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eCD4-immunoglobulin (Ig) is an HIV entry inhibitor that mimics the engagement of both CD4 and CCR5 with the HIV envelope (Env) protein, a property that imbues it with remarkable potency and breadth. However, env is exceptionally genetically malleable and can evolve to escape a wide variety of entry inhibitors. Here we document the evolution of partial eCD4-Ig resistance in SHIV-AD8 EO -infected rhesus macaques (RMs) treated with adeno-associated virus vectors encoding eCD4-Ig. In one RM, setpoint viremia plateaued at 1,000 vRNA copies/mL, despite concomitant serum concentrations of eCD4-Ig in the 60-110 μg/mL range, implying that the virus had gained partial eCD4-Ig resistance. Env mutations occurring prominently in this animal were cloned and further characterized. Three of these mutations (R315G, A436T, and G471E) were sufficient to confer substantial resistance to eCD4-Ig-mediated neutralization onto the parental Env, accompanied by a marked loss of viral fitness. This resistance was not driven by decreased CD4 affinity, subverted sulfopeptide mimicry, changes to co-receptor tropism, or by a gain of CD4 independence. Rather, our data argue that the Env evolving in this animal attained eCD4-Ig resistance by decreasing triggerability, stabilizing the triggered state, and changing the nature of its relationship to the host CD4., Competing Interests: Declaration of interests M.A.M., M.D.A., M.R.G., and M.F. have significant financial interests in Emmune, Inc., a company that is developing HIV immunotherapies based on the immunoadhesin eCD4-Ig, and each serve in a consulting capacity for the company. These potential conflicts of interest are being managed by the authors’ respective institutions. None of the other authors or their immediate family members have any conflicts of interest to declare., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

For use in prevention and treatment, HIV-1 broadly neutralizing antibodies (bnAbs) have to overcome Env conformational heterogeneity of viral quasispecies and neutralize with constant high potency. Comparative analysis of neutralization data from the CATNAP database revealed a nuanced relationship between bnAb activity and Env conformational flexibility, with substantial epitope-specific variation of bnAb potency ranging from increased to decreased activity against open, neutralization-sensitive Env. To systematically investigate the impact of variability in Env conformation on bnAb potency we screened 126 JR-CSF point mutants for generalized neutralization sensitivity to weakly neutralizing antibodies (weak-nAbs) depending on trimer opening and plasma from people with chronic HIV-1 infection. 23 mutations resulted in a highly neutralization sensitive phenotype, which was associated with de-stabilization of the closed, prefusion conformation. Including 19 of these mutants into a Sensitivity Env mutant panel (SENSE-19), we classified bnAbs according to potency variations in response to trimer opening. To verify that these sensitivity patterns are independent of the in vitro assay system, replication-competent SENSE-19 mutant viruses were tested on primary CD4 T cells. While loss of potency on SENSE-19 was registered for bnAbs from several classes recognizing quaternary epitopes on pre-triggered Env, structural destabilization benefitted MPER bnAbs and other inhibitors known to have post-CD4 attachment neutralization activity. Importantly, for a subset of CD4bs bnAbs, and the interface bnAb PGT151, particularly low potency variation was noted, suggesting that Env conformational tolerance can be achieved but is not the rule. In summary, SENSE-19 screens revealed distinct tolerance levels to Env conformational intermediates between bnAbs that provide mechanistic insights in their function and broaden current neutralization breadth assessments., Competing Interests: HFG has been an advisor/consultant for Merck, Gilead Sciences, ViiV Healthcare, GSK, Johnson and Johnson, Janssen and Novartis and has received unrestricted research grants and a travel grant from Gilead Sciences outside of this work. HFG is member of data safety monitoring boards outside of the submitted work. AT has been a consultant for Roche outside of the submitted work. All other authors declare no competing interests., (Copyright: © 2025 Foulkes et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In the past three decades, human immunodeficiency virus type 1 (HIV-1)-derived vectors were evolved and became indispensable to transduce therapeutic genes into a range of different target cell types to facilitate a variety of gene therapeutic strategies. To achieve this, i) the biosafety profile of the vectors was incrementally enhanced and ii) the CD4-restricted tropism mediated by the envelope proteins (Env) of the parental virus needed to be directed towards recruitment of other receptors expressed on the desired target cells. Here, a closer look is first taken at the development of vector components and the mechanisms of Env incorporation into particles. While envelope proteins originating from a broad range of very diverse virus species were successfully utilized, members of the Retroviridae family most frequently provided Env or further engineered variants thereof to form transduction-competent HIV-1 pseudotype vector particles. The development of these vectors is reviewed and anticipated to further contribute to the future progression of somatic gene therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author. Published by Elsevier Inc. All rights reserved.)

During the process by which human immunodeficiency virus (HIV-1) enters cells, the envelope glycoprotein (Env) trimer on the virion surface engages host cell receptors. Binding to the receptor CD4 induces Env to undergo transitions from a pretriggered, "closed" (State-1) conformation to more "open" (State 2/3) conformations. Most broadly neutralizing antibodies (bNAbs), which are difficult to elicit, recognize the pretriggered (State-1) conformation. More open Env conformations are recognized by poorly neutralizing antibodies (pNAbs), which are readily elicited during natural infection and vaccination with current Env immunogens. Env heterogeneity likely contributes to HIV-1 persistence by skewing antibody responses away from the pretriggered conformation. The conformationally flexible gp160 Env precursor on the infected cell or virion surface potentially presents multiple pNAb epitopes to the host immune system. Although proteolytic cleavage to produce the functional, mature Env trimer [(gp120/gp41) 3 ] stabilizes State-1, many primary HIV-1 Envs spontaneously sample more open conformations. Here, we establish inducible cell lines that produce replication-defective HIV-1 particles with Env trimers stabilized in a pretriggered conformation. The mature Env is enriched on virus-like particles (VLPs). Using complementary approaches, we estimate an average of 25-50 Env trimers on each VLP. The stabilizing changes in Env limit the natural conformational heterogeneity of the VLP Env trimers, allowing recognition by bNAbs but not pNAbs. These defective VLPs provide a more homogeneous source of pretriggered Env trimers in a native membrane environment. Thus, these VLPs may facilitate the characterization of this functionally important Env conformation and its interaction with the immune system.IMPORTANCEA major impediment to the development of an effective HIV/AIDS vaccine is the inefficiency with which human immunodeficiency virus (HIV-1) envelope glycoproteins elicit antibodies that neutralize multiple virus strains. Neutralizing antibodies recognize a particular shape of the envelope glycoproteins that resides on the viral membrane before the virus engages the host cell. Here, we report the creation of stable cell lines that inducibly produce non-infectious HIV-like particles. The normally flexible envelope glycoprotein spikes on these virus-like particles have been stabilized in a conformation that is recognized by broadly neutralizing antibodies. These virus-like particles allow the study of the envelope glycoprotein conformation, its modification by sugars, and its ability to elicit desired neutralizing antibodies., Competing Interests: The authors declare no conflict of interest.

HIV-1 envelope glycoprotein (Env) conformation substantially impacts antibody-dependent cellular cytotoxicity (ADCC). Envs from primary HIV-1 isolates adopt a prefusion "closed" conformation, which is targeted by broadly neutralizing antibodies (bnAbs). CD4 binding drives Env into more "open" conformations, which are recognized by non-neutralizing Abs (nnAbs). To better understand Env-Ab and Env-CD4 interaction in CD4+ T cells infected with HIV-1, we simultaneously measured antibody binding and HIV-1 mRNA expression using multiparametric flow cytometry and RNA flow fluorescent in situ hybridization (FISH) techniques. We observed that env mRNA is almost exclusively expressed by HIV-1 productively infected cells that already downmodulated CD4. This suggests that CD4 downmodulation precedes env mRNA expression. Consequently, productively infected cells express "closed" Envs on their surface, which renders them resistant to nnAbs. Cells recognized by nnAbs were all env mRNA negative, indicating Ab binding through shed gp120 or virions attached to their surface. Consistent with these findings, treatment of HIV-1-infected humanized mice with the ADCC-mediating nnAb A32 failed to lower viral replication or reduce the size of the viral reservoir. These findings confirm the resistance of productively infected CD4+ T cells to nnAbs-mediated ADCC and question the rationale of immunotherapy approaches using this strategy., Importance: Antibody-dependent cellular cytotoxicity (ADCC) represents an effective immune response for clearing virally infected cells, making ADCC-mediating antibodies promising therapeutic candidates for HIV-1 cure strategies. Broadly neutralizing antibodies (bNAbs) target epitopes present on the native "closed" envelope glycoprotein (Env), while non-neutralizing antibodies (nnAbs) recognize epitopes exposed upon Env-CD4 interaction. Here, we provide evidence that env mRNA is predominantly expressed by productively infected cells that have already downmodulated cell-surface CD4. This indicates that CD4 downmodulation by HIV-1 precedes Env expression, making productively infected cells resistant to ADCC mediated by nnAbs but sensitive to those mediated by bnAbs. These findings offer critical insights for the development of immunotherapy-based strategies aimed at targeting and eliminating productively infected cells in people living with HIV., Competing Interests: The authors declare no conflict of interest.

