Soluble CD4 broadens neutralization of V3-directed monoclonal antibodies and guinea pig vaccine sera against HIV-1 subtype B and C reference viruses

To better understand the limits of antigenic reactivity and epitope accessibility of the V3 domain of primary HIV-1 isolates, we evaluated three human anti-V3 monoclonal antibodies (mAbs) and selected guinea pig vaccine sera for neutralization against reference panels of subtype B and C pseudoviruses derived from early stage infections. The mAbs and vaccine sera potently neutralized several prototype viruses, but displayed substantially less neutralization of most reference strains. In the presence of soluble CD4 (sCD4), the breadth of V3-mediated neutralization was increased; up to 80% and 77% of the subtype B and C viruses respectively were sensitive to V3-mediated neutralization. Unlike sCD4, the reaction of CD4-binding site mAbs b12 and F105 with native virus did not lead to full exposure of the V3 domain. These findings confirm that V3 antibodies recognize most primary viral strains, but that the epitope often has limited accessibility in the context of native envelope spike.     

Cross-clade neutralizing antibodies against HIV-1 induced in rabbits by focusing the immune response on a neutralizing epitope

Studies were performed to induce cross-clade neutralizing antibodies (Abs) by testing various combinations of prime and boost constructs that focus the immune response on structurally-conserved epitopes in the V3 loop of HIV-1 gp120. Rabbits were immunized with gp120 DNA containing a V3 loop characterized by the GPGR motif at its tip, and/or with gp120 DNA with a V3 loop carrying the GPGQ motif. Priming was followed by boosts with V3-fusion proteins (V3-FPs) carrying the V3 sequence from a subtype B virus (GPGR motif), and/or with V3 sequences from subtypes A and C (GPGQ motif). The broadest and most consistent neutralizing responses were generated when using a clade C gp120 DNA prime and with the V3_B-FP boost. Immune sera displayed neutralizing activity in three assays against pseudoviruses and primary isolates from subtypes A, AG, B, C, and D. Polyclonal Abs in the immune rabbit sera neutralized viruses that were not neutralized by pools of human anti-V3 monoclonal Abs. Greater than 80% of the neutralizing Abs were specific for V3, showing that the immune response could be focused on a neutralizing epitope and that vaccine-induced anti-V3 Abs have cross-clade neutralizing activity.     

A group M consensus envelope glycoprotein induces antibodies that neutralize subsets of subtype B and C HIV-1 primary viruses

HIV-1 subtype C is the most common HIV-1 group M subtype in Africa and many parts of Asia. However, to date HIV-1 vaccine candidate immunogens have not induced potent and broadly neutralizing antibodies against subtype C primary isolates. We have used a centralized gene strategy to address HIV-1 diversity and generated a group M consensus envelope gene with shortened consensus variable loops (CON-S) for comparative studies with wild-type (WT) Env immunogens. Our results indicate that the consensus HIV-1 group M CON-S Env elicited cross-subtype neutralizing antibodies of similar or greater breadth and titer than the WT Envs tested, indicating the utility of a centralized gene strategy. Our study also shows the feasibility of iterative improvements in Env immunogenicity by rational design of centralized genes.     

人类免疫缺陷病毒包膜糖蛋白V3区对细胞结合能力的研究

目的：研究人类免疫缺陷病毒（ＨＩＶ）不同亲嗜株包膜糖蛋白Ｖ３区结合于靶细胞的能力。方法：合成来源于不同嗜性ＨＩＶ－１Ｖ３区的生物素标记和非标记的多肽。采用流式细胞计数分析生物素化的 Ｖ３多肽对细胞的结合能力以及细胞表面的结合配体。结果：ＨＩＶ－１Ｘ４　亲嗜株ＩＩＩＢＶ３区能结合于多种细胞的表面，包括辅助受体ＣＸＣＲ４；竞争实验结果显示蛋白酶抑制剂能抑制该结合。Ｒ５亲嗜株 ＡＤＡ Ｖ３区只极微弱地结合于外周血单核细胞和表达ＣＣＲ５ 的人星形胶质细胞表面。结论：不同嗜性ＨＩＶ－１Ｖ３区结合于细胞表面的能力不同从亲嗜株Ｖ３区直接结合于细胞表面并被其自身所增强，其靶分子至少包括辅助受体 ＣＸＣＲ４和蛋白酶分子；而Ｒ５亲嗜株 ＡＤＡ Ｖ３区则不结合于 ＣＣＲ５和蛋白酶。     

