Determinants of Tenascin-C and HIV-1 envelope binding and neutralization.

Interactions between innate antiviral factors at mucosal surfaces and HIV-1 virions contribute to the natural inefficiency of HIV-1 transmission and are a platform to inform the development of vaccine and nonvaccine strategies to block mucosal HIV-1 transmission. Tenascin-C (TNC) is a large, hexameric extracellular matrix glycoprotein identified in breast milk and genital fluids that broadly neutralizes HIV-1 via interaction with the HIV-1 Envelope (Env) variable 3 (V3) loop. In this report, we characterize the specific determinants of the interaction between TNC and the HIV-1 Env. We observed that TNC binding and neutralization of HIV-1 is dependent on the TNC fibrinogen-like globe (fbg) and fibronectin-type III (fn) domains, oligomerization, and its newly-mapped glycan structure. Moreover, we observed that TNC-mediated neutralization is also dependent on Env V3 residues 321/322 and 326/327, which surround the IGDIR motif of the V3 loop, as well the N332 glycan, which is critical to the broadly neutralizing activity of glycan-dependent V3-specific antibodies such as PGT128. Our results demonstrate a striking parallel between innate and adaptive immune mechanisms of broad HIV neutralization and provide further insight into the host protein-virus interactions responsible for the natural inefficiency of mucosal HIV-1 transmission.

Cross-Linking of a CD4-Mimetic Miniprotein with HIV-1 Env gp140 Alters Kinetics and Specificities of Antibody Responses against HIV-1 Env in Macaques.

Evaluation of the epitope specificities, locations (systemic or mucosal), and effector functions of antibodies elicited by novel HIV-1 immunogens engineered to improve exposure of specific epitopes is critical for HIV-1 vaccine development. Utilizing an array of humoral assays, we evaluated the magnitudes, epitope specificities, avidities, and functions of systemic and mucosal immune responses elicited by a vaccine regimen containing Env cross-linked to a CD4-mimetic miniprotein (gp140-M64U1) in rhesus macaques. Cross-linking of gp140 Env to M64U1 resulted in earlier increases of both the magnitude and avidity of the IgG binding response than those with Env protein alone. Notably, IgG binding responses at an early time point correlated with antibody-dependent cellular cytotoxicity (ADCC) function at the peak immunity time point, which was higher for the cross-linked Env group than for the Env group. In addition, the cross-linked Env group developed higher IgG responses against a linear epitope in the gp120 C1 region of the HIV-1 envelope glycoprotein. These data demonstrate that structural modification of the HIV-1 envelope immunogen by cross-linking of gp140 with the CD4-mimetic M64U1 elicited an earlier increase of binding antibody responses and altered the specificity of the IgG responses, correlating with the rise of subsequent antibody-mediated antiviral functions.IMPORTANCE The development of an efficacious HIV-1 vaccine remains a global priority to prevent new cases of HIV-1 infection. Of the six HIV-1 efficacy trials to date, only one has demonstrated partial efficacy, and immune correlate analysis of that trial revealed a role for binding antibodies and antibody Fc-mediated effector functions. New HIV-1 envelope immunogens are being engineered to selectively expose the most vulnerable and conserved sites on the HIV-1 envelope, with the goal of eliciting antiviral antibodies. Evaluation of the humoral responses elicited by these novel immunogen designs in nonhuman primates is critical for understanding how to improve upon immunogen design to inform further testing in human clinical trials. Our results demonstrate that structural modifications of Env that aim to mimic the CD4-bound conformation can result in earlier antibody elicitation, altered epitope specificity, and increased antiviral function postimmunization.

The Presence and Anti-HIV-1 Function of Tenascin C in Breast Milk and Genital Fluids.