Vaccine development targeting rapidly evolving pathogens such as HIV-1 requires induction of broadly neutralizing antibodies (bnAbs) with conserved paratopes and mutations, and in some cases, the same Ig-heavy chains. The current trial-and-error search for immunogen modifications that improve selection for specific bnAb mutations is imprecise. Here, to precisely engineer bnAb boosting immunogens, we use molecular dynamics simulations to examine encounter states that form when antibodies collide with the HIV-1 Envelope (Env). By mapping how bnAbs use encounter states to find their bound states, we identify Env mutations predicted to select for specific antibody mutations in two HIV-1 bnAb B cell lineages. The Env mutations encode antibody affinity gains and select for desired antibody mutations in vivo. These results demonstrate proof-of-concept that Env immunogens can be designed to directly select for specific antibody mutations at residue-level precision by vaccination, thus demonstrating the feasibility of sequential bnAb-inducing HIV-1 vaccine design., (© 2024. The Author(s).)

Guanylate-binding protein (GBP) 5 is an interferon-inducible cellular factor with broad anti-viral activity. Recently, GBP5 has been shown to antagonize the glycoproteins of a number of enveloped viruses, in part by disrupting the host enzyme furin. Here we show that GBP5 strongly impairs the infectivity of virus particles bearing not only viral glycoproteins that depend on furin cleavage for infectivity-the envelope (Env) glycoproteins of HIV-1 and murine leukemia virus and the spike (S) glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-but also viral glycoproteins that do not depend on furin cleavage: vesicular stomatitis virus glycoprotein and SARS-CoV S. We observe that GBP5 disrupts proper N -linked protein glycosylation and reduces the incorporation of viral glycoproteins into virus particles. The glycosylation of the cellular protein CD4 is also altered by GBP5 expression. Flow cytometry analysis shows that GBP5 expression reduces the cell-surface levels of HIV-1 Env and the S glycoproteins of SARS-CoV and SARS-CoV-2. Our data demonstrate that, under the experimental conditions used, inhibition of furin-mediated glycoprotein cleavage is not the primary anti-viral mechanism of action of GBP5. Rather, the antagonism appears to be related to impaired trafficking of glycoproteins to the plasma membrane. These results provide novel insights into the broad antagonism of viral glycoprotein function by the cellular host innate immune response., Importance: The surface of enveloped viruses contains viral envelope glycoproteins, an important structural component facilitating virus attachment and entry while also acting as targets for the host adaptive immune system. In this study, we show that expression of GBP5 in virus-producer cells alters the glycosylation, cell-surface expression, and virion incorporation of viral glycoproteins across several virus families. This research provides novel insights into the broad impact of the host cell anti-viral factor GBP5 on protein glycosylation and trafficking., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Human immunodeficiency virus (HIV)-1 assembly is initiated by Gag binding to the inner leaflet of the plasma membrane (PM). Gag targeting is mediated by its N-terminally myristoylated matrix (MA) domain and PM phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ]. Upon Gag assembly, envelope (Env) glycoproteins are recruited to assembly sites; this process depends on the MA domain of Gag and the Env cytoplasmic tail. To investigate the dynamics of Env recruitment, we applied a chemical dimerizer system to manipulate HIV-1 assembly by reversible PI(4,5)P 2 depletion in combination with super resolution and live-cell microscopy. This approach enabled us to control and synchronize HIV-1 assembly and track Env recruitment to individual nascent assembly sites in real time. Single virion tracking revealed that Gag and Env are accumulating at HIV-1 assembly sites with similar kinetics. PI(4,5)P 2 depletion prevented Gag PM targeting and Env cluster formation, confirming Gag dependence of Env recruitment. In cells displaying pre-assembled Gag lattices, PI(4,5)P 2 depletion resulted in the disintegration of the complete assembly domain, as not only Gag but also Env clusters were rapidly lost from the PM. These results argue for the existence of a Gag-induced and -maintained membrane micro-environment, which attracts Env. Gag cluster dissociation by PI(4,5)P 2 depletion apparently disrupts this micro-environment, resulting in the loss of Env from the former assembly domain.IMPORTANCEHuman immunodeficiency virus (HIV)-1 assembles at the plasma membrane of infected cells, resulting in the budding of membrane-enveloped virions. HIV-1 assembly is a complex process initiated by the main structural protein of HIV-1, Gag. Interestingly, HIV-1 incorporates only a few envelope (Env) glycoproteins into budding virions, although large Env accumulations surrounding nascent Gag assemblies are detected at the plasma membrane of HIV-expressing cells. The matrix domain of Gag and the Env cytoplasmatic tail play a role in Env recruitment to HIV-1 assembly sites and its incorporation into nascent virions. However, the regulation of these processes is incompletely understood. By combining a chemical dimerizer system to manipulate HIV-1 assembly with super resolution and live-cell microscopy, our study provides new insights into the interplay between Gag, Env, and host cell membranes during viral assembly and into Env incorporation into HIV-1 virions., Competing Interests: The authors declare no conflict of interest.

Nucleoside-modified mRNA technology has revolutionized vaccine development with the success of mRNA COVID-19 vaccines. We used modified mRNA technology for the design of envelopes (Env) to induce HIV-1 broadly neutralizing antibodies (bnAbs). However, unlike SARS-CoV-2 neutralizing antibodies that are readily made, HIV-1 bnAb induction is disfavored by the immune system because of the rarity of bnAb B cell precursors and the cross-reactivity of bnAbs targeting certain Env epitopes with host molecules, thus requiring optimized immunogen design. The use of protein nanoparticles (NPs) has been reported to enhance B cell germinal center responses to HIV-1 Env. Here, we report our experience with the expression of Env-ferritin NPs compared with membrane-bound Env gp160 when encoded by modified mRNA. We found that well-folded Env-ferritin NPs were a minority of the protein expressed by an mRNA design and were immunogenic at 20 µg but minimally immunogenic in mice at 1 µg dose in vivo and were not expressed well in draining lymph nodes (LNs) following intramuscular immunization. In contrast, mRNA encoding gp160 was more immunogenic than mRNA encoding Env-NP at 1 µg dose and was expressed well in draining LN following intramuscular immunization. Thus, analysis of mRNA expression in vitro and immunogenicity at low doses in vivo are critical for the evaluation of mRNA designs for optimal immunogenicity of HIV-1 immunogens.IMPORTANCEAn effective HIV-1 vaccine that induces protective antibody responses remains elusive. We have used mRNA technology for designs of HIV-1 immunogens in the forms of membrane-bound full-length envelope gp160 and envelope ferritin nanoparticle. Here, we demonstrated in a mouse model that the membrane-bound form induced a better response than envelope ferritin nanoparticle because of higher in vivo protein expression. The significance of our research is in highlighting the importance of analysis of mRNA design expression and low-dose immunogenicity studies for HIV-1 immunogens before moving to vaccine clinical trials., Competing Interests: D.W., K.O.S., and B.F.H. are inventors on U.S. patent number 11,318,197 and U.S. application numbers 17/703,332 and 16/977,408 (Stabilization of Human Immunodeficiency Virus [HIV] Envelopes). B.F.H. and K.O.S. are inventors on international patent application number PCT/US2019/020436 (Compositions and Methods for Inducing HIV-1 Antibodies). M.T. is a contract employee for 3M Healthcare. All other authors declare no conflicts of interest.

BACKGROUNDThe HIV Organ Policy Equity (HOPE) Act allows individuals living with HIV to accept organs from donors with HIV. This practice widens the pool of available organs, but also presents important virological issues, including the potential for HIV superinfection of the recipient, viral persistence in the kidney, and loss of virological control.METHODSWe addressed these issues by performing in-depth longitudinal viral sequence analyses on urine, blood, and urine-derived renal epithelial cells from 12 recipients of HIV+ kidney allografts.RESULTSWe amplified donor-derived HIV-1 env sequences in 5 out of 12 recipients after transplant. These donor-derived env sequences were amplified from recipient urine, urine-derived renal epithelial cells, and plasma between 12 and 96 hours after transplant and remained detectable up to 16 days after transplant. Env sequences were also detected in kidney biopsies taken from the allografts before implantation in 6 out of the 12 transplant cases, indicating the presence of donor virus within the organ. One recipient had a viremic episode 3.5 years after transplantation as a result of antiretroviral therapy (ART) interruption. Only recipient strain viral sequences were detected in blood, suggesting that the donor virus, if still present, was not reactivated during the temporary ART withdrawal.CONCLUSIONSThis study demonstrates that the HIV env sequences in a donor kidney can be amplified from biopsies taken from the allograft before implantation and can be detected transiently in blood and urine samples collected from the organ recipients after transplantation.FUNDINGNational Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) grant number R01DK131497.