Permissive residues within the minimal epitopes of neutralizing monoclonal antibodies to the V3 loop of HIV-1

Neutralizing monoclonal antibodies (mAb) specific for the third variable (V3) domain of gp120, the HIV-1 surface envelope protein, are mainly isolate specific. We have studied the composition and the permissivity of the minimal epitopes interacting with two of these, the 110-A and 19.26.4 mAb, which are strictly LAI isolate specific. Screening a hexapeptide phage library displayed on the surface of filamentous phage with the 110-A mAb has allowed selection of 49 phage sequences, permitting the definition of a consensus sequence. Based on this sequence, substituted synthetic peptides were prepared and used in binding assays. Our results show that both mAb interact with the same narrow region (316–320) of the V3 domain. The minimal epitope of the 110-A mAb was identified as a five amino acid sequence, Hy x R G p, where Hy represents any non-aromatic hydrophobic amino acid. By contrast, the minimal epitope of the 19.26.4 mAb was identified as x Q Pos G P, where Pos is any positively charged amino acid. Core residues of the epitope, critical for the binding to the mAb (written in uppercase letters), were set apart from permissive amino acid positions that tolerate substitutions (written in lowercase letters). Interestingly, the identified core residues Q_2/317 (19.26.4 mAb) and R_3/318 (110-A mAb) do not tolerate substitution and correspond to the QR insertion in the V3 domain, characteristic of the LAI isolate as compared to other isolates. This result may explain the strict isolate specificity of most anti-V3 LAI mAb. The two epitopes have totally different patterns of permissivity; thus, the effect of substitutions will differ depending on the mAb involved in the interaction. This suggests that the diversity of the antibody response is high enough to delay the emergence of HIV-1 variants resistant to neutralization by V3-specific antibodies.     

Functional and genetic analysis of coreceptor usage by dualtropic HIV-1 subtype C isolates

It is widely documented that a complete switch from the predominant CCR5 (R5) to CXCR4 (X4) phenotype is less common for HIV-1 subtype C (HIV-1C) compared to other major subtypes. We investigated whether dualtropic HIV-1C isolates represented dualtropic, mixed R5 and X4 clones or both. Thirty of 35 functional HIV-1 env clones generated by bulk PCR amplification from peripheral blood mononuclear cells (PBMCs) infected with seven dualtropic HIV-1C isolates utilized CXCR4 exclusively. Five of 35 clones displayed dualtropism. Endpoint dilution of one isolate did not yield a substantial proportion of R5-monotropic env clones. Sequence-based predictive algorithms showed that env sequences from PBMCs, CXCR4 or CCR5-expressing cell lines were indistinguishable and all possessed X4/dualtropic characteristics. We describe HIV-1C CXCR4-tropic env sequence features. Our results suggest a dramatic loss of CCR5 monotropism as dualtropism emerges in HIV-1C which has important implications for the use of coreceptor antagonists in therapeutic strategies for this subtype.     

Molecular and structural characterization of HIV-1 subtype B Brazilian isolates with GWGR tetramer at the tip of the V3-loop

One of most intriguing features of the HIV-1 subtype B epidemic in Brazil is the high frequency of isolates exhibiting tryptophan (W) in the tetramer (GWGR) at the tip of the V3 loop. We observed that the frequencies of glutamic and aspartic acids at site 25 of the V3 loop are quite distinct in GWGR isolates compared with viruses with other tetramers. The basic amino acids at sites 11 and 25 of V3 are strongly linked with CCR5-to-CXCR4 coreceptor shift. We therefore predicted phenotype usage and found that GWGR isolates are exclusively CCR5-using. Further evidence of this came from intrahost sequences, where basic amino acid substitutions at sites 11 and 25 emerged only in isolates presenting a tryptophan-to-glycine replacement at the tetramer of the V3. In addition, modeled 3D-structures of the V3 loop of GWGR and GGGR in intrahost viruses differ essentially in the binding region of the coreceptor.     

