Primary HIV-1 Isolates Research Articles

Virus-Like Particle Based Vaccines Elicit Neutralizing Antibodies against the HIV-1 Fusion Peptide.

Broadly neutralizing antibodies (bnAbs) isolated from HIV-infected individuals delineate vulnerable sites on the HIV envelope glycoprotein that are potential vaccine targets. A linear epitope within the N-terminal region of the HIV-1 fusion peptide (FP8) is the primary target of VRC34.01, a bnAb that neutralizes ~50% of primary HIV isolates. FP8 has attracted attention as a potential HIV vaccine target because it is a simple linear epitope. Here, platform technologies based on RNA bacteriophage virus-like particles (VLPs) were used to develop multivalent vaccines targeting the FP8 epitope. Both recombinant MS2 VLPs displaying the FP8 peptide and Qβ VLPs displaying chemically conjugated FP8 peptide induced high titers of FP8-specific antibodies in mice. Moreover, a heterologous prime-boost-boost regimen employing the two FP8-VLP vaccines and native envelope trimer was the most effective approach for eliciting HIV-1 neutralizing antibodies. Given the potent immunogenicity of VLP-based vaccines, this vaccination strategy—inspired by bnAb-guided epitope mapping, VLP bioengineering, and prime-boost immunization approaches—may be a useful strategy for eliciting bnAb responses against HIV.

C-C Chemokine receptor-like 2 (CCRL2) acts as coreceptor for human immunodeficiency virus-2.

Most of the typical chemokine receptors (CKRs) have been identified as coreceptors for a variety of human and simian immunodeficiency viruses (HIVs and SIVs). This study evaluated CCRL2 to examine if it was an HIV/SIV coreceptor. The Human glioma cell line, NP-2, is normally resistant to infection by HIV and SIV. The cell was transduced with amplified cluster of differentiation 4 (CD4) as a receptor and CCR5, CXCR4 and CCRL2 as coreceptor candidates to produce NP-2/CD4/coreceptor cells (). The cells were infected with multiplicity of infection (MOI) 1.0. Infected cells were detected by indirect immunofluorescence assay (IFA). Multinucleated giant cells (MGC) in syncytia were quantified by Giemsa staining. Proviral DNA was detected by polymerase chain reaction (PCR), and reverse transcriptase (RT) activity was measured. IFA detected viral antigens of the primary isolates, HIV-1HAN2 and HIV-2MIR in infected NP-2/CD4/CCRL2 cells, indicated CCRL2 as a functional coreceptor. IFA results were confirmed by the detection of proviral DNA and measurement of RT-activity in the spent cell supernatants. Additionally, MGC was detected in HIV-2MIR-infected NP-2/CD4/CCCRL2 cells. HIV-2MIR were found more potent users of CCRL2 than HIV-1HAN2. Moreover, GWAS studies, gene ontology and cell signaling pathways of the HIV-associated genes show interaction of CCRL2 with HIV/SIV envelope protein. In vitro experiments showed CCRL2 to function as a newly identified coreceptor for primary HIV-2 isolates conveniently. The findings contribute additional insights into HIV/SIV transmission and pathogenesis. However, its in vivo relevance still needs to be evaluated. Confirming in vivo relevance, ligands of CCRL2 can be investigated as potential targets for HIV entry-inhibitor drugs.

TZM-gfp cells: a tractable fluorescent tool for analysis of rare and early HIV-1 infection

Here we describe TZM-gfp, a novel HIV-1 reporter cell derived from the same parental clone JC.53, used previously to generate the widely-utilized indicator cell line TZM-bl. We re-engineered JC.53 cells to express GFP under regulation of HIV Tat and Rev. We characterize the new reporter cell line to show that TZM-gfp cells are equally susceptible to HIV infection, exhibit minimal background signal, and can report HIV infection in rare cells from a bulk population of experimentally-infected human monocyte-derived macrophages. We demonstrate the utility and sensitivity of the cells in detection of even a single HIV-positive macrophage by fluorescence-assisted correlative electron microscopy, using the GFP signal to guide imaging of HIV virions in primary co-culture. Finally, we used TZM-gfp cells for viral capture during co-culture with human peripheral blood mononuclear cells, showing that TZM-gfp can support outgrowth and analyses of patient-derived primary HIV-1 isolates.

MxB sensitivity of HIV-1 is determined by a highly variable and dynamic capsid surface.