Background: Coinfection with two phylogenetically distinct Human Immunodeficiency Virus-1 (HIV-1) variants might provide an opportunity for rapid viral expansion and the emergence of fit variants that drive disease progression. However, autologous neutralising immune responses are known to drive Envelope (Env) diversity which can either enhance replicative capacity, have no effect, or reduce viral fitness. This study investigated whether in vivo outgrowth of coinfecting variants was linked to pseudovirus and infectious molecular clones' infectivity to determine whether diversification resulted in more fit virus with the potential to increase disease progression., Results: For most participants, emergent recombinants displaced the co-transmitted variants and comprised the major population at 52 weeks postinfection with significantly higher entry efficiency than other co-circulating viruses. Our findings suggest that recombination within gp41 might have enhanced Env fusogenicity which contributed to the increase in pseudovirus entry efficiency. Finally, there was a significant correlation between pseudovirus entry efficiency and CD4 + T cell count, suggesting that the enhanced replicative capacity of recombinant variants could result in more virulent viruses., Conclusion: Coinfection provides variants with the opportunity to undergo rapid recombination that results in more infectious virus. This highlights the importance of monitoring the replicative fitness of emergent viruses., (© 2024. The Author(s).)

HLA class I variation has the strongest effect genome-wide on outcome after HIV infection, and as such, an understanding of the impact of HLA polymorphism on response to HIV vaccination may inform vaccine design. We sought HLA associations with HIV-directed immunogenicity in the phase 1/2a APPROACH vaccine trial, which tested vaccine regimens containing mosaic inserts in Ad26 and MVA vectors, with or without a trimeric gp140 protein. While there were no HLA allelic associations with the overall cellular immune response to the vaccine assessed by ELISpot (Gag, Pol, and Env combined), significant associations with differential response to Gag compared to Env antigens were observed. Notably, HLA class I alleles known to associate with disease susceptibility in HIV natural history cohorts are associated with stronger Env-directed responses, whereas protective alleles are associated with stronger Gag-directed responses. Mean viral loads determined for each HLA allele in untreated individuals correlated negatively with the strength of the Gag response minus the Env response in Black vaccinees based on both ELISpot and CD8 + T cell ICS responses. As the association of T cell responses to conserved Gag epitopes with lower viral load in untreated individuals is well established, our data raise the possibility that the Ad26.Mos.HIV vaccine may induce more effective cellular responses in those with HLA alleles that confer improved virologic control in untreated HIV infection.IMPORTANCENo vaccine tested to date has shown sufficient efficacy against HIV infection. A vaccine that induces robust responses in one individual may fail to do so in another individual due to variation in HLA class I genes, loci central to the immune response. Extensive data have shown the strong effect of HLA variation on outcome after HIV infection, but very little is known about the effect of such variation on HIV vaccine success. Here, we identify a link between the effect of HLA variation on HIV disease outcome and immune responses to an HIV vaccine. HLA variants associated with better HIV control after infection also induce stronger responses against the HIV Gag protein relative to the Env protein after vaccination. Given the virologic control conferred by responses to Gag in natural history of HIV infection, these data suggest that HLA alleles conferring protection after HIV infection may also support a more effective cellular response to HIV vaccination., Competing Interests: J.v.D, M.G.P., D.S., and F.T. are employees of Janssen, and L.L. is a consultant with Johnson & Johnson.

The HIV-1 envelope glycoprotein (Env) is the sole neutralizing determinant on the surface of the virus. The Env gp120 and gp41 subunits mediate receptor binding and membrane fusion and are generated from the gp160 precursor by cellular furins. This cleavage event is required for viral entry. One approach to generate HIV-1 neutralizing antibodies following immunization is to express membrane-bound Env anchored on the cell-surface by genetic means using the natural HIV gp41 transmembrane (TM) spanning domain. To simplify the process of Env trimer membrane expression we sought to remove the need for Env precursor cleavage while maintaining native-like conformation following genetic expression. To accomplish these objectives, we selected our previously developed 'native flexibly linked' (NFL) stabilized soluble trimers that are both near-native in conformation and cleavage-independent. We genetically fused the NFL construct to the HIV TM domain by using a short linker or by restoring the native membrane external proximal region, absent in soluble trimers, to express the full HIV Env ectodomain on the plasma membrane. Both forms of cell-surface NFL trimers, without and with the MPER, displayed favorable antigenic profiles by flow cytometry when expressed from plasmid DNA or mRNA. These results were consistent with the presence of well-ordered cell surface native-like trimeric Env, a necessary requirement to generate neutralizing antibodies by vaccination. Inoculation of rabbits with mRNA lipid nanoparticles (LNP) expressing membrane-bound stabilized HIV Env NFL trimers generated tier 2 neutralizing antibody serum titers in immunized animals. Multiple inoculations of mRNA LNPs generated similar neutralizing antibody titers compared to immunizations of matched NFL soluble proteins in adjuvant. Given the recent success of mRNA vaccines to prevent severe COVID, these are important developments for genetic expression of native-like HIV Env trimers in animals and potentially in humans., Competing Interests: Authors PL and YT were employed by company Acuitas Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Guenaga, Alirezaei, Feng, Alameh, Lee, Baboo, Cluff, Wilson, Bale, Ozorowski, Lin, Tam, Diedrich, Yates, Paulson, Ward, Weissman and Wyatt.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Previously obtained highly immunogenic Env-VLPs ensure overcoming the natural resistance of HIV-1 surface proteins associated with their low level of incorporation and inaccessibility of conserved epitopes to induce neutralizing antibodies. We also adopted this technology to modify Env trimers of the ZM53(T/F) strain to produce Env-VLPs by recombinant vaccinia viruses (rVVs). For VLP production, rVVs expressing Env, Gag-Pol (HIV-1/SIV), and the cowpox virus hr gene, which overcomes the restriction of vaccinia virus replication in CHO cells, were used. The CHO Lec1 engineered cell line lacking GlcNAc-TI was used for generating VLPs with Env proteins containing a cytoplasmic (CT) domain affecting the surface subunit (SU) conformation. This has created the opportunity to modulate the glycan composition, and refine the conditions for their production, and optimize approaches to overcoming HIV-1 resistance associated with abundant glycosylation.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

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.

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., Competing Interests: Declaration of interests K.W., K.O.S., and B.F.H. have patent applications on some of the concepts and immunogens discussed in this paper. M.A.T. is an employee of 3M Company. 3M Company had no role in the execution of the study, data collection, or data interpretation. C.B. and Y.K.T. are employees of Acuitas Therapeutics; Y.K.T. is named on patents describing the use of modified mRNA LNP. C.B.F. is an inventor on patents and patent applications held/submitted by AAHI, associated with adjuvant formulations containing GLA or 3M-052., (Copyright © 2024. Published by Elsevier Inc.)

Efficiently cleaved HIV-1 Envs are the closest mimics of functional Envs as they specifically expose only bNAb (broadly neutralizing antibody) epitopes and not non-neutralizing ones, making them suitable for developing vaccine immunogens. We have previously identified several efficiently cleaved Envs from clades A, B, C and B/C. We also described that truncation of the CT (C-terminal tail) of a subset of these Envs, but not others, impairs their ectodomain conformation/antigenicity on the cell surface in a CT conserved hydrophilic domain (CHD) or Kennedy epitope (KE)-dependent manner. Here, we report that those Envs (4 - 2.J41 and JRCSF), whose native-like ectodomain conformation/antigenicity on the cell surface is disrupted upon CT truncation, but not other Envs like JRFL, whose CT truncation does not have an effect on ectodomain integrity on the cell surface, are also defective in retrograde transport from early to late endosomes. Restoration of the CHD/KE in the CT of these Envs restores wild-type levels of distribution between early and late endosomes. In the presence of retrograde transport inhibitor Retro 2, cell surface expression of 4 - 2.J41 and JRCSF Envs increases [as does in the presence of Rab7a DN and Rab7b DN (DN: dominant negative)] but particle formation decreases for 4 - 2.J41 and JRCSF Env pseudotyped viruses. Our results show for the first time a correlation between CT-dependent, CHD/KE regulated retrograde transport and cell surface expression/viral particle formation of these efficiently cleaved Envs. Based on our results we hypothesize that a subset of these efficiently cleaved Envs use a CT-dependent, CHD/KE-mediated mechanism for assembly and release from late endosomes., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zacharopoulou, Lee, Oliveira, Thornhill, Robinson, Brown, Kinloch, Goulder, Fox, Fidler, Ansari and Frater.)

The HIV-1 Envelope (Env) protein cytoplasmic tail (CT) recently has been shown to assemble an unusual trimeric baseplate structure that locates beneath Env ectodomain trimers. Mutations at linchpin residues that help organize the baseplate impair virus replication in restrictive T cell lines but not in permissive cell lines. We have identified and characterized a second site suppressor of these baseplate mutations, located at residue 34 in the viral matrix (MA) protein, that rescues viral replication in restrictive cells. The suppressor mutation was dependent on the CT to exert its activity and did not appear to affect Env protein traffic or fusion functions in restrictive cells. Instead, the suppressor mutation increased Env incorporation into virions 3-fold and virus infectivity in single-round infections 10-fold. We also found that a previously described suppressor of Env-incorporation defects that stabilizes the formation of MA trimers was ineffective at rescuing Env baseplate mutations. Our results support an interpretation in which changes at MA residue 34 induce conformational changes that stabilize MA lattice trimer-trimer interactions and/or direct MA-CT associations.IMPORTANCEHow HIV-1 Env trimers assemble into virus particles remains incompletely understood. In restrictive cells, viral incorporation of Env is dependent on the Env CT and on the MA protein, which assembles lattices composed of hexamers of trimers in immature and mature viruses. Recent evidence indicates that CT assembles trimeric baseplate structures that require membrane-proximal residues to interface with trimeric transmembrane domains and C-terminal helices in the CT. We found that mutations of these membrane-proximal residues impaired replication in restrictive cells. This defect was countered by a MA mutation that does not localize to any obvious interprotein regions but was only inefficiently suppressed by a MA mutation that stabilizes MA trimers and has been shown to suppress other CT-dependent Env defects. Our results suggest that efficient suppression of baseplate mutations involves stabilization of MA inter-trimer contacts and/or direct MA-CT associations. These observations shed new light on how Env assembles into virions., Competing Interests: The authors declare no conflict of interest.