Antigenicity and immunogenicity of HIV-1 consensus subtype B envelope glycoproteins

"Centralized" (ancestral and consensus) HIV-1 envelope immunogens induce broadly cross-reactive T cell responses in laboratory animals; however, their potential to elicit cross-reactive neutralizing antibodies has not been fully explored. Here, we report the construction of a panel of consensus subtype B (ConB) envelopes and compare their biologic, antigenic, and immunogenic properties to those of two wild-type Env controls from individuals with early and acute HIV-1 infection. Glycoprotein expressed from full-length (gp160), uncleaved (gp160-UNC), truncated (gp145), and N-linked glycosylation site deleted (gp160-201N/S) versions of the ConB env gene were packaged into virions and, except for the fusion defective gp160-UNC, mediated infection via the CCR5 co-receptor. Pseudovirions containing ConB Envs were sensitive to neutralization by patient plasma and monoclonal antibodies, indicating the preservation of neutralizing epitopes found in contemporary subtype B viruses. When used as DNA vaccines in guinea pigs, ConB and wild-type env immunogens induced appreciable binding, but overall only low level neutralizing antibodies. However, all four ConB immunogens were significantly more potent than one wild-type vaccine at eliciting neutralizing antibodies against a panel of tier 1 and tier 2 viruses, and ConB gp145 and gp160 were significantly more potent than both wild-type vaccines at inducing neutralizing antibodies against tier 1 viruses. Thus, consensus subtype B env immunogens appear to be at least as good as, and in some instances better than, wild-type B env immunogens at inducing a neutralizing antibody response, and are amenable to further improvement by specific gene modifications.     

Neutralizing antibodies mechanism of neutralization and protective activity against HIV-1

The role of the humoral immune response in prevention against HIV-1 infection is still incompletely understood. However, neutralizing antibodies to certain epitopes on HIV-1 envelope glycoproteins inhibit HIV-1 infection in vitro and in vivo. Passive administration of these antibodies by themselvesor in combination completely protected hu-PBL-SCID mice or macaques from intravenous, vaginal, as well as maternal-fetal mucosal transmission. All these studies provide direct experimental evidence that neutralizing antibodies are potentenough to prevent HIV infection, and strongly suggest that neutralizing-antibody-based vaccines could provide effective protection against HIV-1, despite the potent action of CTLs. Some neutralizing epitopes have been defined in vitro and in vivo. Unfortunately, none of the neutralizing-antibody-based candidate vaccines has been demonstrated to induce enough protective activity. Weak antigenicity and immunogenicity of neutralizing epitopes on native or recombinant proteins and other factors made it difficult to induce neutralizing-epitope-specific antibody responses in vivo enough to prevent against primary isolates. Recent studies indicated that HIV-1 variations resulted in escape from neutralization or the CTL responses, which may be the principal challenge for HIV-1 prevention. Epitope vaccine as a new strategy activating both arms of the immune system, namely, using the “principal neutralizing epitopes” and the CTL epitopes in combination, should provide new hope for developing an effective vaccine to halt the HIV-1 epidemic.     

High titer HIV-1 V3-specific antibodies with broad reactivity but low neutralizing potency in acute infection and following vaccination

