HIV-1 Neutralizing Research Articles

Broad and potent neutralization of HIV-1 by a gp41-specific human antibody.

Characterization of human monoclonal antibodies is providing considerable insight into mechanisms of broad HIV-1 neutralization. Here we report an HIV-1 gp41 membrane-proximal external region (MPER)-specific antibody, named 10E8, which neutralizes ~98% of tested viruses. An analysis of sera from 78 healthy HIV-1-infected donors demonstrated that 27% contained MPER-specific antibodies and 8% contained 10E8-like specificities. In contrast to other neutralizing MPER antibodies, 10E8 did not bind phospholipids, was not autoreactive, and bound cell-surface envelope. The structure of 10E8 in complex with the complete MPER revealed a site-of-vulnerability comprising a narrow stretch of highly conserved gp41-hydrophobic residues and a critical Arg/Lys just prior to the transmembrane region. Analysis of resistant HIV-1 variants confirmed the importance of these residues for neutralization. The highly conserved MPER is a target of potent, non-self-reactive neutralizing antibodies, suggesting that HIV-1 vaccines should aim to induce antibodies to this region of HIV-1 Env.

Cross-clade neutralization of HIV-1 by a monoclonal antibody obtained by immunization with liposomes containing lipid A and a synthetic MPER peptide

Background Development of vaccine formulations that induce cross clade neutralizing antibodies to the membrane proximal external region (MPER) of gp41 similar to 4E10 and 2F5 have been extensively investigated. We previously demonstrated that immunization with liposomes containing MPER and lipid A as an adjuvant could induce antibodies to the MPER and IgM monoclonal antibodies obtained from the mice bound to both lipids and MPER and neutralized HIV-1 (Matyas et al., 2009 AIDS 23, 2069-77). We now report a new IgG monoclonal antibody WR324 obtained from mice immunized with a similar vaccine formulation that binds to MPER and can neutralize multiple clades of HIV-1. Methods Mice were immunized with liposomes containing phosphatidyl inositol-4-phosphate and MPER with lipid A as an adjuvant. The peptide, LELDKWASLWNWFDITNWLWYIK (aa 661-684) was derived from gp41 (HXB2 stain). Spleen cells were fused with SP2/O cells and cloned. WR324 was purified and the binding specificities were assessed by ELISA. HIV-1 neutralization was assessed in PBMC using replication competent, Renila reniformis luciferase (LucR)-expressing HIV-1 reporter viruses (LucRIMC) and in a human monocyte derived macrophage (MDM) assay with purified viruses. Results WR324 is an IgG2b with a kappa light chain and it bound to the MPER peptide, but not to lipids or recombinant gp41. WR324 neutralized clade B, BaL and SF162, clade CRF01_AE, CM235, and clade C, GS 014 IMC viruses in the PBMC assay using two different donors. It also neutralized primary viruses, US-1 ,B aL, and clade CRF01_AE, M066 in the MDM assay using 3 different donors. WR324 did not bind to the surface of MDM cells, but binding to HIV-1 was observed by electron microscopy with immuno-gold labeling. Conclusion

Genetic characterization of HIV-1 subtype G envelope sequences by single genome analysis

Background Subtype G is the sixth most prevalent subtype of HIV-1 and is responsible for an estimated 1,500,000 infections worldwide. Although systematic analyses of a wide range of HIV-1 envelope sequences and neutralization have been performed, subtype G viruses are severely underrepresented in these studies. There is thus an important need to study subtype G envelope sequences and their neutralization capacities.

Cross-group neutralization of HIV-1 and evidence for conservation of the PG9/PG16 epitopes within divergent groups of HIV-1

Cross-group neutralization of HIV-1 and evidence for conservation of the PG9/PG16 epitopes within divergent groups of HIV-1

Broad and potent neutralization of HIV-1 by human-llama fusion antibodies derived from immunized llamas

Results Newly isolated VHH from multiple immunized llamas also have broad and potent HIV-1 neutralization activity. J3 targets HIV-1 via the CD4-binding site and neutralization is seen when J3 is used in combination with VHH targeting other Env epitopes. VHH-human FC fusion heavychain only antibodies (VHH-FC) have been constructed and J3 activity is not only preserved in this context but enhanced.