Understanding how different amino acids affect the HIV-1 envelope (Env) trimer will greatly help the design and development of vaccines that induce broadly neutralizing antibodies (bnAbs). A tryptophan residue at position 375 that opens the CD4 binding site without modifying the trimer apex was identified using our saturation mutagenesis strategy. 375W was introduced into a large panel of 27 transmitted/founder, acute stage, chronic infection, and AIDS macrophage-tropic and non-macrophage-tropic primary envelopes from different clades (A, B, C, D, and G) as well as complex and circulating recombinants. We evaluated soluble CD4 and monoclonal antibody neutralization of WT and mutant Envs together with macrophage infection. The 375W substitution increased sensitivity to soluble CD4 in all 27 Envs and macrophage infection in many Envs including an X4 variant. Importantly, 375W did not impair or abrogate neutralization by potent bnAbs. Variants that were already highly macrophage tropic were compromised for macrophage tropism, indicating that other structural factors are involved. Of note, we observed a macrophage-tropic (clade G) and intermediate macrophage-tropic (clades C and D) primary Envs from the blood and not from the central nervous system (CNS), indicating that such variants could be released from the brain or evolve outside the CNS. Our data also indicate that "intermediate" macrophage-tropic variants should belong to a new class of HIV-1 tropism. These Envs infected macrophages more efficiently than non-macrophage-tropic variants without reaching the high levels of macrophage-tropic brain variants. In summary, we show that 375W is ideal for inclusion into HIV-1 vaccines, increasing Env binding to CD4 for widely diverse Envs from different clades and disease stages.IMPORTANCESubstitutions exposing the CD4 binding site (CD4bs) on HIV-1 trimers but still occluding non-neutralizing, immunogenic epitopes are desirable to develop HIV-1 vaccines. If such substitutions induce similar structural changes in trimers across diverse clades, they could be exploited for the development of multi-clade envelope (Env) vaccines. We show that the 375W substitution increases CD4 affinity for envelopes of all clades, circulating recombinant forms, and complex Envs tested, independent of disease stage. Clade B and C Envs with an exposed CD4bs were described for macrophage-tropic strains from the central nervous system (CNS). Here, we show that intermediate (clades C and D) and macrophage-tropic (clade G) envelopes can be detected outside the CNS. Vaccines targeting the CD4bs will be particularly effective against such strains and CNS disease., Competing Interests: The authors declare no conflict of interest.

Consistent elicitation of serum antibody responses that neutralize diverse clades of HIV-1 remains a primary goal of HIV-1 vaccine research. Prior work has defined key features of soluble HIV-1 Envelope (Env) immunogen cocktails that influence the neutralization breadth and potency of multivalent vaccine-elicited antibody responses including the number of Env strains in the regimen. We designed immunization groups that consisted of different numbers of SOSIP Env strains to be used in a cocktail immunization strategy: the smallest cocktail (group 2) consisted of a set of two Env strains, which were a subset of the three Env strains that made up group 3, which, in turn, were a subset of the six Env strains that made up group 4. Serum neutralizing titers were modestly broader in guinea pigs that were immunized with a cocktail of three Envs compared to cocktails of two and six, suggesting that multivalent Env immunization could provide a benefit but may be detrimental when the cocktail size is too large. We then adapted the LIBRA-seq platform for antibody discovery to be compatible with guinea pigs, and isolated several tier 2 neutralizing monoclonal antibodies. Three antibodies isolated from two separate guinea pigs were similar in their gene usage and CDR3s, establishing evidence for a guinea pig public clonotype elicited through vaccination. Taken together, this work investigated multivalent HIV-1 Env immunization strategies and provides a novel methodology for screening guinea pig B cell receptor antigen specificity at a high-throughput level using LIBRA-seq.IMPORTANCEMultivalent vaccination with soluble Env immunogens is at the forefront of HIV-1 vaccination strategies but little is known about the influence of the number of Env strains included in vaccine cocktails. Our results suggest that adding more strains is sometimes beneficial but may be detrimental when the number of strains is too high. In addition, we adapted the LIBRA-seq platform to be compatible with guinea pig samples and isolated several tier 2 neutralizing monoclonal antibodies, some of which share V and J gene usage and >70% CDR3 identity, thus establishing the existence of public clonotypes in guinea pigs elicited through vaccination., Competing Interests: M.J.V. and I.S.G. are listed as inventors on patents filed describing the antibodies discovered here. I.S.G. is listed as an inventor on the patent applications for the LIBRA-seq technology. I.S.G. is a co-founder of AbSeek Bio. I.S.G. has served as a consultant for Sanofi. The Georgiev laboratory at VUMC has received unrelated funding from Merck and Takeda Pharmaceuticals.

Subtype B HIV-1 has been the primary driver of the HIV-1 epidemic in the United States (U.S.) for over forty years and is also a prominent subtype in the Americas, Europe, Australia, the Middle East and North Africa. In this study, the neutralization profiles of contemporary subtype B Envs from the U.S. were assessed to characterize changes in neutralization sensitivities over time. We generated a panel of 30 contemporary pseudoviruses (PSVs) and demonstrated continued diversification of subtype B Env from the 1980s up to 2018. Neutralization sensitivities of the contemporary subtype B PSVs were characterized using 31 neutralizing antibodies (NAbs) and were compared with strains from earlier in the HIV-1 pandemic. A significant reduction in Env neutralization sensitivity was observed for 27 out of 31 NAbs for the contemporary as compared to earlier-decade subtype B PSVs. A decline in neutralization sensitivity was observed across all Env domains; the NAbs that were most potent early in the pandemic suffered the greatest decline in potency over time. A meta-analysis demonstrated this trend across multiple subtypes. As HIV-1 Env diversification continues, changes in Env antigenicity and neutralization sensitivity should continue to be evaluated to inform the development of improved vaccine and antibody products to prevent and treat HIV-1., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Introduction: The generation of an HIV-1 vaccine able to induce long-lasting protective immunity remains a main challenge. Here, we aimed to modify next-generation soluble, prefusion-stabilized, close-to-native, glycan-engineered clade C gp140 envelope (Env) trimers (sC23v4 KIKO and ConCv5 KIKO) for optimal display on the cell surface following homologous or heterologous vector delivery., Methods: A combination of the following modifications scored best regarding the preservation of closed, native-like Env trimer conformation and antigenicity when using a panel of selected broadly neutralizing (bnAb) and non-neutralizing (nnAb) monoclonal antibodies for flow cytometry: i) replacing the natural cleavage site with a native flexible linker and introducing a single amino acid substitution to prevent CD4 binding (*), ii) fusing a heterologous VSV-G-derived transmembrane moiety to the gp140 C-terminus, and iii) deleting six residues proximal to the membrane., Results: When delivering membrane-tethered sC23v4 KIKO* and ConCv5 KIKO* via DNA, VSV-GP, and NYVAC vectors, the two native-like Env trimers provide differential antigenicity profiles. Whereas such patterns were largely consistent among the different vectors for either Env trimer, the membrane-tethered ConCv5 KIKO* trimer adopted a more closed and native-like structure than sC23v4 KIKO*. In immunized mice, VSV-GP and NYVAC vectors expressing the membrane-tethered ConCv5 KIKO* administered in prime/boost combination were the most effective regimens for the priming of Env-specific CD4 T cells among all tested combinations. The subsequent booster administration of trimeric ConCv5 KIKO* Env protein preserved the T cell activation levels between groups. The evaluation of the HIV-1-specific humoral responses induced in the different immunization groups after protein boosts showed that the various prime/boost protocols elicited broad and potent antibody responses, preferentially of a Th1-associated IgG2a subclass, and that the obtained antibody levels remained high at the memory phase., Discussion: In summary, we provide a feasible strategy to display multiple copies of native-like Env trimers on the cell surface, which translates into efficient priming of sustained CD4 + T cell responses after vector delivery as well as broad, potent, and sustained antibody responses following booster immunizations with the homologous, prefusion-stabilized, close-to-native ConCv5 KIKO* gp140 Env trimer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Perdiguero, Hauser, Gómez, Peterhoff, Sideris, Sorzano, Wilmschen, Schaber, Stengel, Asbach, Ding, Von Laer, Levy, Pantaleo, Kimpel, Esteban and Wagner.)