Identifying the earliest neutralizing antibody specificities that are elicited following infection or vaccination by HIV-1 is an important objective of current HIV/AIDS vaccine research. We have shown previously that transplantation of HIV-1 V3 epitopes into an HIV-2 envelope (Env) scaffold provides a sensitive and specific means to detect and quantify HIV-1 V3 epitope specific neutralizing antibodies (Nabs) in human sera. Here, we employ this HIV-2/HIV-1 V3 scaffolding strategy to study the kinetics of development and breadth of V3-specific Nabs in longitudinal sera from individuals acutely infected with clade C or clade B HIV-1 and in human subjects immunized with clade B HIV-1 immunogens. HIV-2/HIV-1 chimeras containing V3 sequences matched to virus type (HIV-2 or HIV-1), subtype (clade B or C), or strain (autologous or heterologous) were used as test reagents. We found that by 3-8 weeks post infection, 12 of 14 clade C subjects had a median IC₅₀ V3-specific Nab titer of 1:700 against chimeric viruses containing a heterologous clade C V3. By 5 months post-infection, all 14 subjects were positive for V3-specific Nabs with median titers of 1:8000 against heterologous clade C V3 and 1:1300 against clade B V3. Two acutely infected clade B patients developed heterologous clade B V3-specific Nabs at titers of 1:300 and 1:1800 by 13 weeks of infection and 1:5000 and 1:11000 by 7 months of infection. Titers were not different against chimeras containing autologous clade B V3 sequences. Each of 10 uninfected normal human volunteers who were immunized with clade B HIV-1 Env immunogens, but none of five sham immunized control subjects, developed V3-specific Nabs titers as high as 1:3000 (median 1:1300; range 1:700-1:3000). None of the HIV-1 infected or vaccinated subjects had antibodies that neutralized primary HIV-1 virus strains. These results indicate that high-titer, broadly reactive V3-specific antibodies are among the first to be elicited during acute and early HIV-1 infection and following vaccination but these antibodies lack neutralizing potency against primary HIV-1 viruses, which effectively shield V3 from antibody binding to the functional Env trimer.     

Genotypic and phenotypic analysis of the env gene from South African HIV-1 subtype B and C isolates

The objective of the study was to assess the genotypic and phenotypic properties of 18 viral strains from human immunodeficiency virus-1 (HIV-1) positive patients and to identify subtype C isolates for vaccine design strategies. All the isolates were non-syncytium-inducing (NSI) in both the primary and MT-2 cell cultures. The amino acid charge of the V3 loop correlated with the NSI phenotype of the strains. The V3 competitive peptide enzyme immunoassay and DNA sequencing of the partial gp120 region gave concordant results on the 15 subtype C strains, whereas the three B genotypes gave a positive to B, a nonreactive to B, and a dual reaction to the B-D peptides, respectively. Sixteen of the isolates used only CCR5 as coreceptor whereas two isolates made use of additional coreceptors including CXCR4. In summary, all our subtype C isolates are NSI phenotypically and almost all of them use CCR5 exclusively as their coreceptor.     

Structure-based,targeted deglycosylation of HIV-1 gp120 and effects on neutralization sensitivity and antibody recognition

The human immunodeficiency virus (HIV-1) exterior envelope glycoprotein, gp120, mediates receptor binding and is the major target for neutralizing antibodies. Primary HIV-1 isolates are characteristically more resistant to broadly neutralizing antibodies, although the structural basis for this resistance remains obscure. Most broadly neutralizing antibodies are directed against functionally conserved gp120 regions involved in binding to either the primary virus receptor, CD4, or the viral coreceptor molecules that normally function as chemokine receptors. These antibodies are known as CD4 binding site (CD4BS) and CD4-induced (CD4i) antibodies, respectively. Inspection of the gp120 crystal structure reveals that although the receptor-binding regions lack glycosylation, sugar moieties lie proximal to both receptor-binding sites on gp120 and thus in proximity to both the CD4BS and the CD4i epitopes. In this study, guided by the X-ray crystal structure of gp120, we deleted four N-linked glycosylation sites that flank the receptor-binding regions. We examined the effects of selected changes on the sensitivity of two prototypic HIV-1 primary isolates to neutralization by antibodies. Surprisingly, removal of a single N-linked glycosylation site at the base of the gp120 third variable region (V3 loop) increased the sensitivity of the primary viruses to neutralization by CD4BS antibodies. Envelope glycoprotein oligomers on the cell surface derived from the V3 glycan-deficient virus were better recognized by a CD4BS antibody and a V3 loop antibody than were the wild-type glycoproteins. Absence of all four glycosylation sites rendered a primary isolate sensitive to CD4i antibody-mediated neutralization. Thus, carbohydrates that flank receptor-binding regions on gp120 protect primary HIV-1 isolates from antibody-mediated neutralization.     