The type one interferon induced restriction factor Myxovirus resistance B (MxB) restricts HIV-1 nuclear entry evidenced by inhibition of 2-LTR but not linear forms of viral DNA. The HIV-1 capsid is the key determinant of MxB sensitivity and cofactor binding defective HIV-1 capsid mutants P90A (defective for cyclophilin A and Nup358 recruitment) and N74D (defective for CPSF6 recruitment) have reduced dependency on nuclear transport associated cofactors, altered integration targeting preferences and are not restricted by MxB expression. This has suggested that nuclear import mechanism may determine MxB sensitivity. Here we have use genetics to separate HIV-1 nuclear import cofactor dependence from MxB sensitivity. We provide evidence that MxB sensitivity depends on HIV-1 capsid conformation, rather than cofactor recruitment. We show that depleting CPSF6 to change nuclear import pathway does not impact MxB sensitivity, but mutants that recapitulate the effect of Cyclophilin A binding on capsid conformation and dynamics strongly impact MxB sensitivity. We demonstrate that HIV-1 primary isolates have different MxB sensitivities due to cytotoxic T lymphocyte (CTL) selected differences in Gag sequence but similar cofactor dependencies. Overall our work demonstrates a complex relationship between cyclophilin dependence and MxB sensitivity likely driven by CTL escape. We propose that cyclophilin binding provides conformational flexibility to HIV-1 capsid facilitating simultaneous evasion of capsid-targeting restriction factors including TRIM5 as well as MxB.

Slow Receptor Binding of the Noncytopathic HIV-2UC1 Envs Is Balanced by Long-Lived Activation State and Efficient Fusion Activity

Many HIV strains downregulate the levels of CD4 receptor on the surface of infected cells to prevent superinfection. In contrast, the rare HIV-2UC1 strain is noncytopathic and has no effect on CD4 expression in infected cells but still replicates as efficiently as more cytopathic strains in peripheral blood mononuclear cells (PBMCs). Here, we show that HIV-2UC1 Env interactions with the CD4 receptor exhibit slow association kinetics, whereas the dissociation kinetics is within the range of cytopathic strains. Despite the resulting 10- to 100-fold decrease in binding affinity, HIV-2UC1 Envs exhibit long-lived activation state and efficient fusion activity. These observations suggest that HIV-2UC1 Envs evolved to balance low affinity with an improved and readily triggerable molecular machinery to mediate entry. Resistance to cold exposure, similar to many primary HIV-1 isolates, and to sCD4 neutralization suggests that HIV-2UC1 Envs preferentially sample a closed Env conformation. Our data provide insights into the mechanism of HIV entry.

A Prime-Boost Immunization Strategy with Vaccinia Virus Expressing Novel gp120 Envelope Glycoprotein from a CRF02_AG Isolate Elicits Cross-Clade Tier 2 HIV-1 Neutralizing Antibodies.

Development of new immunogens eliciting broadly neutralizing antibodies (bNAbs) is a main priority for the HIV-1 vaccine field. Envelope glycoproteins from non-B-non-C HIV-1clades have not been fully explored as components of a vaccine. We produced Vaccinia viruses expressing a truncated version of gp120 (gp120t) from HIV-1 clades CRF02_AG, H, J, B, and C and examined their immunogenicity in mice and rabbits. Mice primed with the recombinant Vaccinia viruses and boosted with the homologous gp120t or C2V3C3 polypeptides developed antibodies that bind potently to homologous and heterologous envelope glycoproteins. Notably, a subset of mice immunized with the CRF02_AG-based envelope immunogens developed a cross-reactive neutralizing response against tier 2 HIV-1 Env-pseudoviruses and primary isolates. Rabbits vaccinated with the CRF02_AG-based envelope immunogens also generated potent binding antibodies, and one animal elicited antibodies that neutralized almost all (13 of 16, 81.3%) tier 2 HIV-1 isolates tested. Overall, the results suggest that the novel CRF02_AG-based envelope immunogens and prime-boost immunization strategy elicit the type of immune responses required for a preventive HIV-1 vaccine.

Novel Transmitted/Founder Simian-Human Immunodeficiency Viruses for Human Immunodeficiency Virus Latency and Cure Research.

