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Introduction: A limited subset of HIV-1 infected adult individuals typically after at least 2-3 years of chronic infection, develop broadly neutralizing antibodies (bnAbs), suggesting that highly conserved neutralizing epitopes on the HIV-1 envelope glycoprotein are difficult for B cell receptors to effectively target, during natural infection. Recent studies have shown the evolution of bnAbs in HIV-1 infected infants. Methods: We used bulk BCR sequencing (BCR-seq) to profile the B cell receptors from longitudinal samples (3 time points) collected from a rare pair of antiretroviralnaïve, HIV-1 infected pediatric monozygotic twins (AIIMS_329 and AIIMS_330) who displayed elite plasma neutralizing activity against HIV-1. Results: BCR-seq of both twins revealed convergent antibody characteristics including V-gene use, CDRH3 lengths and somatic hypermutation (SHM). Further, antibody clonotypes with genetic features similar to highly potent bnAbs isolated from adults showed ongoing development in donor AIIMS_330 but not in AIIMS_329, corroborating our earlier findings based on plasma bnAbs responses. An increase in SHM was observed in sequences of the IgA isotype from AIIMS_330. Discussion: This study suggests that children living with chronic HIV-1 can develop clonotypes of HIV-1 bnAbs against multiple envelope epitopes similar to those isolated from adults, highlighting that such B cells could be steered to elicit bnAbs responses through vaccines aimed to induce bnAbs against HIV-1 in a broad range of people including children. [ABSTRACT FROM AUTHOR]

The development of HIV prophylactic vaccines is facing an impasse, since all phase IIb/III clinical trials were halted in 2023 without demonstrating efficacy. Thus, the field is in need of developing novel immunogens and vaccination strategies that induce broadly neutralising antibodies together with potent Fc-dependent effector functions, as well as protective cross-reactive CD4+ and CD8+ T cell responses. Nucleic acid vaccines, particularly mRNA vaccines, have been one of the major groundbreaking advances in the current decade. Nucleic acid vaccines may help recalibrate the HIV vaccine field towards the use of delivery systems that allow the proper expression of immunogens as a sole antigen (i.e., membrane-bound trimeric envelope glycoproteins) or even to be displayed in a multiantigen platform that will be synthesised by the host. In this review, we will summarise how the multiple HIV vaccine strategies pursued in the last 40 years of HIV research have driven current vaccine development, which are the most relevant immunogens identified so far to induce balanced adaptive immune responses, and how they can benefit from the acceptance of nucleic acid vaccines in the market by reducing the limitations of previous delivery systems. The incorporation of nucleic acid vaccines into the current heterogeneous repertoire of vaccine platforms may represent an invaluable opportunity to reignite the fight against HIV. [ABSTRACT FROM AUTHOR]

HIV-1 envelope glycoproteins (Envs) mediate viral entry and represent a target of choice for small molecule inhibitors. One of them, temsavir (BMS-626529) prevents the interaction of the host cell receptor CD4 with Env by binding the pocket under the β20–β21 loop of the Env subunit gp120. Along with its capacity to prevent viral entry, temsavir stabilizes Env in its "closed" conformation. We recently reported that temsavir affects glycosylation, proteolytic processing, and overall conformation of Env. Here, we extend these results to a panel of primary Envs and infectious molecular clones (IMCs), where we observe a heterogeneous impact on Env cleavage and conformation. Our results suggest that the effect of temsavir on Env conformation is associated with its capacity to decrease Env processing. Indeed, we found that the effect of temsavir on Env processing affects the recognition of HIV-1-infected cells by broadly neutralizing antibodies and correlates with their capacity to mediate antibody-dependent cellular cytotoxicity (ADCC). [ABSTRACT FROM AUTHOR]

In the early 2000s, novel humanized mouse models based on the transplantation of human hematopoietic stem and progenitor cells (HSPCs) into immunocompromised mice were introduced (hu mice). The human HSPCs gave rise to a lymphoid system of human origin. The HIV research community has greatly benefitted from these hu mice. Since human immunodeficiency virus (HIV) type 1 infection results in a high-titer disseminated HIV infection, hu mice have been of great value for all types of HIV research from pathogenesis to novel therapies. Since the first description of this new generation of hu mice, great efforts have been expended to improve humanization by creating other immunodeficient mouse models or supplementing mice with human transgenes to improve human engraftment. Many labs have their own customized hu mouse models, making comparisons quite difficult. Here, we discuss the different hu mouse models in the context of specific research questions in order to define which characteristics should be considered when determining which hu mouse model is appropriate for the question posed. We strongly believe that researchers must first define their research question and then determine whether a hu mouse model exists, allowing the research question to be studied. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 Gly4Ser 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 IC50 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. [ABSTRACT FROM AUTHOR]