Tenascin-C (TNC) is a newly identified innate HIV-1-neutralizing protein present in breast milk, yet its presence and potential HIV-inhibitory function in other mucosal fluids is unknown. In this study, we identified TNC as a component of semen and cervical fluid of HIV-1-infected and uninfected individuals, although it is present at a significantly lower concentration and frequency compared to that of colostrum and mature breast milk, potentially due to genital fluid protease degradation. However, TNC was able to neutralize HIV-1 after exposure to low pH, suggesting that TNC could be active at low pH in the vaginal compartment. As mucosal fluids are complex and contain a number of proteins known to interact with the HIV-1 envelope, we further studied the relationship between the concentration of TNC and neutralizing activity in breast milk. The amount of TNC correlated only weakly with the overall innate HIV-1-neutralizing activity of breast milk of uninfected women and negatively correlated with neutralizing activity in milk of HIV-1 infected women, indicating that the amount of TNC in mucosal fluids is not adequate to impede HIV-1 transmission. Moreover, the presence of polyclonal IgG from milk of HIV-1 infected women, but not other HIV-1 envelope-binding milk proteins or monoclonal antibodies, blocked the neutralizing activity of TNC. Finally, as exogenous administration of TNC would be necessary for it to mediate measurable HIV-1 neutralizing activity in mucosal compartments, we established that recombinantly produced TNC has neutralizing activity against transmitted/founder HIV-1 strains that mimic that of purified TNC. Thus, we conclude that endogenous TNC concentration in mucosal fluids is likely inadequate to block HIV-1 transmission to uninfected individuals.

Structural analysis of the unmutated ancestor of the HIV-1 envelope V2 region antibody CH58 isolated from an RV144 vaccine efficacy trial vaccinee.

Human monoclonal antibody CH58 isolated from an RV144 vaccinee binds at Lys169 of the HIV-1 Env gp120 V2 region, a site of vaccine-induced immune pressure. CH58 neutralizes HIV-1 CRF_01 AE strain 92TH023 and mediates ADCC against CD4 + T cell targets infected with CRF_01 AE tier 2 virus. CH58 and other antibodies that bind to a gp120 V2 epitope have a second light chain complementarity determining region (LCDR2) bearing a glutamic acid, aspartic acid (ED) motif involved in forming salt bridges with polar, basic side amino acid side chains in V2. In an effort to learn how V2 responses develop, we determined the crystal structures of the CH58-UA antibody unliganded and bound to V2 peptide. The structures showed an LCDR2 structurally pre-conformed from germline to interact with V2 residue Lys169. LCDR3 was subject to conformational selection through the affinity maturation process. Kinetic analyses demonstrate that only a few contacts were responsible for a 2000-fold increase in KD through maturation, and this effect was predominantly due to an improvement in off-rate. This study shows that preconformation and preconfiguration can work in concert to produce antibodies with desired immunogenic properties.

Maternal HIV-1 envelope-specific antibody responses and reduced risk of perinatal transmission.

Despite the wide availability of antiretroviral drugs, more than 250,000 infants are vertically infected with HIV-1 annually, emphasizing the need for additional interventions to eliminate pediatric HIV-1 infections. Here, we aimed to define humoral immune correlates of risk of mother-to-child transmission (MTCT) of HIV-1, including responses associated with protection in the RV144 vaccine trial. Eighty-three untreated, HIV-1-transmitting mothers and 165 propensity score-matched nontransmitting mothers were selected from the Women and Infants Transmission Study (WITS) of US nonbreastfeeding, HIV-1-infected mothers. In a multivariable logistic regression model, the magnitude of the maternal IgG responses specific for the third variable loop (V3) of the HIV-1 envelope was predictive of a reduced risk of MTCT. Neutralizing Ab responses against easy-to-neutralize (tier 1) HIV-1 strains also predicted a reduced risk of peripartum transmission in secondary analyses. Moreover, recombinant maternal V3-specific IgG mAbs mediated neutralization of autologous HIV-1 isolates. Thus, common V3-specific Ab responses in maternal plasma predicted a reduced risk of MTCT and mediated autologous virus neutralization, suggesting that boosting these maternal Ab responses may further reduce HIV-1 MTCT.

Antibody light-chain-restricted recognition of the site of immune pressure in the RV144 HIV-1 vaccine trial is phylogenetically conserved.

In HIV-1, the ability to mount antibody responses to conserved, neutralizing epitopes is critical for protection. Here we have studied the light chain usage of human and rhesus macaque antibodies targeted to a dominant region of the HIV-1 envelope second variable (V2) region involving lysine (K) 169, the site of immune pressure in the RV144 vaccine efficacy trial. We found that humans and rhesus macaques used orthologous lambda variable gene segments encoding a glutamic acid-aspartic acid (ED) motif for K169 recognition. Structure determination of an unmutated ancestor antibody demonstrated that the V2 binding site was preconfigured for ED motif-mediated recognition prior to maturation. Thus, light chain usage for recognition of the site of immune pressure in the RV144 trial is highly conserved across species. These data indicate that the HIV-1 K169-recognizing ED motif has persisted over the diversification between rhesus macaques and humans, suggesting an evolutionary advantage of this antibody recognition mode.