A robust simian-human immunodeficiency virus (SHIV)-macaque model of latency is critical to investigate eradicative and suppressive strategies that target HIV-1 Env. To this end, we previously reported a novel strategy for constructing SHIVs that bear primary or transmitted/founder (TF) Envs with modifications at Env residue 375 that enable efficient replication in Indian rhesus macaques (RM). Such TF SHIVs, however, have not been examined for their suitability for HIV-1 latency and cure research. Here, we evaluate two promising TF SHIVs, SHIV.D.191859 and SHIV.C.CH848, which encode TF subtype D and C HIV-1 Envs, respectively, for their viral kinetics and persistence during suppressive combination antiretroviral therapy (cART) and treatment interruption in RM. Our results suggest that the viral kinetics of these SHIVs in RM during acute, early, and chronic infection, and upon cART initiation, maintenance and discontinuation, mirror those of HIV-1 infection. We demonstrate consistent early peak and set point viremia, rapid declines in viremia to undetectable plasma titers following cART initiation, infection of long-lived cellular subsets and establishment of viral latency, and viral rebound with return to pretreatment set point viremia following treatment interruption. The viral dynamics and reservoir biology of SHIV.D.191859, and to a lesser extent SHIV.C.CH848, during chronic infection, cART administration, and upon treatment interruption suggest that these TF SHIVs are promising reagents for a SHIV model of HIV-1 latency and cure.IMPORTANCE Simian-human immunodeficiency viruses (SHIVs) have been successfully used for over 2 decades to study virus-host interactions, transmission, and pathogenesis in rhesus macaques. The majority of Env trimers of most previously studied SHIVs, however, do not recapitulate key properties of transmitted/founder (TF) or primary HIV-1 isolates, such as CCR5 tropism, tier 2 neutralization resistance, and native trimer conformation. Here, we test two recently generated TF SHIVs, SHIV.D.191859 and SHIV.C.CH848, which were designed to address these issues as components of a nonhuman primate model of HIV-1 latency. We conclude that the TF SHIV-macaque model reflects several hallmarks of HIV and SIV infection and latency. Results suggest that this model has broad applications for evaluating eradicative and suppressive strategies against the HIV reservoir, including Env-specific interventions, therapeutic vaccines, and engineered T cells.

In vitro anti-HIV activity of some Indian medicinal plant extracts

BackgroundHuman Immunodeficiency Virus (HIV) persists to be a significant public health issue worldwide. The current strategy for the treatment of HIV infection, Highly Active Antiretroviral Therapy (HAART), has reduced deaths from AIDS related disease, but it can be an expensive regime for the underdeveloped and developing countries where the supply of drugs is scarce and often not well tolerated, especially in persons undergoing long term treatment. The present therapy also has limitations of development of multidrug resistance, thus there is a need for the discovery of novel anti-HIV compounds from plants as a potential alternative in combating HIV disease.MethodsTen Indian medicinal plants were tested for entry and replication inhibition against laboratory adapted strains HIV-1IIIB, HIV-1Ada5 and primary isolates HIV-1UG070, HIV-1VB59 in TZM-bl cell lines and primary isolates HIV-1UG070, HIV-1VB59 in PM1 cell lines. The plant extracts were further evaluated for toxicity in HEC-1A epithelial cell lines by transwell epithelial model.ResultsThe methanolic extracts of Achyranthes aspera, Rosa centifolia and aqueous extract of Ficus benghalensis inhibited laboratory adapted HIV-1 strains (IC80 3.6–118 μg/ml) and primary isolates (IC80 4.8–156 μg/ml) in TZM-bl cells. Methanolic extract of Strychnos potatorum, aqueous extract of Ficus infectoria and hydroalcoholic extract of Annona squamosa inhibited laboratory adapted HIV-1 strains (IC80 4.24–125 μg/ml) and primary isolates (IC80 18–156 μg/ml) in TZM-bl cells. Methanolic extracts of Achyranthes aspera and Rosa centifolia, (IC801-9 μg/ml) further significantly inhibited HIV-1 primary isolates in PM1cells. Methanolic extracts of Tridax procumbens, Mallotus philippinensis, Annona reticulate, aqueous extract of Ficus benghalensis and hydroalcoholic extract of Albizzia lebbeck did not exhibit anti-HIV activity in all the tested strains. Methanolic extract of Rosa centifolia also demonstrated to be non-toxic to HEC-1A epithelial cells and maintained epithelial integrity (at 500 μg/ml) when tested in transwell dual-chamber.ConclusionThese active methanolic extracts of Achyranthes aspera and Rosa centifolia, could be further subjected to chemical analysis to investigate the active moiety responsible for the anti-HIV activity. Methanolic extract of Rosa centifolia was found to be well tolerated maintaining the epithelial integrity of HEC-1A cells in vitro and thus has potential for investigating it further as candidate microbicide.