NK cells are well‐known for their antiviral functions. Also, their role in HIV has been well established, with rapid responses elicited during early HIV infection. Most immune cells including CD4+ T cells, monocytes, Mϕs, and dendritic cells are readily infected by HIV. Recent evidence from multiple studies has suggested that similar to these cells, in chronic conditions like HIV, NK cells also undergo functional exhaustion with impaired cytotoxicity, altered cytokine production, and impaired ADCC. NK‐based immunotherapy aims to successfully restore, boost, and modify their activity as has been already demonstrated in the field of cancer immunotherapy. The utilization of NK cell‐based strategies for the eradication of HIV from the body provides many advantages over classical ART. The literature search consisted of manually selecting the most relevant studies from databases including PubMed, Embase, Google Scholar, and ClinicalTrial.gov. Some of the treatments currently under consideration are CAR‐NK cell therapy, facilitating ADCC, TLR agonists, bNAbs, and BiKEs/TriKEs, blocking inhibitory NK receptors during infection, IL‐15 and IL‐15 superagonists (eg: ALT‐803), and so on. This review aims to discuss the NK cell‐based therapies currently under experimentation against HIV infection and finally highlight the challenges associated with NK cell‐based immunotherapies. [ABSTRACT FROM AUTHOR]

Recent studies showed that immunological tolerance may restrict the development of Env-specific autoreactive broadly neutralizing antibodies. This evidence is consistent with the finding that Env immunization of a systemic lupus erythematosus (SLE) murine model produced antibodies that neutralize tier 2 HIV-1 strains. In this study, we address the possibility of eliciting neutralizing anti-Env antibodies in other autoimmune diseases such as multiple sclerosis (MS). While, as reported for SLE, we showed for the first time that a small number of HIV-1 negative, relapsing remitting MS patients exhibited antibodies with neutralizing properties, our attempts at inducing those antibodies in a EAE mouse model of MS failed. The success in eliciting Env-specific neutralizing antibodies might be related to the specific characteristics of the autoimmune disease, or it might rely in improving the vaccination design. Studies using mouse models are useful to gain insight in how HIV-specific neutralizing antibody responses are regulated in order to develop a protective HIV-1 vaccine. [ABSTRACT FROM AUTHOR]

The potential of immunotherapy strategies utilizing broadly neutralizing antibodies (BNAbs), such as 3BNC117 and 10‐1074, to limit viral replication while also facilitating clearance of HIV infected cells has heightened interest in identifying the predominant NK effector subset(s) capable of mediating antibody dependent cellular cytotoxicity (ADCC). Utilizing advanced polychromatic flow cytometry, we identified that CD57 positive NK cells from ART‐suppressed in People Living With HIV (PLWH) expressed significantly higher levels of the CD16 FcγR receptor, 2B4 ADCC coreceptor, and HLA‐DR activation marker while NKG2C positive NK cells expressed significantly higher levels of the CD2 ADCC coreceptor (p < 0.001, n = 32). Functionally, CD57 positive NK cells from ART‐suppressed PLWH with either high or low NKG2C expansion exhibited significantly enhanced degranulation and IFN‐γ production against heterologous gp120‐coated ADCC targets coated with HIV reference plasma compared to CD57 negative NK cells (p = 0.0029, n = 11). CD57 positive NK cells from control donors lacking NKG2C expansion also exhibited significantly more degranulation and IFN‐γ production at every timepoint tested against both heterologous ADCC targets (p = 0.019, n = 9) and HIV‐1 infected autologous CD4+ primary T cells coated with BNAbs. Together, our data support CD57 positive and NKG2C positive NK cells as the predominant ADCC effector subsets capable of targeting HIV‐infected CD4+ cells in the presence of 3BNC117 and 10‐1074 immunotherapy. [ABSTRACT FROM AUTHOR]