Purpose of Review: Explore whether immunization with germline-targeting Env immunogens during ART, followed by ATI, leads to the identification of viral envelope glycoproteins (Envs) that promote and guide the full maturation of broadly neutralizing antibody responses., Recent Findings: The HIV-1 envelope glycoprotein (Env) does not efficiently engage the germline precursors of broadly neutralizing antibodies (bnAbs). However, Env-derived proteins specifically designed to precisely do that, have been recently developed. These 'germline-targeting' Env immunogens activate naïve B cells that express the germline precursors of bnAbs but by themselves cannot guide their maturation towards their broadly neutralizing forms. This requires sequential immunizations with heterologous sets of Envs. These 'booster' Envs are currently unknown., Summary: Combining germline-targeting Env immunization approaches during ART with ATI could lead to the identification of natural Envs that are responsible for the maturation of broadly neutralizing antibody responses during infection. Such Envs could then serve as booster immunogens to guide the maturation of glBCRs that have become activated by germline-targeting immunogens in uninfected subjects., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Stabilized trimers of the HIV-1 envelope glycoprotein Env are capable of inducing a potent and sustained broadly neutralizing antibody response in laboratory animals and therefore are attractive targets for anti-HIV vaccine development. In this work, a stable producer of the trimer Env recombinant form CRF63_02A6 of HIV-1 was derived from the CHO-K1 cell line. Using immunochemical assays, the trimers synthesized in CHO-K1 cells were shown to be recognized by both monoclonal broadly neutralizing antibodies and sera from HIV-positive patients. The resulting trimers of the recombinant form CRF63_02A6 of HIV-1 can be used both for structural studies and as a candidate vaccine immunogen against HIV-1., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Importance: Pathogenesis of HIV-1 is enhanced through several viral-encoded proteins that counteract a range of host restriction molecules. HIV-1 Nef counteracts the cell membrane protein SERINC5 by downregulating it from the cell surface, thereby enhancing virion infectivity. Some subtype B reference Envelope sequences have shown the ability to bypass SERINC5 infectivity restriction independent of Nef. However, it is not clear if and to what extent circulating HIV-1 strains can exhibit resistance to SERINC5 restriction. Using a panel of Envelope sequences isolated from 50 Tanzanians infected with non-B HIV-1 subtypes, we show that the lentiviral reporters pseudotyped with patient-derived Envelopes have reduced sensitivity to SERINC5 and that this sensitivity differed among viral subtypes. Moreover, we found that SERINC5 sensitivity within patient-derived Envelopes can be modulated by separate regions, highlighting the complexity of viral/host interactions., Competing Interests: The authors declare no conflict of interest.

Background: An approach to a preventive HIV vaccine is induction of effective broadly neutralizing antibodies (bnAbs) and effector binding antibodies (bAbs). Preclinical studies suggest that trimeric envelope (Env) proteins may elicit nAbs, which led to the development of the recombinant gp145 subtype C Env protein (gp145 C.6980) immunogen. HVTN 122 was a Phase 1 trial that evaluated the safety, tolerability, and immunogenicity of gp145 C.6980 in adults., Methods: Healthy, HIV-1 seronegative adults received three intramuscular injections of gp145 C.6980 with aluminum hydroxide (alum) at months 0, 2, and 6 at either 300 mcg (high dose, n = 25) or 100 mcg (low dose, n = 15), or placebo/saline (placebo, n = 5). Participants were followed for 12 months., Results: Forty-five participants were enrolled. High and low doses of the study protein were well-tolerated, with mild or moderate reactogenicity commonly reported. Only one adverse event (mild injection site pruritis) in one participant (low dose) was considered product-related; there were no dose-limiting toxicities. High and low dose recipients demonstrated robust bAb responses to vaccine-matched consensus gp140 Env and subtype-matched gp120 Env proteins two weeks post-last vaccination (response rates >90 %), while no responses were detected to a heterologous subtype-matched V1V2 antigen. No significant differences were seen between high and low dose groups. Participants in both experimental arms demonstrated nAb response rates of 76.5 % to a tier 1 virus (MW9635.26), but no responses to tier 2 isolates. Env-specific CD4 + T-cell responses were elicited in 36.4 % of vaccine recipients, without significant differences between groups; no participants demonstrated CD8 + T-cell responses., Conclusions: Three doses of novel subtype C gp145 Env protein with alum were safe and well-tolerated. Participants demonstrated bAb, Env-specific CD4 + T-cell, and tier 1 nAb responses, but the regimen failed to induce tier 2 or heterologous nAb responses., Clinical Trials Registration: NCT03382418., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Understanding the specificities of human serum antibodies that broadly neutralize HIV can inform prevention and treatment strategies. Here, we describe a deep mutational scanning system that can measure the effects of combinations of mutations to HIV envelope (Env) on neutralization by antibodies and polyclonal serum. We first show that this system can accurately map how all functionally tolerated mutations to Env affect neutralization by monoclonal antibodies. We then comprehensively map Env mutations that affect neutralization by a set of human polyclonal sera that neutralize diverse strains of HIV and target the site engaging the host receptor CD4. The neutralizing activities of these sera target different epitopes, with most sera having specificities reminiscent of individual characterized monoclonal antibodies, but one serum targeting two epitopes within the CD4-binding site. Mapping the specificity of the neutralizing activity in polyclonal human serum will aid in assessing anti-HIV immune responses to inform prevention strategies., Competing Interests: Declaration of interests J.D.B. is on the scientific advisory boards of Apriori Bio, Invivyd, Aerium Therapeutics, and the Vaccine Company. J.D.B., K.H.D.C., B.D., A.S.D., and C.E.R. receive royalty payments as inventors on Fred Hutch licensed patents related to viral deep mutational scanning. A.S.D. is currently an employee of Apriori Bio, although his contributions to this manuscript were performed when he was an employee of Fred Hutch before he started work at Apriori Bio. A patent application encompassing aspects of this work has been filed by the University of Cologne and lists P.S. and F.K. as inventors. P.S. and F.K. received payments from the University of Cologne for licensed antibodies., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

HIV reservoirs persist in anatomic compartments despite antiretroviral therapy (ART). Characterizing archival HIV DNA in the central nervous system (CNS) and other tissues is crucial to inform cure strategies. We evaluated paired autopsy brain-frontal cortex (FC), occipital cortex (OCC), and basal ganglia (BG)-and peripheral lymphoid tissues from 63 people with HIV. Participants passed away while virally suppressed on ART at the last visit and without evidence of CNS opportunistic disease. We quantified total HIV DNA in all participants and obtained full-length HIV-envelope (FL HIV- env ) sequences from a subset of 14 participants. We detected HIV DNA ( gag ) in most brain (65.1%) and all lymphoid tissues. Lymphoid tissues had higher HIV DNA levels than the brain ( P  < 0.01). Levels of HIV gag between BG and FC were similar ( P  > 0.2), while OCC had the lowest levels ( P  = 0.01). Females had higher HIV DNA levels in tissues than males ( gag , P  = 0.03; 2-LTR, P  = 0.05), suggesting possible sex-associated mechanisms for HIV reservoir persistence. Most FL HIV- env sequences ( n  = 143) were intact, while 42 were defective. Clonal sequences were found in 8 out of 14 participants, and 1 participant had clonal defective sequences in the brain and spleen, suggestive of cell migration. From 10 donors with paired brain and lymphoid sequences, we observed evidence of compartmentalized sequences in 2 donors. Our data further the idea that the brain is a site for archival HIV DNA during ART where compartmentalized provirus may occur in a subset of people. Future studies assessing FL HIV-provirus and replication competence are needed to further evaluate the HIV reservoirs in tissues. IMPORTANCE HIV infection of the brain is associated with adverse neuropsychiatric outcomes, despite efficient antiretroviral treatment. HIV may persist in reservoirs in the brain and other tissues, which can seed virus replication if treatment is interrupted, representing a major challenge to cure HIV. We evaluated reservoirs and genetic features in postmortem brain and lymphoid tissues from people with HIV who passed away during suppressed HIV replication. We found a differential distribution of HIV reservoirs across brain regions which was lower than that in lymphoid tissues. We observed that most HIV reservoirs in tissues had intact envelope sequences, suggesting they could potentially generate replicative viruses. We found that women had higher HIV reservoir levels in brain and lymphoid tissues than men, suggesting possible sex-based mechanisms of maintenance of HIV reservoirs in tissues, warranting further investigation. Characterizing the archival HIV DNA in tissues is important to inform future HIV cure strategies., Competing Interests: The authors declare no conflict of interest.