Comparative multi-assay evaluation of Determine\u2122 HIV-1/2 Ag/Ab Combo rapid diagnostic tests in acute and chronic HIV infection

In resource-limited or point-of-care settings, rapid diagnostic tests (RDTs), that aim to simultaneously detect HIV antibodies and p24 capsid (p24CA) antigen with high sensitivity, can pose important alternatives to screen for early infections. We evaluated the performance of the antibody and antigen components of the old and novel version of the Determine™ HIV-1/2 Ag/Ab Combo RDTs in parallel to quantifications in a fourth-generation antigen/antibody immunoassay (4G-EIA), p24CA antigen immunoassay (p24CA-EIA), immunoblots, and nucleic acid quantification. We included plasma samples of acute, treatment-naïve HIV-1 infections (Fiebig stages I–VI, subtypes A1, B, C, F, CRF02_AG, CRF02_AE, URF) or chronic HIV-1 and HIV-2 infections. The tests’ antigen component was evaluated also for a panel of subtype B HIV-1 transmitted/founder (T/F) viruses, HIV-2 strains and HIV-2 primary isolates. Furthermore, we assessed the analytical sensitivity of the RDTs to detect p24CA using a highly purified HIV-1NL4-3 p24CA standard. We found that 77% of plasma samples from acutely infected, immunoblot-negative HIV-1 patients in Fiebig stages II–III were identified by the new RDT, while only 25% scored positive in the old RDT. Both RDTs reacted to all samples from chronically HIV-1-infected and acutely HIV-1-infected patients with positive immunoblots. All specimens from chronically infected HIV-2 patients scored positive in the new RDT. Of note, the sensitivity of the RDTs to detect recombinant p24CA from a subtype B virus ranged between 50 and 200 pg/mL, mirrored also by the detection of HIV-1 T/F viruses only at antigen concentrations tenfold higher than suggested by the manufacturer. The RTD failed to recognize any of the HIV-2 viruses tested. Our results indicate that the new version of the Determine™ HIV-1/2 Ag/Ab Combo displays an increased sensitivity to detect HIV-1 p24CA-positive, immunoblot-negative plasma samples compared to the precursor version. The sensitivity of 4G-EIA and p24CA-EIA to detect the major structural HIV antigen, and thus to diagnose acute infections prior to seroconversion, is still superior.

Novel Central Nervous System (CNS)-Targeting Protease Inhibitors for Drug-Resistant HIV Infection and HIV-Associated CNS Complications.

There is currently no specific therapeutics for the HIV-1-related central nervous system (CNS) complications. Here we report that three newly designed CNS-targeting HIV-1 protease inhibitors (PIs), GRL-083-13, GRL-084-13, and GRL-087-13, which contain a P1-3,5-bis-fluorophenyl or P1-para-monofluorophenyl ring, and P2-bis-tetrahydrofuran (bis-THF) or P2-tetrahydropyrano-tetrahydrofuran (Tp-THF), with a sulfonamide isostere, are highly active against wild-type HIV-1 strains and primary clinical isolates (50% effective concentration [EC50], 0.0002 to ∼0.003 μM), with minimal cytotoxicity. These CNS-targeting PIs efficiently suppressed the replication of HIV-1 variants (EC50, 0.002 to ∼0.047 μM) that had been selected to propagate at high concentrations of conventional HIV-1 PIs. Such CNS-targeting PIs maintained their antiviral activity against HIV-2ROD as well as multidrug-resistant clinical HIV-1 variants isolated from AIDS patients who no longer responded to existing antiviral regimens after long-term therapy. Long-term drug selection experiments revealed that the emergence of resistant-HIV-1 against these CNS-targeting PIs was substantially delayed. In addition, the CNS-targeting PIs showed the most favorable CNS penetration properties among the tested compounds, including various FDA-approved anti-HIV-1 drugs, as assessed with the in vitro blood-brain barrier reconstruction system. Crystallographic analysis demonstrated that the bicyclic rings at the P2 moiety of the CNS-targeting PIs form strong hydrogen-bond interactions with HIV-1 protease (PR) active site. Moreover, both the P1-3,5-bis-fluorophenyl and P1-para-monofluorophenyl rings sustain greater van der Waals contacts with PR than in the case of darunavir (DRV). The data suggest that the present CNS-targeting PIs have desirable features for treating patients infected with wild-type and/or multidrug-resistant HIV-1 strains and might serve as promising preventive and/or therapeutic candidates for HIV-1-associated neurocognitive disorders (HAND) and other CNS complications.