Background: HIV remains one of the most important health issues worldwide, with almost 40 million people living with HIV. Although patients develop antibodies against the virus, its high mutation rate allows evasion of immune responses. Some patients, however, produce antibodies that are able to bind to, and neutralise different strains of HIV. One such 'broadly neutralising' antibody is 'N6'. Identified in 2016, N6 can neutralise 98% of HIV-1 isolates with a median IC50 of 0.066 µg/mL. This neutralisation breadth makes N6 a very promising therapeutic candidate. Results: N6 was expressed in a glycoengineered line of N. benthamiana plants (pN6) and compared to the mammalian cell-expressed equivalent (mN6). Expression at 49 mg/kg (fresh leaf tissue) was achieved in plants, although extraction and purification are more challenging than for most plant-expressed antibodies. N-glycoanalysis demonstrated the absence of xylosylation and a reduction in α(1,3)-fucosylation that are typically found in plant glycoproteins. The N6 light chain contains a potential N-glycosylation site, which was modified and displayed more α(1,3)-fucose than the heavy chain. The binding kinetics of pN6 and mN6, measured by surface plasmon resonance, were similar for HIV gp120. pN6 had a tenfold higher affinity for FcγRIIIa, which was reflected in an antibody-dependent cellular cytotoxicity assay, where pN6 induced a more potent response from effector cells than that of mN6. pN6 demonstrated the same potency and breadth of neutralisation as mN6, against a panel of HIV strains. Conclusions: The successful expression of N6 in tobacco supports the prospect of developing a low-cost, low-tech production platform for a monoclonal antibody cocktail to control HIV in low-to middle income countries. [ABSTRACT FROM AUTHOR]

At present, HIV-1 infection is treated with combined antiretroviral therapy (cART). cART greatly improves the life quality and outcome of infected individuals but cannot eradicate the virus from the hosts. Therefore, new treatment methods that can clear the virus are needed. In this review, we summarized four novel approaches that have the potentials to cure HIV infection, which are dendritic cell targeting, CCR5 editing, LASER ART and CRISPR-Cas9 dual therapy and broad neutralization antibodies. [ABSTRACT FROM AUTHOR]

Natural environments can present diverse challenges, but some genotypes remain fit across many environments. Such "generalists" can be hard to evolve, outcompeted by specialists fitter in any particular environment. Here, inspired by the search for broadly neutralizing antibodies during B cell affinity maturation, we demonstrate that environmental changes on an intermediate timescale can reliably evolve generalists, even when faster or slower environmental changes are unable to do so. We find that changing environments on timescales comparable with evolutionary transients in a population enhance the rate of evolving generalists from specialists, without enhancing the reverse process. The yield of generalists is further increased in more complex dynamic environments, such as a "chirp" of increasing frequency. Our work offers design principles for how nonequilibrium fitness "seascapes" can dynamically funnel populations to genotypes unobtainable in static environments. [ABSTRACT FROM AUTHOR]

In the last decade, over a dozen potent broadly neutralizing antibodies (bnAbs) to several HIV envelope protein epitopes have been identified, and their in vitro neutralization profiles have been defined. Many have demonstrated prevention efficacy in preclinical trials and favorable safety and pharmacokinetic profiles in early human clinical trials. The first human prevention efficacy trials using 10 sequential, every-two-month administrations of a single anti-HIV bnAb are anticipated to conclude in 2020. Combinations of complementary bnAbs and multi-specific bnAbs exhibit improved breadth and potency over most individual antibodies and are entering advanced clinical development. Genetic engineering of the Fc regions has markedly improved bnAb half-life, increased mucosal tissue concentrations of antibodies (especially in the genital tract), and enhanced immunomodulatory and Fc effector functionality, all of which improve antibodies' preventative and therapeutic potential. Human-derived monoclonal antibodies are likely to enter the realm of primary care prevention and therapy for viral infections in the near future. [ABSTRACT FROM AUTHOR]

Abstract: Here, we describe a one‐step, in vivo CRISPR/Cas9 nuclease‐mediated strategy to generate knock‐in mice. We produced knock‐in (KI) mice wherein a 1.9‐kb DNA fragment bearing a pre‐arranged human B‐cell receptor heavy chain was recombined into the native murine immunoglobulin locus. Our methodology relies on Cas9 nuclease‐induced double‐stranded breaks directed by two sgRNAs to occur within the specific target locus of fertilized oocytes. These double‐stranded breaks are subsequently repaired via homology‐directed repair by a plasmid‐borne template containing the pre‐arranged human immunoglobulin heavy chain. To validate our knock‐in mouse model, we examined the expression of the KI immunoglobulin heavy chains by following B‐cell development and performing single B‐cell receptor sequencing. We optimized this strategy to generate immunoglobulin KI mice in a short amount of time with a high frequency of homologous recombination (30–50%). In the future, we envision that such knock‐in mice will provide much needed vaccination models to evaluate immunoresponses against immunogens specific for various infectious diseases. [ABSTRACT FROM AUTHOR]