Understanding the dynamics of early immune responses to HIV-1 infection, including the evolution of initial neutralizing and antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies, will inform HIV vaccine design. In this study, we assess the development of autologous neutralizing antibodies (ANAbs) against founder envelopes (Envs) from 18 participants with HIV-1 CRF01&#95;AE acute infection. The timing of ANAb development directly associated with the magnitude of the longitudinal ANAb response. Participants that developed ANAbs within 6 months of infection had significantly higher ANAb responses at 1 year (50% inhibitory concentration [IC 50 ] geometric mean titer [GMT] = 2,010 versus 184; P  = 0.001) and 2 years (GMT = 3,479 versus 340; P  = 0.015), compared to participants that developed ANAb responses after 6 months. Participants with later development of ANAb tended to develop an earlier, potent heterologous tier 1 (92TH023) neutralizing antibody (NAb) response ( P  = 0.049). CRF01&#95;AE founder Env V1V2 loop lengths correlated indirectly with the timing ( P  = 0.002, r = -0.675) and directly with magnitude ( P  = 0.005, r  = 0.635) of ANAb responses; Envs with longer V1V2 loop lengths elicited earlier and more potent ANAb responses. While ANAb responses did not associate with viral load, the viral load set point correlated directly with neutralization of the heterologous 92TH023 strain ( P  = 0.007, r  = 0.638). In contrast, a striking inverse correlation was observed between viral load set point and peak ADCC against heterologous 92TH023 Env strain ( P  = 0.0005, r = -0.738). These data indicate that specific antibody functions can be differentially related to viral load set point and may affect HIV-1 pathogenesis. Exploiting Env properties, such as V1V2 length, could facilitate development of subtype-specific vaccines that elicit more effective immune responses and improved protection. IMPORTANCE Development of an effective HIV-1 vaccine will be facilitated by better understanding the dynamics between the founder virus and the early humoral responses. Variations between subtypes may influence the evolution of immune responses and should be considered as we strive to understand these dynamics. In this study, autologous founder envelope neutralization and heterologous functional humoral responses were evaluated after acute infection by HIV-1 CRF01&#95;AE, a subtype that has not been thoroughly characterized. The evolution of these humoral responses was assessed in relation to envelope characteristics, magnitude of elicited immune responses, and viral load. Understanding immune parameters in natural infection will improve our understanding of protective responses and aid in the development of immunogens that elicit protective functional antibodies. Advancing our knowledge of correlates of positive clinical outcomes should lead to the design of more efficacious vaccines.

Objectives: Despite suppressive antiretroviral therapy (ART), HIV can persist in a diverse range of CD4+ T-cell subsets. Through longitudinal env sampling from people with HIV (PWH) on ART, we characterized the persistence and phenotypic properties of HIV envs over two time-points (T1 and T2)., Methods: Longitudinal blood and lymphoid tissue samples were obtained from eight PWH on suppressive ART. Single genome amplification (SGA) was performed on env to understand the genetic diversity and degree of clonal expansions over time. A subset of envs were used to generate pseudovirus particles to assess sensitivity to autologous plasma IgG and broadly neutralizing antibodies (bNAbs)., Results: Identical env sequences indicating clonal expansion persisted between T1 and T2 and within multiple T-cell subsets. At both time-points, CXCR4-tropic (X4) Envs were more prevalent in naive and central memory cells; the proportion of X4 Envs did not significantly change in each subset between T1 and T2. Autologous purified plasma IgG showed variable neutralization of Envs, with no significant difference in neutralization between R5 and X4 Envs. X4 Envs were more sensitive to neutralization with clinical bNAbs, with CD4-binding site bNAbs demonstrating high breadth and potency against Envs., Conclusion: Our data suggest the viral reservoir in PWH on ART was predominantly maintained over time through proliferation and potentially differentiation of infected cells. We found the humoral immune response to Envs within the latent reservoir was variable between PWH. Finally, we identified coreceptor usage can influence bNAb sensitivity and may need to be considered for future bNAb immunotherapy approaches., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc.)

The envelope glycoprotein (Env) is the main focus of human immunodeficiency virus type 1 (HIV-1) vaccine development due to its critical role in viral entry. Despite advances in protein engineering, many Env proteins remain recalcitrant to recombinant expression due to their inherent metastability, making biochemical and immunological experiments impractical or impossible. Here, we report a novel proline stabilization strategy to facilitate the production of prefusion Env trimers. This approach, termed "2P," works synergistically with previously described SOSIP mutations and dramatically increases the yield of recombinantly expressed Env ectodomains without altering the antigenic or conformational properties of near-native Env. We determined that the 2P mutations function by enhancing the durability of the prefusion conformation and that this stabilization strategy is broadly applicable to evolutionarily and antigenically diverse Env constructs. These findings provide a new Env stabilization platform to facilitate biochemical research and expand the number of Env variants that can be developed as future HIV-1 vaccine candidates. IMPORTANCE Recent estimates have placed the number of new human immunodeficiency virus type 1 (HIV-1) infections at approximately 1.5 million per year, emphasizing the ongoing and urgent need for an effective vaccine. The envelope (Env) glycoprotein is the main focus of HIV-1 vaccine development, but, due to its inherent metastability, many Env variants are difficult to recombinantly express in the relatively large quantities that are required for biochemical studies and animal trials. Here, we describe a novel structure-based stabilization strategy that works synergistically with previously described SOSIP mutations to increase the yield of prefusion HIV-1 Env.

Broadly neutralizing antibodies (bNAbs) for HIV-1 prevention or cure strategies must inhibit transmitted/founder and reservoir viruses. Establishing sensitivity of circulating viruses to bNAbs and genetic patterns affecting neutralization variability may guide rational bNAbs selection for clinical development. We analyzed 326 single env genomes from nine individuals followed longitudinally following acute HIV-1 infection, with samples collected at ~1 week after the first detection of plasma viremia; 300 to 1,709 days postinfection but prior to initiating antiretroviral therapy (ART) (median = 724 days); and ~1 year post ART initiation. Sequences were assessed for phylogenetic relatedness, potential N- and O-linked glycosylation, and variable loop lengths (V1 to V5). A total of 43 env amplicons (median = 3 per patient per time point) were cloned into an expression vector and the TZM-bl assay was used to assess the neutralization profiles of 15 bNAbs targeting the CD4 binding site, V1/V2 region, V3 supersite, MPER, gp120/gp41 interface, and fusion peptide. At 1 μg/mL, the neutralization breadths were as follows: VRC07-LS and N6.LS (100%), VRC01 (86%), PGT151 (81%), 10-1074 and PGT121 (80%), and less than 70% for 10E8, 3BNC117, CAP256.VRC26, 4E10, PGDM1400, and N123-VRC34.01. Features associated with low sensitivity to V1/V2 and V3 bNAbs were higher potential glycosylation sites and/or relatively longer V1 and V4 domains, including known "signature" mutations. The study shows significant variability in the breadth and potency of bNAbs against circulating HIV-1 subtype C envelopes. VRC07-LS, N6.LS, VRC01, PGT151, 10-1074, and PGT121 display broad activity against subtype C variants, and major determinants of sensitivity to most bNAbs were within the V1/V4 domains. IMPORTANCE Broadly neutralizing antibodies (bNAbs) have potential clinical utility in HIV-1 prevention and cure strategies. However, bNAbs target diverse epitopes on the HIV-1 envelope and the virus may evolve to evade immune responses. It is therefore important to identify antibodies with broad activity in high prevalence settings, as well as the genetic patterns that may lead to neutralization escape. We investigated 15 bNAbs with diverse biophysical properties that target six epitopes of the HIV-1 Env glycoprotein for their ability to inhibit viruses that initiated infection, viruses circulating in plasma at chronic infection before antiretroviral treatment (ART), or viruses that were archived in the reservoir during ART in subtype C infected individuals in South Africa, a high burden country. We identify the antibodies most likely to be effective for clinical use in this setting and describe mutational patterns associated with neutralization escape from these antibodies.

A minority of HIV-1-infected patients produce broadly neutralizing antibodies (bNAbs). Identification of viral and host correlates of bNAb production may help develop vaccines. We aimed to characterize the neutralizing response and viral and host-associated factors in Angola, which has one of the oldest, most dynamic, and most diverse HIV-1 epidemics in the world. Three hundred twenty-two HIV-1-infected adults from Angola were included in this retrospective study. Phylogenetic analysis of C2V3C3 env gene sequences was used for virus subtyping. Env-binding antibody reactivity was tested against polypeptides comprising the C2, V3, and C3 regions. Neutralizing-antibody responses were determined against a reference panel of tier 2 Env pseudoviruses in TZM-bl cells; neutralizing epitope specificities were predicted using ClustVis. All subtypes were found, along with untypeable strains and recombinant forms. Notably, 56% of the patients developed cross neutralizing, broadly neutralizing, or elite neutralizing responses. Broad and elite neutralization was associated with longer infection time, subtype C, lower CD4 + T cell counts, higher age, and higher titer of C2V3C3-specific antibodies relative to failure to develop bNAbs. Neutralizing antibodies targeted the V3-glycan supersite in most patients. V3 and C3 regions were significantly less variable in elite neutralizers than in weak neutralizers and nonneutralizers, suggesting an active role of V3C3-directed bNAbs in controlling HIV-1 replication and diversification. In conclusion, prolonged and low-level envelope V3C3 stimulation by highly diverse and ancestral HIV-1 isolates promotes the frequent elicitation of bNAbs. These results provide important clues for the development of an effective HIV-1 vaccine. IMPORTANCE Studies on neutralization by antibodies and their determinants in HIV-1-infected individuals have mostly been conducted in relatively recent epidemics caused by subtype B and C viruses. Results have suggested that elicitation of broadly neutralizing antibodies (bNAbs) is uncommon. The mechanisms underlying the elicitation of bNAbs are still largely unknown. We performed the first characterization of the plasma neutralizing response in a cohort of HIV-1-infected patients from Angola. Angola is characterized by an old and dynamic epidemic caused by highly diverse HIV-1 variants. Remarkably, more than half of the patients produced bNAbs, mostly targeting the V3-glycan supersite in HIV-1. This was associated with higher age, longer infection time, lower CD4 + T cell counts, subtype C infection, or higher titer of C2V3C3-specific antibodies relative to patients that did not develop bNAbs. These results may help develop the next generation of vaccine candidates for HIV-1.