HIV-1 envelope gp41 antibodies can originate from terminal ileum B cells that share cross-reactivity with commensal bacteria.

Monoclonal antibodies derived from blood plasma cells of acute HIV-1-infected individuals are predominantly targeted to the HIV Env gp41 and cross-reactive with commensal bacteria. To understand this phenomenon, we examined anti-HIV responses in ileum B cells using recombinant antibody technology and probed their relationship to commensal bacteria. The dominant ileum B cell response was to Env gp41. Remarkably, a majority (82%) of the ileum anti-gp41 antibodies cross-reacted with commensal bacteria, and of those, 43% showed non-HIV-1 antigen polyreactivity. Pyrosequencing revealed shared HIV-1 antibody clonal lineages between ileum and blood. Mutated immunoglobulin G antibodies cross-reactive with both Env gp41 and microbiota could also be isolated from the ileum of HIV-1 uninfected individuals. Thus, the gp41 commensal bacterial antigen cross-reactive antibodies originate in the intestine, and the gp41 Env response in HIV-1 infection can be derived from a preinfection memory B cell pool triggered by commensal bacteria that cross-react with Env.

Vaccine-induced HIV-1 envelope gp120 constant region 1-specific antibodies expose a CD4-inducible epitope and block the interaction of HIV-1 gp140 with galactosylceramide.

UNLABELLED: Mucosal epithelial cell surface galactosylceramide (Galcer) has been postulated to be a receptor for HIV-1 envelope (Env) interactions with mucosal epithelial cells. Disruption of the HIV-1 Env interaction with such alternate receptors could be one strategy to prevent HIV-1 entry through the mucosal barrier. To study antibody modulation of HIV-1 Env-Galcer interactions, we used Galcer-containing liposomes to assess whether natural- and vaccine-induced monoclonal antibodies can block HIV-1 Env binding to Galcer. HIV-1 Env gp140 proteins bound to Galcer liposomes with Kds (dissociation constants) in the nanomolar range. Several HIV-1 ALVAC/AIDSVAX vaccinee-derived monoclonal antibodies (MAbs) specific for the gp120 first constant (C1) region blocked Galcer binding of a transmitted/founder HIV-1 Env gp140. Among the C1-specific MAbs that showed Galcer blocking, the antibody-dependent cellular cytotoxicity-mediating CH38 IgG and its natural IgA isotype were the most potent blocking antibodies. C1-specific IgG monoclonal antibodies that blocked Env binding to Galcer induced upregulation of the gp120 CD4-inducible (CD4i) epitope bound by MAb 17B, demonstrating that a conformational change in gp120 may be required for Galcer blocking. However, the MAb 17B itself did not block Env-Galcer binding, suggesting that the C1 antibody-induced gp120 conformational changes resulted in alteration in a Galcer binding site distant from the CD4i 17B MAb binding site. IMPORTANCE: Galactosyl ceramide, a glycosphingolipid, has been postulated to be a receptor for the HIV-1 envelope glycoprotein (Env) interaction with mucosal epithelial cells. Here, we have mimicked this interaction by using an artificial membrane containing synthetic Galcer and recombinant HIV-1 Env proteins to identify antibodies that would block the HIV-1 Env-Galcer interaction. Our study revealed that a class of vaccine-induced human antibodies potently blocks HIV-1 Env-Galcer binding by perturbing the HIV-1 Env conformation.

Modulation of nonneutralizing HIV-1 gp41 responses by an MHC-restricted TH epitope overlapping those of membrane proximal external region broadly neutralizing antibodies.