Use of broadly neutralizing antibodies for HIV-1 prevention

Antibodies have a long history in antiviral therapy, but until recently, they have not been actively pursued for HIV-1 due to modest potency and breadth of early human monoclonal antibodies (MAbs) and perceived insurmountable technical, financial, and logistical hurdles. Recent advances in the identification and characterization of MAbs with the ability to potently neutralize diverse HIV-1 isolates have reinvigorated discussion and testing of these products in humans, since new broadly neutralizing MAbs (bnMAbs) are more likely to be effective against worldwide strains of HIV-1. In animal models, there is abundant evidence that bnMAbs can block infection in a dose-dependent manner, and the more potent bnMAbs will allow clinical testing at infusion doses that are practically achievable. Moreover, recent advances in antibody engineering are providing further improvements in MAb potency, breadth, and half-life. This review summarizes the current state of the field of bnMAb protection in animal models as well as a review of variables that are critical for antiviral activity. Several bnMAbs are currently in clinical testing, and we offer perspectives on their use as pre-exposure prophylaxis (PrEP), potential benefits beyond sterilizing immunity, and a discussion of future approaches to engineer novel molecules.     

Isolation and characterization of monoclonal antibodies elicited by trimeric HIV-1 Env gp140 protein immunogens

Eleven anti-HIV Env monoclonal antibodies (MAbs) were isolated from mice immunized with soluble Env proteins derived from the clade B Env, SF162, or DeltaV2 (a derivative of SF162 lacking the V2 loop). All six anti-gp120 MAbs studied, neutralized SF162 and their activities were dependent by the glycosylation patterns of the V1, V2 or V3 loops. Only one anti-gp120 MAb (an anti-V3 MAb) displayed cross-neutralizing activity, which was influenced by the type of V1 loop present on the target heterologous viruses. None of the five anti-gp41 MAbs studied displayed anti-SF162 neutralizing activity. Our studies indicate that the current limitation of soluble HIV Env gp140 immunogens to elicit robust cross-reactive neutralizing antibody responses is not only due to the elicitation of high titers of homologous antibodies but also due to the elicitation of antibodies whose epitopes are naturally occluded, or not present, on the virion-associated Env.     

Structure-guided design and immunological characterization of immunogens presenting the HIV-1 gp120 V3 loop on a CTB scaffold

V3 loop is a major neutralizing determinant of the HIV-1 gp120. Using 3D structures of cholera toxin B subunit (CTB), complete V3 in the gp120 context, and V3 bound to a monoclonal antibody (mAb), we designed two V3-scaffold immunogen constructs (V3-CTB). The full-length V3-CTB presenting the complete V3 in a structural context mimicking gp120 was recognized by the large majority of our panel of 24 mAbs. The short V3-CTB presenting a V3 fragment in the conformation observed in the complex with the 447-52D Fab, exhibited high-affinity binding to this mAb. The immunogens were evaluated in rabbits using DNA-prime/protein-boost protocol. Boosting with the full-length V3-CTB induced high anti-V3 titers in sera that potently neutralize multiple HIV virus strains. The short V3-CTB was ineffective. The results suggest that very narrow antigenic profile of an immunogen is associated with poor Ab response. An immunogen with broader antigenic activity elicits robust Ab response.     

Characterization and large production of human monoclonal antibodies against the HIV-1 envelope.

Peripheral blood lymphocytes from a volunteer immunized with a recombinant vaccinia virus VSC-25 expressing the gp160 env protein of HTLV-IIIB strain and from an asymptomatic HIV-infected individual were immortalized by Epstein-Barr (EBV). Clones which secrete human monoclonal antibodies from the two individuals (DZ, IgG1, lambda and C31, IgG1, kappa) were obtained and were stable for more than 2 years. The two monoclonals were directed against the gp160 env protein of HIV, DZ directed against the gp41 and C31 directed against the gp120. C31 was group-specific, whereas DZ was directed against the HTLV-IIIB and HTLV-RF strains. The epitope recognized by DZ was mapped to the carboxy terminus of the gp41, by expression of HIV DNA fragments in a yeast system and peptide analysis. The C31 epitope was not expressed by the yeast library and not present among the peptides which were tested. Monoclonal antibodies had no inhibitory effect in an HIV-induced cell fusion assay, but DZ showed a weak neutralizing activity against the HTLV-IIIB strain. Cloned EBV-transformed cell lines were fused to a murine myeloma, which allowed the heteromyeloma to be cultivated in serum-free medium. The monoclonal antibodies were produced in large quantity in a hollow-fibre reactor at defined culture conditions and purification procedures.     