The immense global diversity of HIV-1 is a significant obstacle to developing a safe and effective vaccine. We recently showed that infections established with multiple founder variants are associated with the development of neutralization breadth years later. We propose a novel vaccine design strategy that integrates the variability observed in acute HIV-1 infections with multiple founder variants. We developed a probabilistic model to simulate this variability, yielding a set of sequences that present the minimal diversity seen in an infection with multiple founders. We applied this model to a subtype C consensus sequence for the Envelope (Env) (used as input) and showed that the simulated Env sequences mimic the mutational landscape of an infection with multiple founder variants, including diversity at antibody epitopes. The derived set of multi-founder-variant-like, minimally distant antigens is designed to be used as a vaccine cocktail specific to a HIV-1 subtype or circulating recombinant form and is expected to promote the development of broadly neutralizing antibodies., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: A patent application on invention disclosed in this publication is filed. MR and EL are the co-inventors.

Background: Human immunodeficiency virus type 1 (HIV-1) sequence diversity and the presence of archived epitope muta-tions in antibody binding sites are a major obstacle for the clinical application of broadly neutralizing antibodies (bNAbs) against HIV-1. Specifically, it is unclear to what degree the viral reservoir is compartmentalized and if virus susceptibility to antibody neutralization differs across tissues., Methods: The Last Gift cohort enrolled 7 people with HIV diagnosed with a terminal illness and collected antemortem blood and postmortem tissues across 33 anatomical compartments for near full-length env HIV genome sequencing. Using these data, we applied a Bayesian machine-learning model (Markov chain Monte Carlo-support vector machine) that uses HIV-1 envelope sequences and approximated glycan-occupancy information to quantitatively predict the half-maximal inhib-itory concentrations (IC50) of bNAbs, allowing us to map neutralization resistance pattern across tissue reservoirs., Results: Predicted mean susceptibilities across tissues within participants were relatively homogenous, and the susceptibility pattern observed in blood often matched what was predicted for tissues. However, selected tissues, such as the brain, showed ev-idence of compartmentalized viral populations with distinct neutralization susceptibilities in some participants. Additionally, we found substantial heterogeneity in the range of neutralization susceptibilities across tissues within and between indi-viduals, and between bNAbs within individuals (standard deviation of log2(IC50) >3.4)., Conclusions: Blood-based screening methods to determine viral susceptibility to bNAbs might underestimate the presence of resistant viral variants in tissues. The extent to which these resistant viruses are clinically relevant, that is, lead to bNAb therapeutic failure, needs to be further explored., Competing Interests: Potential conflicts of interest. B. J. reports stock or stock options in Gilead Sciences. D. M. S. reports consulting fees from Arena Pharmaceuticals, Bayer, Matrix Biomed, and Model Medicines; payment or honoraria from IAS USA, Medscape, and Kiadis; and stock or stock options from Linear Therapies (Vx Biosciences), Model Medicines, and Fluxergy. C. S. T. reports an NIH grant (MH112360) unrelated to the study. All remaining authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Binding to the host cell receptors CD4 and CCR5/CXCR4 triggers conformational changes in the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer that promote virus entry. CD4 binding allows the gp120 exterior Env to bind CCR5/CXCR4 and induces a short-lived prehairpin intermediate conformation in the gp41 transmembrane Env. Small-molecule CD4-mimetic compounds (CD4mcs) bind within the conserved Phe-43 cavity of gp120, near the binding site for CD4. CD4mcs like BNM-III-170 inhibit HIV-1 infection by competing with CD4 and by prematurely activating Env, leading to irreversible inactivation. In cell culture, we selected and analyzed variants of the primary HIV-1 AD8 strain resistant to BNM-III-170. Two changes (S375N and I424T) in gp120 residues that flank the Phe-43 cavity each conferred an ~5-fold resistance to BNM-III-170 with minimal fitness cost. A third change (E64G) in layer 1 of the gp120 inner domain resulted in ~100-fold resistance to BNM-III-170, ~2- to 3-fold resistance to soluble CD4-Ig, and a moderate decrease in viral fitness. The gp120 changes additively or synergistically contributed to BNM-III-170 resistance. The sensitivity of the Env variants to BNM-III-170 inhibition of virus entry correlated with their sensitivity to BNM-III-170-induced Env activation and shedding of gp120. Together, the S375N and I424T changes, but not the E64G change, conferred >100-fold and 33-fold resistance to BMS-806 and BMS-529 (temsavir), respectively, potent HIV-1 entry inhibitors that block Env conformational transitions. These studies identify pathways whereby HIV-1 can develop resistance to CD4mcs and conformational blockers, two classes of entry inhibitors that target the conserved gp120 Phe-43 cavity. IMPORTANCE CD4-mimetic compounds (CD4mcs) and conformational blockers like BMS-806 and BMS-529 (temsavir) are small-molecule inhibitors of human immunodeficiency virus (HIV-1) entry into host cells. Although CD4mcs and conformational blockers inhibit HIV-1 entry by different mechanisms, they both target a pocket on the viral envelope glycoprotein (Env) spike that is used for binding to the receptor CD4 and is highly conserved among HIV-1 strains. Our study identifies changes near this pocket that can confer various levels of resistance to the antiviral effects of a CD4mc and conformational blockers. We relate the antiviral potency of a CD4mc against this panel of HIV-1 variants to the ability of the CD4mc to activate changes in Env conformation and to induce the shedding of the gp120 exterior Env from the spike. These findings will guide efforts to improve the potency and breadth of small-molecule HIV-1 entry inhibitors.

Two-component self-assembling virus-like particles (VLPs) are promising scaffolds for achieving high-density display of HIV-1 envelope (gp140) trimers, which can improve the induction of neutralising antibodies (NAbs). In this study gp140 was displayed on the surface of VLPs formed by the AP205 phage coat protein. The CAP256 SU gp140 antigen was selected as the patient who this virus was isolated from developed broadly neutralising antibodies (bNAbs) shortly after superinfection with this virus. The CAP256 SU envelope is also sensitive to several bNAbs and has shown enhanced reactivity for certain bNAb precursors. A fusion protein comprising the HIV-1 CAP256 SU gp140 and the SpyTag (ST) (gp140-ST) was produced in HEK293 cells, and trimers were purified to homogeneity using gel filtration. SpyCatcher (SC)-AP205 VLPs were produced in Escherichia coli and purified by ultracentrifugation. The gp140-ST trimers and the SC-AP205 VLPs were mixed in varying molar ratios to generate VLPs displaying the glycoprotein (AP205-gp140-ST particles). Dynamic light scattering, negative stain electron microscopy and 2D classification indicated that gp140-ST was successfully bound to the VLPs, although not all potential binding sites were occupied. The immunogenicity of the coupled VLPs was evaluated in a pilot study in rabbits. One group was injected four times with coupled VLPs, and the second group was primed with DNA vaccines expressing Env and a mosaic Gag, followed by modified vaccinia Ankara expressing the same antigens. The animals were then boosted twice with coupled VLPs. Encouragingly, gp140-ST displayed on SC-AP205 VLPs was an effective boost to heterologously primed rabbits, leading to induction of autologous Tier 2 neutralising antibodies in 2/5 rabbits. However, four inoculations of coupled VLPs alone failed to elicit any Tier 2 antibodies. These results demonstrate that the native-like structure of HIV-1 envelope trimers and selection of a geometrically-suitable nanoparticle scaffold to achieve a high-density display of the trimers are important considerations that could improve the effect of nanoparticle-displayed gp140., (© 2022 IOP Publishing Ltd.)