Recognition and penetration of the HIV-1 Env glycan shield by potent broadly neutralizing antibodies

Conclusion Structural characterization and biochemical analysis of these antibodies have uncovered novel specificities to new glycan-dependent epitopes and reveal further mechanisms for viral neutralization. These new epitopes provide additional insights for neutralization of HIV-1 and how antibodies can bind and penetrate the glycan shield, a novel framework for structure-assisted vaccine design. This study was supported by the International AIDS Vaccine Initiative (IAVI), Ragon Institute of MGH, MIT and Harvard, and NIH AI84817.

Deep sequencing with longitudinal sampling of a VRC01-like-antibody response in a chronically infected individual

Background VRC01-like antibodies use heavy chain mimicry of the CD4-receptor to achieve effective neutralization of HIV-1. The VRC01-like antibodies that have been observed in a number of HIV-1-infected individuals (i) display extensive somatic changes (70-100 nucleotide changes in VH-gene), (ii) can be detected only after several years of infection, (iii) derive from VH1-2, and (iv) are compatible with several different heavy J chains and different light chains. Methods To understand the persistence, evolution, and lineage of VRC01-like antibodies, we sampled PBMCs from donor 45, the source of VRC01 and VRC03 antibodies, at approximately yearly intervals over a 15-year period, and performed deep sequencing on the heavy and light chain variable portions of expressed antibodies. Anti-idiotypic antibodies were used to correlate mRNA levels of antibodies identified by the deep sequencing with expressed levels of these antibodies in serum. Results High expression levels of VRC01-like antibody sequences persisted over the entire 15-year period. Multiple lineages of VRC01-like antibodies were detected at each time point, and some of these, in particular the lineages that include VRC01 and VRC03, persisted over multiple time points, and displayed extensive branching in their evolution. Conclusion Deep sequencing provides a means to define the genetic record of the lineage and maturation of antibodies effective at neutralizing HIV-1. Precise definition of the natural ontogeny of broadly neutralizing antibodies may be essential in defining appropriate strategies to elicit such antibodies in vaccine settings.

HIV-1 neutralizing antibodies display dual specificity for the primary and coreceptor binding sites and preferential recognition of fully-cleaved Env

Background The gp120 CD4 binding site (CD4bs) and coreceptor binding site (CoRbs) are two functionally conserved elements of the HIV-1 envelope glycoproteins (Env). We previously defined the presence of CD4bs neutralizing antibodies (nAbs) in the serum of an HIV-1 infected individual and subsequently isolated the CD4bs-specific monoclonal antibodies (mAbs) VRC01 and VRC03 from the memory B cell population. From the same patient sera, we detected that there was also a fraction of potent nAbs specific for elements of the Env CoRbs by differential protein adsorption followed by neutralization analysis.

Isolation of broadly neutralizing HIV-1 antibodies from high-throughput single B cell culture

