infection using simian/human immunodeficiency virus (SHIV) and combination antiretroviral therapy (cART) in pigtail macaques. We demonstrate that transplantation of CCR5 gene-edited hematopoietic stem/progenitor cells (HSPCs) persist in infected and suppressed animals, and that protected cells expand through virus-dependent positive selection. CCR5 gene-edited cells are readily detectable in tissues, namely those closely associated with viral reservoirs such as lymph nodes and gastrointestinal tract. Following autologous transplantation, tissue-associated SHIV DNA and RNA levels in suppressed animals are significantly reduced (p ≤ 0.05), relative to suppressed, untransplanted control animals. In contrast, the size of the peripheral reservoir, measured by QVOA, is variably impacted by transplantation. Our studies demonstrate that CCR5 gene editing is equally feasible in infected and uninfected animals, that edited cells persist, traffic to, and engraft in tissue reservoirs, and that this approach significantly reduces secondary lymphoid tissue viral reservoir size. Our robust NHP model of HIV gene therapy and viral persistence can be immediately applied to the investigation of combinatorial approaches that incorporate anti-HIV gene therapy, immune modulators, therapeutic vaccination, and latency reversing agents.

Development of broad neutralization activity in simian/human immunodeficiency virus-infected rhesus macaques after long-term infection.

OBJECTIVE: Nonhuman primates (NHPs) are the only animal model that can be used to evaluate protection efficacy of HIV-1 envelope vaccines. However, whether broadly neutralizing antibodies (bnAbs) can be elicited in NHPs infected with simian/human immunodeficiency virus (SHIV) has not been fully understood. The objective of this study is to investigate whether broad neutralization activities were developed in SHIV-infected macaques after long-term infection as in humans. DESIGN: Neutralization breadth and specificities in plasmas from SHIV-infected macaques were determined by analyzing a panel of tier 2 viruses and their mutants. METHODS: Forty-four Chinese macaques infected with SHIV1157ipd3N4, SHIVSF162P3 or SHIVCHN19P4 were followed for 54-321 weeks. Archived plasmas from 19 macaques were used to determine neutralization breadth and specificities against 17 tier 2 envelope-pseudoviruses. RESULTS: Longitudinal plasma from three SHIVSF162P3-infected macaques and three SHIV1157ipd3N4-infected macaques rarely neutralized viruses (<25%) within 1 year of infection. The neutralization breadth in two SHIV1157ipd3N4-infected macaques significantly increased (≥65%) by year 6. Four of six SHIV1157ipd3N4-infected macaques could neutralize 50-75% viruses, whereas none of macaques infected with SHIVSF162P3 or SHIVCHN19P4 could neutralize more than 25% of viruses after 6 years of infection (P = 0.035). Neutralization specificity analysis showed mutations resistant to bnAbs in V2, V3 or CD4bs regions could abrogate neutralization by year-6 plasma from three SHIV1157ipd3N4-infected macaques. CONCLUSION: These results demonstrate that bnAbs targeting common HIV-1 epitopes can be elicited in SHIV1157ipd3N4-infected macaques as in humans after 4-6 years of infection, and SHIV/NHP can serve as an ideal model to study bnAb maturation.

Increased surface expression of HIV-1 envelope is associated with improved antibody response in vaccinia prime/protein boost immunization.

HIV-1 envelope (Env)-based vaccines have so far largely failed to induce antibodies that prevent HIV-1 infection. One factor proposed to limit the immunogenicity of cell-associated Env is its low level of expression on the cell surface, restricting accessibility to antibodies. Using a vaccinia prime/protein boost protocol in mice, we explored the immunologic effects of mutations in the Env cytoplasmic tail (CT) that increased surface expression, including partial truncation and ablation of a tyrosine-dependent endocytosis motif. After vaccinia primes, CT-modified Envs induced up to 7-fold higher gp120-specific IgG, and after gp120 protein boosts, they elicited up to 16-fold greater Tier-1 HIV-1 neutralizing antibody titers, although results were variable between isolates. These data indicate that the immunogenicity of HIV-1 Env in a prime/boost vaccine can be enhanced in a strain-dependent manner by CT mutations that increase Env surface expression, thus highlighting the importance of the prime in this vaccine format.

Extracellular Matrix Proteins Mediate HIV-1 gp120 Interactions with α4ß7.