Anti-retroviral therapy (ART) generally suppresses HIV replication to undetectable levels in peripheral blood, but immune activation associated with increased morbidity and mortality is sustained during ART, and infection rebounds when treatment is interrupted. To identify drivers of immune activation and potential sources of viral rebound, we modified RNAscope in situ hybridization to visualize HIV-producing cells as a standard against which to compare the following assays of potential sources of immune activation and virus rebound following treatment interruption: (i) envelope detection by induced transcription-based sequencing (EDITS) assay; (ii) HIV-Flow; (iii) Flow-FISH assays that can scan tissues and cell suspensions to detect rare cells expressing env mRNA, gag mRNA/Gag protein and p24; and (iv) an ultrasensitive immunoassay that detects p24 in cell/tissue lysates at subfemtomolar levels. We show that the sensitivities of these assays are sufficient to detect one rare HIV-producing/env mRNA + /p24 + cell in one million uninfected cells. These high-throughput technologies provide contemporary tools to detect and characterize rare cells producing virus and viral antigens as potential sources of immune activation and viral rebound. IMPORTANCE Anti-retroviral therapy (ART) has greatly improved the quality and length of life for people living with HIV, but immune activation does not normalize during ART, and persistent immune activation has been linked to increased morbidity and mortality. We report a comparison of assays of two potential sources of immune activation during ART: rare cells producing HIV and the virus' major viral protein, p24, benchmarked on a cell model of active and latent infections and a method to visualize HIV-producing cells. We show that assays of HIV envelope mRNA (EDITS assay), gag mRNA, and p24 (Flow-FISH, HIV-Flow. and ultrasensitive p24 immunoassay) detect HIV-producing cells and p24 at sensitivities of one infected cell in a million uninfected cells, thereby providing validated tools to explore sources of immune activation during ART in the lymphoid and other tissue reservoirs.

HIV-1 envelope glycoprotein (Env) interacts with cellular receptors and mediates virus entry into target cells. Blocking Env-receptor interactions represents an effective interventional strategy for developing HIV-1 entry inhibitors. We previously designed a panel of CD4-linker-DC-SIGN (CLD) constructs by fusing the extracellular CD4 and DC-SIGN domains with various linkers. Such CLDs produced by the prokaryotic system efficiently inhibited HIV-1 infection and dissemination in vitro and ex vivo. In this study, following the construction and identification of the most promising candidate with a linker of 8 Gly 4 Ser repeats (named CLD), we further designed an improved form (named CLDmut) by back mutating Cys to Ser at amino acid 60 of CD4. Both CLD and CLDmut were produced in mammalian (293F) cells for better protein translation and modification. The anti-HIV-1 activity of CLD and CLDmut was assessed against the infection of a range of HIV-1 isolates, including transmitted and founder (T/F) viruses. While both CLD and CLDmut efficiently neutralized the tested HIV-1 isolates, CLDmut demonstrated much higher neutralizing activity than CLD, with an IC 50 up to one log lower. The neutralizing activity of CLDmut was close to or more potent than those of the highly effective HIV-1 broadly neutralizing antibodies (bNAbs) reported to date. Findings in this study indicate that mammalian cell-expressed CLDmut may have the potential to be used as prophylaxis or/and therapeutics against HIV-1 infection.

Binding to the receptor, CD4, drives the pretriggered, "closed" (state-1) conformation of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer into more "open" conformations (states 2 and 3). Broadly neutralizing antibodies, which are elicited inefficiently, mostly recognize the state-1 Env conformation, whereas the more commonly elicited poorly neutralizing antibodies recognize states 2/3. HIV-1 Env metastability has created challenges for defining the state-1 structure and developing immunogens mimicking this labile conformation. The availability of functional state-1 Envs that can be efficiently cross-linked at lysine and/or acidic amino acid residues might assist these endeavors. To that end, we modified HIV-1 AD8 Env, which exhibits an intermediate level of triggerability by CD4. We introduced lysine/acidic residues at positions that exhibit such polymorphisms in natural HIV-1 strains. Env changes that were tolerated with respect to gp120-gp41 processing, subunit association, and virus entry were further combined. Two common polymorphisms, Q114E and Q567K, as well as a known variant, A582T, additively rendered pseudoviruses resistant to cold, soluble CD4, and a CD4-mimetic compound, phenotypes indicative of stabilization of the pretriggered state-1 Env conformation. Combining these changes resulted in two lysine-rich HIV-1 AD8 Env variants (E.2 and AE.2) with neutralization- and cold-resistant phenotypes comparable to those of natural, less triggerable tier 2/3 HIV-1 isolates. Compared with these and the parental Envs, the E.2 and AE.2 Envs were cleaved more efficiently and exhibited stronger gp120-trimer association in detergent lysates. These highly cross-linkable Envs enriched in a pretriggered conformation should assist characterization of the structure and immunogenicity of this labile state. IMPORTANCE The development of an efficient vaccine is critical for combating HIV-1 infection worldwide. However, the instability of the pretriggered shape (state 1) of the viral envelope glycoprotein (Env) makes it difficult to raise neutralizing antibodies against HIV-1. Here, by introducing multiple changes in Env, we derived two HIV-1 Env variants that are enriched in state 1 and can be efficiently cross-linked to maintain this shape. These Env complexes are more stable in detergent, assisting their purification. Thus, our study provides a path to a better characterization of the native pretriggered Env, which should assist vaccine development.

Eliciting broadly neutralizing antibodies (bnAbs) is a cornerstone of HIV-1 vaccine strategies. Comparing HIV-1 envelope (env) sequences from the first weeks of infection to the breadth of antibody responses observed several years after infection can help define viral features critical to vaccine design. We investigated the relationship between HIV-1 env genetics and the development of neutralization breadth in 70 individuals enrolled in a prospective acute HIV-1 cohort. Half of the individuals who developed bnAbs were infected with multiple HIV-1 founder variants, whereas all individuals with limited neutralization breadth had been infected with single HIV-1 founders. Accordingly, at HIV-1 diagnosis, env diversity was significantly higher in participants who later developed bnAbs compared to those with limited breadth (p = 0.012). This association between founder multiplicity and the subsequent development of neutralization breadth was also observed in 56 placebo recipients in the RV144 vaccine efficacy trial. In addition, we found no evidence that neutralization breath was heritable when analyzing env sequences from the 126 participants. These results demonstrate that the presence of slightly different HIV-1 variants in acute infection could promote the induction of bnAbs, suggesting a novel vaccine strategy, whereby an initial immunization with a cocktail of minimally distant antigens would be able to initiate bnAb development towards breadth., Competing Interests: The authors have declared that no competing interests exist.

The success of nucleoside-modified mRNAs in lipid nanoparticles (mRNA-LNP) as COVID-19 vaccines heralded a new era of vaccine development. For HIV-1, multivalent envelope (Env) trimer protein nanoparticles are superior immunogens compared with trimers alone for priming of broadly neutralizing antibody (bnAb) B cell lineages. The successful expression of complex multivalent nanoparticle immunogens with mRNAs has not been demonstrated. Here, we show that mRNAs can encode antigenic Env trimers on ferritin nanoparticles that initiate bnAb precursor B cell expansion and induce serum autologous tier 2 neutralizing activity in bnAb precursor V H  + V L knock-in mice. Next-generation sequencing demonstrates acquisition of critical mutations, and monoclonal antibodies that neutralize heterologous HIV-1 isolates are isolated. Thus, mRNA-LNP can encode complex immunogens and may be of use in design of germline-targeting and sequential boosting immunogens for HIV-1 vaccine development., Competing Interests: Declaration of interests B.F.H., K.O.S., and K.W. have patent applications on some of the concepts and immunogens discussed in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The sequence diversity of HIV-1 is the biggest hurdle for the design of a prophylactic vaccine. Mosaic (Mos) antigens consisting of synthetically shuffled epitopes from various HIV-1 strains are currently tested in the clinical vaccine trial Mosaico (NCT03964415). Besides adenovirus vectors encoding variants of Mos.Gag-Pol and soluble Mos.Env proteins, the Mosaico vaccine entails vectors mediating gene transfer and expression of the membrane-anchored Env-variant Mos2S.Env. We thus examined whether the expression of mosaic Gag mediates the formation of virus-like particles (VLPs). Mos1.Gag- and Mos2.Gag-VLP-formation was readily detected using Western blot- and electron microscopic-analysis. Upon co-expression of both mosaic Gag variants with Mos2S.Env, incorporation of Env into Gag-formed VLPs was observed. The display of the respective neutralization-sensitive target epitopes on Mos2S.Env-decorated VLPs was demonstrated employing a panel of broadly neutralizing antibodies (bNAbs) in a VLP-capture assay. This opens new perspectives for future HIV vaccine designs., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Human immunodeficiency virus type-1 (HIV-1) antigenic variation poses a great challenge for vaccine immunogen design to elicit broadly neutralizing antibodies (bNAbs). Over the last 10-15 years, great progress has been made to understand the conserved sites of sensitivity on HIV envelope glycoprotein spikes targeted by bNAbs. Plasma neutralization mapping and monoclonal antibody isolation efforts have revealed five major conserved epitope clusters. Most of this work has focused on subtype B and C-infected Caucasian or African donors. It is not clear if the same epitopes and epitope rank order preferences are also true in donors infected with different HIV-1 subtypes and with different racial backgrounds. To investigate this point, in this study we report the first attempt to profile the bNAb specificities of CRF01&#95;AE-infected Malaysian plasmas. We first measured neutralization titers of 21 plasmas against a subtype A, B, and AE pseudovirus panel. This revealed that 14% (3 of 21) plasmas had cross-clade breadth. Focusing on the cross-neutralizing plasma P9, we used AE and JR-FL mutant pseudoviruses, gp120 monomer interference, and native polyacrylamide gel electrophoresis to better understand the neutralization specificity. P9 demonstrates CD4-binding-site specificity with trimer dependence and D368 independence.