HIV-1 determinants of thrombocytopenia at the stage of CD34+ progenitor cell differentiation in vivo lie in the viral envelope gp120 V3 loop region

HIV-1 V3 loop clones of virus isolates derived from patients suffering from thrombocytopenia were used for infection of the human thymus/liver conjoint hematopoietic organ that developed in the severe combined immunodeficient mouse (SCID-hu Thy/Liv). The V3 loop clones showed a significantly greater degree of inhibition of megakaryopoiesis than myelopoiesis and erythropoiesis of the human CD34+ progenitor cells, in vivo. Inhibition of megakaryopoiesis occurs through reduction in c-Mpl expression and consequent decrease in STAT5 activation. Therefore HIV-1 V3 loop sequences of thrombocytopenic patients exhibit preferential inhibition of megakaryocyte lineage-specific differentiation of CD34+ progenitor cells, thus reflecting the patients' clinical condition.     

Mutations in the V3 stem versus the V3 crown and C4 region have different effects on the binding and fusion steps of human immunodeficiency virus type 1 gp120 interaction with the CCR5 coreceptor

The current model for HIV-1 envelope-coreceptor interaction depicts the V3 stem and bridging sheet binding to the CCR5 N-terminus while the V3 crown interacts with the second extracellular loop, which is the coreceptor domain that appears to be relatively more important for fusion and infection. Our prediction based on this model is that mutations in the V3 crown might consequently have more effects on cell-cell fusion and virus entry than mutations introduced in the V3 stem and C4 region. We performed alanine-scanning of the V3 loop and selected C4 residues in the JRFL envelope and tested the capacity of the resulting mutants for CCR5 binding, cell-cell fusion, and virus infection. Our cross comparison analysis revealed that residues in C4 and in both the V3 stem and crown were important for CCR5 binding of gp120 subunits. Contrary to our prediction, mutations in the V3 crown had less effect on membrane fusion than mutations in the V3 stem. The V3 stem thus appears to be the most important region for CCR5 utilization since it affected both coreceptor binding and subsequent fusion and viral entry. Our data raises the possibility that some residues in the V3 crown and in C4 may play distinct roles in the binding and fusion steps of envelope-coreceptor interaction.     

Focusing the immune response on the V3 loop, a neutralizing epitope of the HIV-1 gp120 envelope

Rabbits were immunized with a novel regimen designed to focus the immune response on a single neutralizing epitope of HIV-1 gp120 and thereby preferentially induce neutralizing antibodies (Abs). Animals were primed with gp120 DNA from a clade A Env bearing the GPGR V3 motif and/or a clade C Env bearing the GPGQ V3 motif, and boosted with one or more fusion proteins containing V3 sequences from clades A, B and/or C. Immune sera neutralized three of four Tier 1 primary isolates, including strains heterologous to the immunizing strains, and potent cross-clade-neutralizing activity was demonstrated against V3 chimeric pseudoviruses carrying in a Tier 1 Env, the consensus V3 sequences from clades A1, AG, B, AE, or F. The broadest and most potent neutralizing responses were elicited with the clade C gp120 DNA and a combination of V3-fusion proteins from clades A, B and C. Neutralizing activity was primarily due to V3-specific Abs. The results demonstrate that the immune response can be focused on a neutralizing epitope and show that the anti-V3 Abs induced recognize a diverse set of V3 loops.     

Selective regimes and evolutionary rates of HIV-1 subtype B V3 variants in the Brazilian epidemic

Half of subtype B Brazilian HIV-1 harbors the V3 tip GWGR instead of the GPGR. To investigate the evolution of GW variants, we analyzed 81 env sequences and 5 full-length GW genomes from antiretroviral-naïve individuals sampled between 1983 and 1999. Phylogenetic analysis indicated that GW strains intermingle in the tree with other subtype B sequences. The mean d_N/d_S values of GW strains were proximal to those of the other sequences, regardless of sampling years or clinical status. In sequences from patients with CD4+ T cell counts ≥ 200 cells/μL, the mean d_N/d_S ratio was greater than one, suggesting a positive selection. The prevalence of GW variants was lower among individuals in whom disease progressed. This is probably attributable to the fact that tryptophan is replaced by other amino acids over time, whereas the GP motif does not evolve as rapidly.