Gut-homing α4ß7high CD4+ T lymphocytes have been shown to be preferentially targeted by human immunodeficiency virus type 1 (HIV-1) and are implicated in HIV-1 pathogenesis. Previous studies demonstrated that HIV-1 envelope protein gp120 binds and signals through α4ß7 and that this likely contributes to the infection of α4ß7high T cells and promotes cell-to-cell virus transmission. Structures within the second variable loop (V2) of gp120, including the tripeptide motif LDV/I, are thought to mediate gp120-α4ß7 binding. However, lack of α4ß7 binding has been reported in gp120 proteins containing LDV/I, and the precise determinants of gp120-α4ß7 binding are not fully defined. In this work, we report the novel finding that fibronectins mediate indirect gp120-α4ß7 interactions. We show that Chinese hamster ovary (CHO) cells used to express recombinant gp120 produced fibronectins and other extracellular matrix proteins that copurified with gp120. CHO cell fibronectins were able to mediate the binding of a diverse panel of gp120 proteins to α4ß7 in an in vitro cell binding assay. The V2 loop was not required for fibronectin-mediated binding of gp120 to α4ß7, nor did V2-specific antibodies block this interaction. Removal of fibronectin through anion-exchange chromatography abrogated V2-independent gp120-α4ß7 binding. Additionally, we showed a recombinant human fibronectin fragment mediated gp120-α4ß7 interactions similarly to CHO cell fibronectin. These findings provide an explanation for the apparently contradictory observations regarding the gp120-α4ß7 interaction and offer new insights into the potential role of fibronectin and other extracellular matrix proteins in HIV-1 biology.IMPORTANCE Immune tissues within the gut are severely damaged by HIV-1, and this plays an important role in the development of AIDS. Integrin α4ß7 plays a major role in the trafficking of lymphocytes, including CD4+ T cells, into gut lymphoid tissues. Previous reports indicate that some HIV-1 gp120 envelope proteins bind to and signal through α4ß7, which may help explain the preferential infection of gut CD4+ T cells. In this study, we demonstrate that extracellular matrix proteins can mediate interactions between gp120 and α4ß7 This suggests that the extracellular matrix may be an important mediator of HIV-1 interaction with α4ß7-expressing cells. These findings provide new insight into the nature of HIV-1-α4ß7 interactions and how these interactions may represent targets for therapeutic intervention.

Pentavalent HIV-1 vaccine protects against simian-human immunodeficiency virus challenge.

The RV144 Thai trial HIV-1 vaccine of recombinant poxvirus (ALVAC) and recombinant HIV-1 gp120 subtype B/subtype E (B/E) proteins demonstrated 31% vaccine efficacy. Here we design an ALVAC/Pentavalent B/E/E/E/E vaccine to increase the diversity of gp120 motifs in the immunogen to elicit a broader antibody response and enhance protection. We find that immunization of rhesus macaques with the pentavalent vaccine results in protection of 55% of pentavalent-vaccine-immunized macaques from simian-human immunodeficiency virus (SHIV) challenge. Systems serology of the antibody responses identifies plasma antibody binding to HIV-infected cells, peak ADCC antibody titres, NK cell-mediated ADCC and antibody-mediated activation of MIP-1ß in NK cells as the four immunological parameters that best predict decreased infection risk that are improved by the pentavalent vaccine. Thus inclusion of additional gp120 immunogens to a pox-prime/protein boost regimen can augment antibody responses and enhance protection from a SHIV challenge in rhesus macaques.

A spatio-temporal assessment of simian/human immunodeficiency virus (SHIV) evolution reveals a highly dynamic process within the host.

The process by which drug-resistant HIV-1 arises and spreads spatially within an infected individual is poorly understood. Studies have found variable results relating how HIV-1 in the blood differs from virus sampled in tissues, offering conflicting findings about whether HIV-1 throughout the body is homogeneously distributed. However, most of these studies sample only two compartments and few have data from multiple time points. To directly measure how drug resistance spreads within a host and to assess how spatial structure impacts its emergence, we examined serial sequences from four macaques infected with RT-SHIVmne027, a simian immunodeficiency virus encoding HIV-1 reverse transcriptase (RT), and treated with RT inhibitors. Both viral DNA and RNA (vDNA and vRNA) were isolated from the blood (including plasma and peripheral blood mononuclear cells), lymph nodes, gut, and vagina at a median of four time points and RT was characterized via single-genome sequencing. The resulting sequences reveal a dynamic system in which vRNA rapidly acquires drug resistance concomitantly across compartments through multiple independent mutations. Fast migration results in the same viral genotypes present across compartments, but not so fast as to equilibrate their frequencies immediately. The blood and lymph nodes were found to be compartmentalized rarely, while both the blood and lymph node were more frequently different from mucosal tissues. This study suggests that even oft-sampled blood does not fully capture the viral dynamics in other parts of the body, especially the gut where vRNA turnover was faster than the plasma and vDNA retained fewer wild-type viruses than other sampled compartments. Our findings of transient compartmentalization across multiple tissues may help explain the varied results of previous compartmentalization studies in HIV-1.