Summary Recent efforts toward HIV vaccine development include the design of immunogens that can engage B cell receptors with the potential to affinity mature into broadly neutralizing antibodies (bnAbs). V2-apex bnAbs, which bind a protein-glycan region on HIV envelope glycoprotein (Env) trimer, are among the most broad and potent described. We show here that a rare “glycan hole” at the V2 apex is enriched in HIV isolates neutralized by inferred precursors of prototype V2-apex bnAbs. To investigate whether this feature could focus neutralizing responses onto the apex bnAb region, we immunized wild-type rabbits with soluble trimers adapted from these Envs. Potent autologous tier 2 neutralizing responses targeting basic residues in strand C of the V2 region, which forms the core epitope for V2-apex bnAbs, were observed. Neutralizing monoclonal antibodies (mAbs) derived from these animals display features promising for subsequent broadening of the response. [ABSTRACT FROM AUTHOR]

HIV-1 envelope (Env) conformation determines the susceptibility of infected CD4+ T cells to antibody-dependent cellular cytotoxicity (ADCC). Upon interaction with CD4, Env adopts more "open" conformations, exposing ADCC epitopes. HIV-1 limits Env-CD4 interaction and protects infected cells against ADCC by downregulating CD4 via Nef, Vpu, and Env. Limited data exist, however, of the role of these proteins in downmodulating CD4 on infected macrophages and how this impacts Env conformation. While Nef, Vpu, and Env are all required to efficiently downregulate CD4 on infected CD4+ T cells, we show here that any one of these proteins is sufficient to downmodulate most CD4 from the surface of infected macrophages. Consistent with this finding, Nef and Vpu have a lesser impact on Env conformation and ADCC sensitivity in infected macrophages compared with CD4+ T cells. However, treatment of infected macrophages with small CD4 mimetics exposes vulnerable CD4-induced Env epitopes and sensitizes them to ADCC. [Display omitted] • Nef, Vpu, and Env are required to fully downregulate CD4 from infected CD4+ T cells • Any combination of two of these proteins are sufficient in infected macrophages • Infected macrophages are resistant to ADCC mediated by HIV+ plasma • Small CD4 mimetics sensitize infected macrophages to ADCC mediated by HIV+ plasma In this study, Laumaea et al. characterize CD4 downregulation, Env conformation, and ADCC responses in infected macrophages and autologous CD4+ T cells. They report that Nef and Vpu protect infected macrophages from ADCC responses mediated by HIV+ plasma and that small CD4 mimetics sensitize them to ADCC. [ABSTRACT FROM AUTHOR]

Introduction: Within monocyte-derived macrophages, HIV-1 accumulates in intracellular virus-containing compartments (VCCs) that are inaccessible to the external environment, which implicate these cells as latently infected HIV-1 reservoirs. During mother-to-child transmission of HIV-1, human placental macrophages (Hofbauer cells (HCs)) are viral targets, and have been shown to be infected in vivo and sustain low levels of viral replication in vitro; however, the risk of in utero transmission is less than 7%. The role of these primary macrophages as viral reservoirs is largely undefined. The objective of this study is to define potential sites of viral assembly, accumulation and neutralization in HCs given the pivotal role of the placenta in preventing HIV-1 infection in the mother-infant dyad. Methods: Term placentae from 20 HIV-1 seronegative women were obtained following caesarian section. VCCs were evaluated by 3D confocal and electron microscopy. Colocalization R values (Pearson's correlation) were quantified with colocalization module of Volocity 5.2.1. Replication kinetics and neutralization studies were evaluated using p24 ELISA. Results: We demonstrate that primary HCs assemble and sequester HIV-1BaL in intracellular VCCs, which are enriched in endosomal/lysosomal markers, including CD9, CD81, CD63 and LAMP-1. Following infection, we observed HIV-1 accumulation in potentially acidic compartments, which stained intensely with Lysotracker-Red. Remarkably, these compartments are readily accessible via the cell surface and can be targeted by exogenously applied small molecules and HIV-1-specific broadly neutralizing antibodies. In addition, broadly neutralizing antibodies (4E10 and VRC01) limited viral replication by HIV-1-infected HCs, which may be mediated by FcγRI. Conclusions: These findings suggest that placental HCs possess intrinsic adaptations facilitating unique sequestration of HIV-1, and may serve as a protective viral reservoir to permit viral neutralization and/or antiretroviral drug entry in utero. [ABSTRACT FROM AUTHOR]