Background The isolation of broadly neutralizing HIV-1 antibodies that arise during infection has provided insights into the design of vaccine immunogens capable of eliciting similar antibody response. The use of HIV-specific sorting probes resulted in the isolation of antibodies to vulnerable viral epitopes, such as the CD4-binding site, but the use of such probes does not explore the subject’s immunoglobulin repertoire breadth. Methods To identify new HIV-1 monoclonal antibodies (mAbs), we developed a B-cell culture system to isolate and screen thousands of B cells. Memory B cells were isolated by negative selection and individually cultured in 384 well plates with irradiated feeder cells expressing CD40 ligand. The addition of cytokines IL-2 and IL-21 stimulated proliferation and immunoglobulin secretion. Results After 14 days in culture, approximately 35% of the B cell clones secreted >100 ng/ul of IgG which met the sensitivity threshold to screen each clone in an automated micro-neutralization assay. B cell clonal expansion allowed recovery of the immunoglobulin heavy and light chains by RT-PCR, with subsequent cloning into expression vectors and mAb testing against a large panel of viruses. In one experiment screening approximately 8600 B cells, 9 clones were identified as potential contributors to the neutralization detected in the patient’s serum. One of the isolated clones produced mAb VRC22 with 30% neutralization breadth and moderate potency. Further studies revealed that VRC22 was sensitive to JRCSF glycan mutants N332A and N301A but not N160K. VRC22 utilizes VH4-34 with a single amino acid CDR1 deletion and a mutation frequency of 7% which is a lower level of affinity maturation than observed for most known HIV-1 neutralizing antibodies. Conclusion This B-cell culture system allows efficient screening of thousands of individual B cells and the recovery of antigen specific mAbs. This approach can be used to isolate human mAbs to diverse pathogens.

Analysis of gp41 epitopes in model viral membranes to study HIV-1 neutralization

Background The membrane proximal external region (MPER) of the HIV surface glycoprotein gp41 is the binding site for several potent broadly neutralizing antibodies. These antibodies recognize highly conserved linear peptide epitopes and antigens derived from this region have the potential to be important components of a vaccine directed against HIV-1. Highly conserved peptide epitopes for these antibodies are sterically obscured from the immune system by an intimate association with the viral membrane and by the presence of a large trimeric glycoprotein spike. As a result, previous attempts to immunize with linear peptides have failed to elicit broadly neutralizing antibodies.

The impact of differences in viral entry and trafficking on the mechanism of HIV-1 neutralization

Background Cellular entry of HIV-1 occurs through fusion at the plasma membrane (PM) or via endocytosis. PM fusion, resulting in deposit of nucleocapsid into the cytoplasm, typically establishes productive infection. In contrast, entry through endocytosis results in either lysosomal degredation, or productive infection through endosomal membrane fusion. While viral entry and permissivity are known to differ between host cells, the influence of these differences on antibody-mediate neutralization is unknown. Here we explore the relationships between HIV-1 entry, neutralization sensitivity, and the mechanisms by which antibodies act.

Antibody-dependent cellular cytotoxicity-mediating antibodies from an HIV-1 vaccine efficacy trial preferentially use the VH1 gene family

Background The ALVAC-HIV/AIDSVAX-B/E RV144 vaccine efficacy trial showed an estimated efficacy of 31%. The immune correlates analysis raised the hypothesis that the observed protection in RV144 may be partially due to AntibodyDependent Cellular Cytotoxicity (ADCC)-mediating antibodies in the presence of low levels of Env IgA antibodies. In this study we analyzed the Ig VH family usage of vaccine-induced ADCC mAbs isolated from memory B cells of vaccinees. Methods From a total of 321,945 memory B-cells of 6 vaccinees we obtained 23 mAbs that mediated ADCC using IgG+ memory B-cell cultures (n=9) and Env-specific flow cytometric single memory B-cell sorting (n=14). ADCC activity was measured using both E.CM243 gp120-coated and E. CM235-infected target cells in a flow-based assay. Results ADCC-mediating mAbs displayed a disproportionate usage of VH1 family genes (17/23; 74%), in particular the VH1-2 gene segment (10/17; 59%), as recently observed for CD4bs broadly neutralizing antibodies (HAAD bNAbs). In contrast, only 17.1% of 111 heavy chains isolated from cultures that did not mediate ADCC used the VH1 gene. VH1 ADCC-mediating mAbs showed a high degree of V(D)J amino acid similarity to both the VH (68-84%) and VL (70-87%) HAAD motifs. V(D)J rearrangements displayed modest levels of affinity maturation (0.5-5.1% for heavy chains and 0.4-4.3% for light chains). While none of the VH1 ADCC-mediating mAbs was capable of mediating HIV-1 neutralization, the strength of their ADCC activity correlated with the levels of heavy chain somatic mutations (p=0.02). We produced the reverted unmutated ancestor antibodies of two VH1 ADCC-mediating mAbs: one bound to B.MN Env and both reacted against autoantigens. Conclusion ADCC-mediating antibodies induced by the ALVAC-HIV/ AIDSVAX-B/E vaccine underwent limited affinity maturation, and preferentially used VH1 gene segments which share the HAAD motif with CD4bs bNAbs. These observations raise the hypothesis that HIV-1 Env preferentially selects VH1 family usage for distinct subsets of antibodies with different functions.