Loss of immune homeostasis dictates SHIV rebound after stem-cell transplantation.

The conditioning regimen used as part of the Berlin patient's hematopoietic cell transplant likely contributed to his eradication of HIV infection. We studied the impact of conditioning in simian-human immunodeficiency virus-infected (SHIV-infected) macaques suppressed by combination antiretroviral therapy (cART). The conditioning regimen resulted in a dramatic, but incomplete depletion of CD4+ and CD8+ T cells and CD20+ B cells, increased T cell activation and exhaustion, and a significant loss of SHIV-specific Abs. The disrupted T cell homeostasis and markers of microbial translocation positively correlated with an increased viral rebound after cART interruption. Quantitative viral outgrowth and Tat/rev-induced limiting dilution assays showed that the size of the latent SHIV reservoir did not correlate with viral rebound. These findings identify perturbations of the immune system as a mechanism for the failure of autologous transplantation to eradicate HIV. Thus, transplantation strategies may be improved by incorporating immune modulators to prevent disrupted homeostasis, and gene therapy to protect transplanted cells.

Design and In Vivo Characterization of Immunoconjugates Targeting HIV gp160.

The envelope (Env) glycoprotein of HIV is expressed on the surface of productively infected cells and can be used as a target for cytotoxic immunoconjugates (ICs), in which cell-killing moieties, including toxins, drugs, or radionuclides, are chemically or genetically linked to monoclonal antibodies (MAbs) or other targeting ligands. Such ICs could be used to eliminate persistent reservoirs of HIV infection. We have found that MAbs which bind to the external loop of gp41, e.g., MAb 7B2, make highly effective ICs, particularly when used in combination with soluble CD4. We evaluated the toxicity, immunogenicity, and efficacy of the ICs targeted with 7B2 in mice and in simian-human immunodeficiency virus-infected macaques. In the macaques, we tested immunotoxins (ITs), consisting of protein toxins bound to the targeting agent. ITs were well tolerated and initially efficacious but were ultimately limited by their immunogenicity. In an effort to decrease immunogenicity, we tested different toxic moieties, including recombinant toxins, cytotoxic drugs, and tubulin inhibitors. ICs containing deglycosylated ricin A chain prepared from ricin toxin extracted from castor beans were the most effective in killing HIV-infected cells. Having identified immunogenicity as a major concern, we show that conjugation of IT to polyethylene glycol limits immunogenicity. These studies demonstrate that cytotoxic ICs can target virus-infected cells in vivo but also highlight potential problems to be addressed. IMPORTANCE: It is not yet possible to cure HIV infection. Even after years of fully effective antiviral therapy, a persistent reservoir of virus-infected cells remains. Here we propose that a targeted conjugate consisting of an anti-HIV antibody bound to a toxic moiety could function to kill the HIV-infected cells that constitute this reservoir. We tested this approach in HIV-infected cells grown in the lab and in animal infections. Our studies demonstrated that these immunoconjugates are effective both in vitro and in test animals. In particular, ITs constructed with the deglycosylated A chain prepared from native ricin were the most effective in killing cells, but their utility was blunted because they provoked immune reactions that interfered with the therapeutic effects. We then demonstrated that coating of the ITs with polyethylene glycol minimized the immunogenicity, as has been demonstrated with other protein therapies.

Changes in Structure and Antigenicity of HIV-1 Env Trimers Resulting from Removal of a Conserved CD4 Binding Site-Proximal Glycan.

UNLABELLED: The envelope glycoprotein (Env) is the major target for HIV-1 broadly neutralizing antibodies (bNAbs). One of the mechanisms that HIV has evolved to escape the host's immune response is to mask conserved epitopes on Env with dense glycosylation. Previous studies have shown that the removal of a particular conserved glycan at N197 increases the neutralization sensitivity of the virus to antibodies targeting the CD4 binding site (CD4bs), making it a site of significant interest from the perspective of vaccine design. At present, the structural consequences that result from the removal of the N197 glycan have not been characterized. Using native-like SOSIP trimers, we examine the effects on antigenicity and local structural dynamics resulting from the removal of this glycan. A large increase in the binding of CD4bs and V3-targeting antibodies is observed for the N197Q mutant in trimeric Env, while no changes are observed with monomeric gp120. While the overall structure and thermostability are not altered, a subtle increase in the flexibility of the variable loops at the trimeric interface of adjacent protomers is evident in the N197Q mutant by hydrogen-deuterium exchange mass spectrometry. Structural modeling of the glycan chains suggests that the spatial occupancy of the N197 glycan leads to steric clashes with CD4bs antibodies in the Env trimer but not monomeric gp120. Our results indicate that the removal of the N197 glycan enhances the exposure of relevant bNAb epitopes on Env with a minimal impact on the overall trimeric structure. These findings present a simple modification for enhancing trimeric Env immunogens in vaccines. IMPORTANCE: The HIV-1 Env glycoprotein presents a dense patchwork of host cell-derived N-linked glycans. This so-called glycan shield is considered to be a major protective mechanism against immune recognition. While the positions of many N-linked glycans are isolate specific, some are highly conserved and are believed to play key functional roles. In this study, we examine the conserved, CD4 binding site-proximal N197 glycan and demonstrate that its removal both facilitates neutralizing antibody access to the CD4 binding site and modestly impacts the structural dynamics at the trimer crown without drastically altering global Env trimer stability. This indicates that surgical glycosylation site modification may be an effective way of sculpting epitope presentation in Env-based vaccines.