Broadly neutralizing monoclonal antibodies (bNAbs) against conserved domains in the influenza hemagglutinin are in clinical trials. Several next generation influenza vaccines designed to elicit such bNAbs are also in clinical development. One of the common features of the isolated bNAbs is the use of restricted IgVH repertoire. More than 80% of stem-targeting bNAbs express IgVH1-69, which may indicate genetic constraints on the evolution of such antibodies. In the current study, we evaluated a panel of influenza virus bNAbs in comparison with HIV-1 MAb 4E10 and anti-RSV MAb Palivizumab (approved for human use) for autoreactivity using 30 normal human tissues microarray and human protein (>9000) arrays. We found that several human bNAbs (CR6261, CR9114, and F2603) reacted with human tissues, especially with pituitary gland tissue. Importantly, protein array analysis identified high-affinity interaction of CR6261 with the autoantigen "Enhancer of mRNA decapping 3 homolog" (EDC3), which was not previously described. Moreover, EDC3 competed with hemagglutinin for binding to bNAb CR6261. These autoreactivity findings underscores the need for careful evaluation of such bNAbs for therapeutics and stem-based vaccines against influenza virus. [ABSTRACT FROM AUTHOR]

The HIV-1 envelope glycoprotein (Env) maintains a delicate balance between mediating viral entry and escaping antibody neutralization. Adaptation during transmission of neutralization-sensitive Envs with an "open" conformation remains poorly understood. By passaging a replication-competent simian-human immunodeficiency virus carrying a highly neutralization-sensitive Env (SHIV CNE40) in rhesus macaques, we show that SHIV CNE40 develops enhanced replication kinetics associated with neutralization resistance against antibodies and autologous serum. A gp41 substitution, E658K, functions as the major determinant for these properties. Structural modeling and functional verification indicate that the substitution disrupts an intermolecular salt bridge with the neighboring protomer, thereby promoting fusion and facilitating immune evasion. This effect is applicable across diverse HIV-1 subtypes. Our results highlight the critical role of gp41 in shaping the neutralization profile and the overall conformation of Env during viral adaptation. The unique intermolecular salt bridge could potentially be utilized for rational vaccine design involving more stable HIV-1 envelope trimers. • SHIV with "open" envelope adapts into "closed" conformation during passages in monkeys • E658K substitution in the gp41 HR2 region is a major cause of the conformational change • E658K disrupts an intermolecular salt bridge with E601 and renders antibody resistance • gp41 plays a critical role in shaping Env conformation and neutralization profile Wang et al. find that simian-human immunodeficiency virus (SHIV) with an "open" envelope adapts into a "closed" conformation in monkeys. This is largely due to a single E658K substitution in gp41, which disrupts an intermolecular salt bridge and improves viral entry efficiency. In vivo adaptation at gp41 impacts envelope conformation and the neutralization profile. [ABSTRACT FROM AUTHOR]

HIV-1 spreads through contacts between infected and target cells. Polarized viral budding at the contact site forms the virological synapse. Additional cellular processes, such as nanotubes, filopodia, virus accumulation in endocytic or phagocytic compartments promote efficient viral propagation. Cell-to-cell transmission allows immune evasion and likely contributes to HIV-1 spread in vivo. Anti-HIV-1 broadly neutralizing antibodies (bNAbs) defeat the majority of circulating viral strains by binding to the viral envelope glycoprotein (Env). Several bNAbs have entered clinical evaluation during the last years. It is thus important to understand their mechanism of action and to determine how they interact with infected cells. In experimental models, HIV-1 cell-to-cell transmission is sensitive to neutralization, but the effect of antibodies is often less marked than during cell-free infection. This may be due to differences in the conformation or accessibility of Env at the surface of virions and cells. In this review, we summarize the current knowledge on HIV-1 cell-to-cell transmission and discuss the role of bNAbs during this process. [ABSTRACT FROM AUTHOR]