Optimization and validation of the HIV-1 neutralizing antibody assay in A3R5 cells

Background A3R5 is a highly sensitive cell line for the detection of neutralizing antibodies (Nabs) against tier 2 strains of HIV-1. This cell line is particularly useful for the detection of weak Nab responses in preclinical and clinical trials of candidate HIV-1 vaccines. All methods used for endpoint analyses in clinical trials should be validated and demonstrably fit for purpose, in compliance with ICH Q2 (R1) guidelines. Here we describe the optimization/qualification and validation of the HIV-1 Nab Assay in A3R5 cells.

A gp41 MPER-specific llama VHH requires a hydrophobic CDR3 determinant for neutralization but not for antigen recognition

The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10.

Impact of IgA constant domain on HIV-1 neutralizing function of monoclonal antibody F425-A1g8

Impact of IgA constant domain on HIV-1 neutralizing function of monoclonal antibody F425-A1g8

Potency of an HIV-SAM™ vaccine in a heterologous prime-boost vaccination regimen

Results We evaluated systemic and mucosal immune responses in mice and rabbits using the SAMTM platform expressing HIV-1 gp140 (HIV-SAMTM vaccine) prime, protein/MF59 vaccine boost regimen for both HIV-1 Clade B and C Env antigens. In mice, the primed Env-specific IgG response to 1 μg of the HIV-SAMTM vaccine was comparable to a 10 μg dose of an identically formulated DNA vaccine, 10(7) IU of VRP, and 10 μg protein/MF59 vaccines. The HIVSAMTM vaccine primed response could be boosted robustly by a protein/MF59 vaccine and resulted in a balanced IgG1, IgG2a subclass response, similar to that seen with the VRP vaccine, but unlike the dominant IgG1 response to protein/MF59 only vaccinations. Both Envspecific CD4+ and CD8+ T-cell responses were detectable after two HIV-SAMTM vaccinations. A TH1 type (IFNg+, IL-5-) profile was demonstrable for the HIV-SAMTM vaccine primed, protein boosted CD4+ T-cell response, similar to that seen with the DNA or VRP primed protein boosted responses, in contrast to a TH2 type (IFNglow, IL-5+) response seen with protein/MF59 vaccination. In rabbits, priming with the 25 or 50 μg of the formulated HIV-SAMTM vaccine induced robust and avid Env-binding IgG and HIV neutralizing antibodies that were superior to 500 μg of an unformulated DNA vaccine and comparable to VRP and protein/MF59 vaccines. In addition, protein/ MF59 boostable Env-specific vaginal wash Ig was consistently demonstrable in both mice and rabbits immunized with the HIV-SAMTM.

Structural definition of a novel CD4-induced epitope that is targeted by a single-headed immunoglobulin to effect broad and potent HIV neutralization

Background HIV-1 enters cells by sequentially binding the CD4 receptor and a coreceptor, either CCR5 or CXCR4. Functional constraints result in a high degree of conservation of the receptor-binding sites making them potential targets for intervention. The coreceptor-binding site on HIV-1 envelope gp120 glycoprotein is protected from the humoral immune system by conformational masking and steric occlusion. The site becomes available after a conformational change in gp120 following CD4 engagement, but at that point in the entry process, the proximity of the viral and cellular membranes makes the site inaccessible to bulky antibody molecules. Thus, in spite of being highly conserved, this region has not been considered a viable vaccine target.