Selective use of primate CD4 receptors by HIV-1

Individuals chronically infected with HIV-1 harbor complex viral populations within their bloodstreams. Recently, it has come to light that when these people infect others, the new infection is typically established by only one or a small number of virions from within this complex viral swarm. An important goal is to characterize the biological properties of HIV-1 virions that seed and exist early in new human infections because these are potentially the only viruses against which a prophylactic HIV-1 vaccine would need to elicit protection. This includes understanding how the Envelope (Env) protein of these virions interacts with the T-cell receptor CD4, which supports attachment and entry of HIV-1 into target cells. We examined early HIV-1 isolates for their ability to infect cells via the CD4 receptor of 15 different primate species. Primates were the original source of HIV-1 and now serve as valuable animal models for studying HIV-1. We find that most primary isolates of HIV-1 from the blood, including early isolates, are highly selective and enter cells through some primate CD4 receptor orthologs but not others. This phenotype is remarkably consistent, regardless of route of transmission, viral subtype, or time of isolation post infection. We show that the weak CD4 binding affinity of blood-derived HIV-1 isolates is what makes them sensitive to the small sequence differences in CD4 from one primate species to the next. To substantiate this, we engineered an early HIV-1 Env to have high, medium, or low binding affinity to CD4, and we show that it loses the ability to enter cells via the CD4 receptor of many primate species as the binding affinity gets weaker. Based on the phenotype of selective use of primate CD4, we find that weak CD4 binding appears to be a nearly universal property of HIV-1 circulating in the bloodstream. Therefore, weak binding to CD4 must be a selected and important property in the biology of HIV-1 in the body. We identify six primate species that encode CD4 receptors that fully support the entry of early HIV-1 isolates despite their low binding affinity for CD4. These findings will help inform long-standing efforts to model HIV-1 transmission and early disease in primates.

Human IgM monoclonal antibodies block HIV-transmission to immune cells in cervico-vaginal tissues and across polarized epithelial cells in vitro

The importance of natural IgM antibodies in protection against infections is still emerging and these antibodies have a potential role in the maintenance of homeostasis through clearance of apoptotic bodies, complement-dependent mechanisms, inflammation and exclusion of misfolded proteins. Natural IgM act as a first line of defence against unknown hazardous factors and are present in most vertebrates. We investigated the functional capacity of anti-HIV-1 IgM monoclonal antibodies, from a combinatorial Fab library derived from healthy individuals, and evaluated their protective role in inhibiting HIV-1 in vitro when passing across the human mucosal epithelial barrier. Primary HIV-1 isolates were efficiently transmitted over the tight polarized epithelial cells when added to their apical surface. Efficient inhibition of HIV-1 transmission was achieved when anti-HIV-1 IgM monoclonal antibodies were added to the basolateral side of the cells. Two of these human IgM MoAbs had the ability to neutralize HIV and reduced infection of dendritic cells in primary cervico-vaginal tissue biopsies in vitro. This indicates a potential role of natural IgM antibodies in the reduction of HIV-1 transmission in mucosal tissues and improve our understanding of how natural IgM antibodies against a neutralizing epitope could interfere with viral transmission.

Epitope-based vaccine design yields fusion peptide-directed antibodies that neutralize diverse strains of HIV-1.

A central goal of HIV-1-vaccine research is the elicitation of antibodies capable of neutralizing diverse primary isolates of HIV-1. Here we show that focusing the immune response to exposed N-terminal residues of the fusion peptide, a critical component of the viral entry machinery and the epitope of antibodies elicited by HIV-1 infection, through immunization with fusion peptide-coupled carriers and prefusion-stabilized envelope trimers, induces cross-clade neutralizing responses. In mice, these immunogens elicited monoclonal antibodies capable of neutralizing up to 31% of a cross-clade panel of 208 HIV-1 strains. Crystal and cryo-electron microscopy structures of these antibodies revealed fusion peptide-conformational diversity as a molecular explanation for the cross-clade neutralization. Immunization of guinea pigs and rhesus macaques induced similarly broad fusion peptide-directed neutralizing responses suggesting translatability. The N terminus of the HIV-1-fusion peptide is thus a promising target of vaccine efforts aimed at eliciting broadly neutralizing antibodies.