Somatic Hypermutation-Induced Changes in the Structure and Dynamics of HIV-1 Broadly Neutralizing Antibodies.

Antibody somatic hypermutation (SHM) and affinity maturation enhance antigen recognition by modifying antibody paratope structure to improve its complementarity with the target epitope. SHM-induced changes in paratope dynamics may also contribute to antibody maturation, but direct evidence of this is limited. Here, we examine two classes of HIV-1 broadly neutralizing antibodies (bNAbs) for SHM-induced changes in structure and dynamics, and delineate the effects of these changes on interactions with the HIV-1 envelope glycoprotein (Env). In combination with new and existing structures of unmutated and affinity matured antibody Fab fragments, we used hydrogen/deuterium exchange with mass spectrometry to directly measure Fab structural dynamics. Changes in antibody structure and dynamics were positioned to improve complementarity with Env, with changes in dynamics primarily observed at the paratope peripheries. We conclude that SHM optimizes paratope complementarity to conserved HIV-1 epitopes and restricts the mobility of paratope-peripheral residues to minimize clashes with variable features on HIV-1 Env.

Epitope-Independent Purification of Native-Like Envelope Trimers from Diverse HIV-1 Isolates.

UNLABELLED: Soluble forms of trimeric HIV-1 envelope glycoprotein (Env) have long been sought as immunogens and as reagents for analysis of Env structure and function. Isolation of trimers that mimic native Env, derived from diverse viruses, however, represents a major challenge. Thus far, the most promising native-like (NL) structures have been obtained by engineering trimer-stabilizing mutations, termed SOSIP, into truncated Env sequences. However, the abundances of NL trimeric conformers vary among Envs, necessitating purification by monoclonal antibodies (MAbs) like PGT145, which target specific epitopes. To surmount this inherent limitation, we developed an approach that uses lectin affinity chromatography, ion-exchange chromatography, hydrophobic-interaction chromatography (HIC), and size exclusion chromatography (SEC) to isolate NL trimers from nonnative Env species. We validated this method with SOSIP trimers from HIV-1 clades A and B. Analyses by SEC, blue native PAGE, SDS-PAGE, and dynamic light scattering indicated that the resulting material was homogeneous (>95% pure), fully cleaved, and of the appropriate molecular weight and size for SOSIP trimers. Negative-stain electron microscopy further demonstrated that our preparations were composed of NL trimeric structures. By hydrogen/deuterium-exchange mass spectrometry, these HIC-pure trimers exhibited structural organization consistent with NL trimers and inconsistent with profiles seen in nonnative Envs. Screened for antigenicity, some Envs, like BS208.b1 and KNH1144 T162A, did not present the glycan/quaternary structure-dependent epitope for PGT145 binding, suggesting that these SOSIPs would be challenging to isolate by existing MAb affinity methods. By selecting based on biochemical rather than antigenic properties, our method offers an epitope-independent alternative to MAbs for isolation of NL Env trimers. IMPORTANCE: The production and purification of diverse soluble Env trimers that maintain native-like (NL) structure present technical challenges that must be overcome in order to advance vaccine development and provide reagents for HIV research. Low levels of NL trimer expression amid heterogeneous Env conformers, even with the addition of stabilizing mutations, have presented a major challenge. In addition, it has been difficult to separate the NL trimers from these heterogeneous mixtures. While MAbs with specificity for quaternary NL trimer epitopes have provided one approach to purifying the desirable species, such methods are dependent on the Env displaying the proper epitope. In addition, MAb affinity chromatography can be expensive, the necessary MAb may be in limited supply, and large-scale purification may not be feasible. Our method based on biochemical separation techniques offers an epitope-independent approach to purification of NL trimers with general application to diverse Envs.