Enhanced Recognition and Neutralization of HIV-1 by Antibody-Derived CCR5-Mimetic Peptide Variants

A tyrosine-sulfated CCR5-mimetic peptide, CCR5mim1, inhibits HIV-1 infection more efficiently than sulfopeptides based on the CCR5 amino terminus. Here we characterized sulfopeptide chimeras of CCR5mim1 and the heavy-chain CDR3 of the antibody PG16. Two chimeras bound a range of envelope glycoproteins and neutralized HIV-1 more efficiently than CCR5mim1. An immunoadhesin form of one of these, CCR5mim2-Ig, synergized with CD4-Ig to neutralize HIV-1. These sulfopeptides are among the broadest and most potent CCR5-mimetic peptides described to date.

Ultrasensitive analysis of binding affinity of HIV receptor and neutralizing antibodies using solution-phase electrochemiluminescence assay

Binding of a few ligand molecules with its receptors on cell surface can initiate cellular signaling transduction pathways, and trigger viral infection of host cells. HIV-1 infects host T-cells by binding its viral envelope protein (gp120) with its receptor (a glycoprotein, CD4) on T cells. Primary strategies to prevent and treat HIV infection is to develop therapies (e.g., neutralizing antibodies) that can block specific binding of CD4 with gp120. The infection often leads to the lower counts of CD4 cells, which makes it an effective biomarker to monitor the AIDS progression and treatment. Despite research over decades, quantitative assays for effective measurements of binding affinities of protein–protein (ligand–receptor, antigen–antibody) interactions remains highly sought. Solid-phase electrochemiluminescence (ECL) immunoassay has been commonly used to capture analytes from the solution for analysis, which involves immobilization of antibody on solid surfaces (micron-sized beads), but it cannot quantitatively measure binding affinities of molecular interactions. In this study, we have developed solution-phase ECL assay with a wide dynamic range (0–2nM) and high sensitivity and specificity for quantitative analysis of CD4 at femtomolar level and their binding affinity with gp120 and monoclonal antibodies (MABs). We found that binding affinities of CD4 with gp120 and MAB (Q4120) are 9.5×108 and 1.2×109M−1, respectively. The results also show that MAB (Q4120) of CD4 can completely block the binding of gp120 with CD4, while MAB (17b) of gp120 can only partially block their interaction. This study demonstrates that the solution-phase ECL assay can be used for ultrasensitive and quantitative analysis of binding affinities of protein–protein interactions in solution for better understating of protein functions and identification of effective therapies to block their interactions.

Structural elements of primary CCR5-using HIV-1 gp120 proteins influencing sensitivity and resistance to the broadly neutralizing monoclonal antibody b12

Structure-guided approaches to HIV-1 vaccine design depend on knowledge of the presentation of neutralizing epitopes on gp120, such as the epitope for the broadly neutralizing mAb b12. Here, we characterized predicted three-dimensional structures of functionally diverse gp120 proteins in their b12-bound conformation, to better understand the gp120 determinants that expose or occlude the b12 epitope. Mapping the gp120–b12 binding interface identified amino acid polymorphisms within the C2, C3, C4 and V5 regions of gp120 associated with augmented b12 binding, and importantly, identified residues in the b12-exclusive binding domain of gp120 that are important for b12 neutralization resistance. Structural studies suggest that these b12 resistance variants promote reduced conformational flexibility in the b12 recognition site, which we show involves structural alterations within the gp120 CD4 binding loop and the V4 loop. Together, our studies provide new mechanistic insights into the gp120 determinants influencing sensitivity and resistance to HIV-1 neutralization by b12.