PH-responsive delivery of Griffithsin from electrospun fibers

Human immunodeficiency virus (HIV-1) affects over 36 million people globally. Current prevention strategies utilize antiretrovirals that have demonstrated protection, but result in antiviral resistance, adverse toxicity, and require frequent administration. A novel biologic, griffithsin (GRFT), has demonstrated outstanding safety and efficacy against laboratory and primary HIV isolates and against intravaginal murine herpes simplex virus 2 (HSV-2) challenge, making it a promising microbicide candidate. However, transient activity and instability remain concerns surrounding biologic delivery, particularly in the harsh environment of the female reproductive tract (FRT). Recently, electrospun fibers (EFs) have demonstrated promise for intravaginal delivery, with the potential to conserve active agent until release is needed. The goal of this study was to fabricate and characterize pH-responsive fibers comprised of poly(lactic-co-glycolic acid) (PLGA) or methoxypolyethylene glycol-b-PLGA (mPEG-PLGA) with varying ratios of poly(n-butyl acrylate-co-acrylic acid) (PBA-co-PAA), to selectively release GRFT under pH-conditions that mimic semen introduction. Fibers comprised of mPEG-PLGA:PBA-co-PAA (90:10 w/w) demonstrated high GRFT loading that was maintained within simulated vaginal fluid (SVF), and pH-dependent release upon exposure to buffered and SVF:simulated semen solutions. Moreover, GRFT fibers demonstrated potent in vitro efficacy against HIV-1 and safety in vaginal epithelial cells, suggesting their future potential for efficacious biologic delivery to the FRT.

Anti-HIV-1 antibody-dependent cellular cytotoxicity: is there more to antibodies than neutralization?

An increasing body of evidence suggests that nonneutralizing Fc effector functions including antibody-dependent cellular cytotoxicity (ADCC) contribute to protection against HIV-1 acquisition. We discuss recent advances in anti-HIV-1 ADCC research with a particular focus on ADCC mediated by Env-specific antibodies in vitro and in vivo, the curative potential of HIV-1-specific ADCC antibodies and the mechanisms of HIV-1 resistance to ADCC. ADCC activities of broadly neutralizing and nonneutralizing monoclonal antibody panels were recently characterized in vitro against several lab-adapted and primary isolates of HIV-1. ADCC activity of these monoclonal antibodies generally correlated with binding to infected cells and were greater against the lab-adapted strains compared with primary HIV-1 isolates. Several recent studies in mouse and macaque models of HIV-1 infection suggest Fc-mediated effector functions contribute to the protective efficacy of broadly neutralizing antibodies and exert immune pressure on HIV-1 in vivo. An increasing body of evidence suggests that ADCC-mediating antibodies, particularly when combined with neutralizing functions, can facilitate prevention and control of HIV-1. The precise mechanisms of partial protection conferred by nonneutralizing antibodies in vivo remain unclear and will need to be fully investigated in order to realize their full potential for HIV-1 vaccines.

Identification of Novel Structural Determinants in MW965 Env That Regulate the Neutralization Phenotype and Conformational Masking Potential of Primary HIV-1 Isolates.

The subtype C HIV-1 isolate MW965.26 is a highly neutralization-sensitive tier 1a primary isolate that is widely used in vaccine studies, but the basis for the sensitive neutralization phenotype of this isolate is not known. Substituting the MW965.26 V1/V2 domain into a neutralization-sensitive SF162 Env clone resulted in high resistance to standard anti-V3 monoclonal antibodies, demonstrating that this region possesses strong masking activity in a standard Env backbone and indicating that determinants elsewhere in MW965.26 Env are responsible for its unusual neutralization sensitivity. Key determinants for this phenotype were mapped by generating chimeric Envs between MW965.26 Env and a typical resistant Env clone, the consensus C (ConC) clone, and localized to two residues, Cys384 in the C3 domain and Asn502 in the C5 domain. Substituting the sensitizing mutations Y384C and K502N at these positions into several resistant primary Envs resulted in conversion to neutralization-sensitive phenotypes, demonstrating the generalizability of this effect. In contrast to the sensitizing effects of these substitutions on normally masked epitopes, these mutations reduced the sensitivity of VRC01-like epitopes overlapping the CD4-binding domain, while they had no effect on several other classes of broadly neutralizing epitopes, including members of several lineages of V2-dependent quaternary epitopes and representatives of N332 glycan-dependent epitopes (PGT121) and quaternary, cleavage-dependent epitopes centered at the gp41-gp120 interface on intact HIV-1 Env trimers (PGT151). These results identify novel substitutions in gp120 that regulate the expression of alternative conformations of Env and differentially affect the exposure of different classes of epitopes, thereby influencing the neutralization phenotype of primary HIV-1 isolates.IMPORTANCE A better understanding of the mechanisms that determine the wide range of neutralization sensitivity of circulating primary HIV-1 isolates would provide important information about the natural structural and conformational diversity of HIV-1 Env and how this affects the neutralization phenotype. A useful way of studying this is to determine the molecular basis for the unusually high neutralization sensitivities of the limited number of available tier 1a viruses. This study localized the neutralization sensitivity of MW965.26, an extremely sensitive subtype C-derived primary isolate, to two rare substitutions in the C3 and C5 domains and demonstrated that the sequences at these positions differentially affect the presentation of epitopes recognized by different classes of standard and conformation-dependent broadly neutralizing antibodies. These results provide novel insight into how these regions regulate the neutralization phenotype and provide tools for controlling the Env conformation that could have applications both for structural studies and in vaccine design.

Neutralizing Activity of Broadly Neutralizing Anti-HIV-1 Antibodies against Clade B Clinical Isolates Produced in Peripheral Blood Mononuclear Cells.

ABSTRACTRecently discovered broadly neutralizing antibodies (bNAbs) against HIV-1 demonstrate extensive breadth and potency against diverse HIV-1 strains and represent a promising approach for the treatment and prevention of HIV-1 infection. The breadth and potency of these antibodies have primarily been evaluated by using panels of HIV-1 Env-pseudotyped viruses produced in 293T cells expressing molecularly cloned Env proteins. Here we report on the ability of five bNAbs currently in clinical development to neutralize circulating primary HIV-1 isolates derived from peripheral blood mononuclear cells (PBMCs) and compare the results to those obtained with the pseudovirus panels used to characterize the bNAbs. The five bNAbs demonstrated significantly less breadth and potency against clinical isolates produced in PBMCs than against Env-pseudotyped viruses. The magnitude of this difference in neutralizing activity varied, depending on the antibody epitope. Glycan-targeting antibodies showed differences of only 3- to 4-fold, while antibody 10E8, which targets the membrane-proximal external region, showed a nearly 100-fold decrease in activity between published Env-pseudotyped virus panels and PBMC-derived primary isolates. Utilizing clonal PBMC-derived primary isolates and molecular clones, we determined that the observed discrepancy in bNAb performance is due to the increased sensitivity to neutralization exhibited by 293T-produced Env-pseudotyped viruses. We also found that while full-length molecularly cloned viruses produced in 293T cells exhibit greater sensitivity to neutralization than PBMC-derived viruses do, Env-pseudotyped viruses produced in 293T cells generally exhibit even greater sensitivity to neutralization. As the clinical development of bNAbs progresses, it will be critical to determine the relevance of each of these in vitro neutralization assays to in vivo antibody performance.IMPORTANCE Novel therapeutic and preventive strategies are needed to contain the HIV-1 epidemic. Antibodies with exceptional neutralizing activity against HIV-1 may provide several advantages to traditional HIV drugs, including an improved side-effect profile, a reduced dosing frequency, and immune enhancement. The activity of these antibodies has been established in vitro by utilizing HIV-1 Env-pseudotyped viruses derived from circulating viruses but produced in 293T cells by pairing Env proteins with a backbone vector. We tested PBMC-produced circulating viruses against five anti-HIV-1 antibodies currently in clinical development. We found that the activity of these antibodies against PBMC isolates is significantly less than that against 293T Env-pseudotyped viruses. This decline varied among the antibodies tested, with some demonstrating moderate reductions in activity and others showing an almost 100-fold reduction. As the development of these antibodies progresses, it will be critical to determine how the results of different in vitro tests correspond to performance in the clinic.

Characterization of Envelope Surface Glycoprotein from HIV-2 Primary Isolates with Different Coreceptor Usage Profile.

The main goal of this work was to identify molecular signatures in envelope surface glycoprotein that may be correlated with coreceptor usage by different human immunodeficiency virus (HIV)-2 strains. From inspection of aligned HIV-2 sequences, we verified that V1/V2 region showed the highest degree of amino acid sequence heterogeneity, including polymorphisms in N-linked glycosylation sites, sequence, and length. Furthermore, we did not find any correlation between the net charge and specific amino acid positions in V3 region with any particular coreceptor usage pattern. In conclusion, we showed that for HIV-2, the genetic determinants for coreceptor usage are distinct from those of HIV-1. More specifically, we did not identify any molecular signature, based on discrete amino acid positions either in V1/V2 or in V3 regions, which could be assigned to the preferential usage of a specific coreceptor.

HIV-1 Cross-Reactive Primary Virus Neutralizing Antibody Response Elicited by Immunization in Nonhuman Primates.

Elicitation of broadly neutralizing antibody (bNAb) responses is a major goal for the development of an HIV-1 vaccine. Current HIV-1 envelope glycoprotein (Env) vaccine candidates elicit predominantly tier 1 and/or autologous tier 2 virus neutralizing antibody (NAb) responses, as well as weak and/or sporadic cross-reactive tier 2 virus NAb responses with unknown specificity. To delineate the specificity of vaccine-elicited cross-reactive tier 2 virus NAb responses, we performed single memory B cell sorting from the peripheral blood of a rhesus macaque immunized with YU2gp140-F trimers in adjuvant, using JR-FL SOSIP.664, a native Env trimer mimetic, as a sorting probe to isolate monoclonal Abs (MAbs). We found striking genetic and functional convergence of the SOSIP-sorted Ig repertoire, with predominant VH4 or VH5 gene family usage and Env V3 specificity. Of these vaccine-elicited V3-specific MAbs, nearly 20% (6/33) displayed cross-reactive tier 2 virus neutralization, which recapitulated the serum neutralization capacity. Substantial similarities in binding specificity, neutralization breadth and potency, and sequence/structural homology were observed between selected macaque cross-reactive V3 NAbs elicited by vaccination and prototypic V3 NAbs derived from natural infections in humans, highlighting the convergence of this subset of primate V3-specific B cell repertories. Our study demonstrated that cross-reactive primary virus neutralizing B cell lineages could be elicited by vaccination as detected using a standardized panel of tier 2 viruses. Whether these lineages could be expanded to acquire increased breadth and potency of neutralization merits further investigation.IMPORTANCE Elicitation of antibody responses capable of neutralizing diverse HIV-1 primary virus isolates (designated broadly neutralizing antibodies [bNAbs]) remains a high priority for the vaccine field. bNAb responses were so far observed only in response to natural infection within a subset of individuals. To achieve this goal, an improved understanding of vaccine-elicited responses, including at the monoclonal Ab level, is essential. Here, we isolated and characterized a panel of vaccine-elicited cross-reactive neutralizing MAbs targeting the Env V3 loop that moderately neutralized several primary viruses and recapitulated the serum neutralizing antibody response. Striking similarities between the cross-reactive V3 NAbs elicited by vaccination in macaques and natural infections in humans illustrate commonalities between the vaccine- and infection-induced responses to V3 and support the feasibility of exploring the V3 epitope as a HIV-1 vaccine target in nonhuman primates.

GRL-09510, a Unique P2-Crown-Tetrahydrofuranylurethane -Containing HIV-1 Protease Inhibitor, Maintains Its Favorable Antiviral Activity against Highly-Drug-Resistant HIV-1 Variants in vitro

We report that GRL-09510, a novel HIV-1 protease inhibitor (PI) containing a newly-generated P2-crown-tetrahydrofuranylurethane (Crwn-THF), a P2′-methoxybenzene, and a sulfonamide isostere, is highly active against laboratory and primary clinical HIV-1 isolates (EC50: 0.0014–0.0028 μM) with minimal cytotoxicity (CC50: 39.0 μM). Similarly, GRL-09510 efficiently blocked the replication of HIV-1NL4-3 variants, which were capable of propagating at high-concentrations of atazanavir, lopinavir, and amprenavir (APV). GRL-09510 was also potent against multi-drug-resistant clinical HIV-1 variants and HIV-2ROD. Under the selection condition, where HIV-1NL4-3 rapidly acquired significant resistance to APV, an integrase inhibitor raltegravir, and a GRL-09510 congener (GRL-09610), no variants highly resistant against GRL-09510 emerged over long-term in vitro passage of the virus. Crystallographic analysis demonstrated that the Crwn-THF moiety of GRL-09510 forms strong hydrogen-bond-interactions with HIV-1 protease (PR) active-site amino acids and is bulkier with a larger contact surface, making greater van der Waals contacts with PR than the bis-THF moiety of darunavir. The present data demonstrate that GRL-09510 has favorable features for treating patients infected with wild-type and/or multi-drug-resistant HIV-1 variants, that the newly generated P2-Crwn-THF moiety confers highly desirable anti-HIV-1 potency. The use of the novel Crwn-THF moiety sheds lights in the design of novel PIs.