A goal of HIV-1 vaccine development is to elicit broadly neutralizing Abs (BnAbs), but current immunization strategies fail to induce BnAbs, and for unknown reasons, often induce nonneutralizing Abs instead. To explore potential host genetic contributions controlling Ab responses to the HIV-1 Envelope, we have used congenic strains to identify a critical role for MHC class II restriction in modulating Ab responses to the membrane proximal external region (MPER) of gp41, a key vaccine target. Immunized H-2(d)-congenic strains had more rapid, sustained, and elevated MPER(+) Ab titers than those bearing other haplotypes, regardless of immunogen, adjuvant, or prime or boost regimen used, including formulations designed to provide T cell help. H-2(d)-restricted MPER(+) serum Ab responses depended on CD4 TH interactions with class II (as revealed in immunized intra-H-2(d/b) congenic or CD154(-/-) H-2(d) strains, and by selective abrogation of MPER restimulated, H-2(d)-restricted primed splenocytes by class II-blocking Abs), and failed to neutralize HIV-1 in the TZM-b/l neutralization assay, coinciding with lack of specificity for an aspartate residue in the neutralization core of BnAb 2F5. Unexpectedly, H-2(d)-restricted MPER(+) responses functionally mapped to a core TH epitope partially overlapping the 2F5/z13/4E10 BnAb epitopes as well as nonneutralizing B cell-Ab binding residues. We propose that class II restriction contributes to the general heterogeneity of nonneutralizing gp41 responses induced by Envelope. Moreover, the proximity of TH and B cell epitopes in this restriction may have to be considered in redesigning minimal MPER immunogens aimed at exclusively binding BnAb epitopes and triggering MPER(+) BnAbs.

Tenascin-C is an innate broad-spectrum, HIV-1-neutralizing protein in breast milk.

Achieving an AIDS-free generation will require elimination of postnatal transmission of HIV-1 while maintaining the nutritional and immunologic benefits of breastfeeding for infants in developing regions. Maternal/infant antiretroviral prophylaxis can reduce postnatal HIV-1 transmission, yet toxicities and the development of drug-resistant viral strains may limit the effectiveness of this strategy. Interestingly, in the absence of antiretroviral prophylaxis, greater than 90% of infants exposed to HIV-1 via breastfeeding remain uninfected, despite daily mucosal exposure to the virus for up to 2 y. Moreover, milk of uninfected women inherently neutralizes HIV-1 and prevents virus transmission in animal models, yet the factor(s) responsible for this anti-HIV activity is not well-defined. In this report, we identify a primary HIV-1-neutralizing protein in breast milk, Tenascin-C (TNC). TNC is an extracellular matrix protein important in fetal development and wound healing, yet its antimicrobial properties have not previously been established. Purified TNC captured and neutralized multiclade chronic and transmitted/founder HIV-1 variants, and depletion of TNC abolished the HIV-1-neutralizing activity of milk. TNC bound the HIV-1 Envelope protein at a site that is induced upon engagement of its primary receptor, CD4, and is blocked by V3 loop- (19B and F39F) and chemokine coreceptor binding site-directed (17B) monoclonal antibodies. Our results demonstrate the ability of an innate mucosal host protein found in milk to neutralize HIV-1 via binding to the chemokine coreceptor site, potentially explaining why the majority of HIV-1-exposed breastfed infants are protected against mucosal HIV-1 transmission.

Recognition of synthetic glycopeptides by HIV-1 broadly neutralizing antibodies and their unmutated ancestors.

Current HIV-1 vaccines elicit strain-specific neutralizing antibodies. Broadly neutralizing antibodies (BnAbs) are not induced by current vaccines, but are found in plasma in ∼20% of HIV-1-infected individuals after several years of infection. One strategy for induction of unfavored antibody responses is to produce homogeneous immunogens that selectively express BnAb epitopes but minimally express dominant strain-specific epitopes. Here we report that synthetic, homogeneously glycosylated peptides that bind avidly to variable loop 1/2 (V1V2) BnAbs PG9 and CH01 bind minimally to strain-specific neutralizing V2 antibodies that are targeted to the same envelope polypeptide site. Both oligomannose derivatization and conformational stabilization by disulfide-linked dimer formation of synthetic V1V2 peptides were required for strong binding of V1V2 BnAbs. An HIV-1 vaccine should target BnAb unmutated common ancestor (UCA) B-cell receptors of naïve B cells, but to date no HIV-1 envelope constructs have been found that bind to the UCA of V1V2 BnAb PG9. We demonstrate herein that V1V2 glycopeptide dimers bearing Man5GlcNAc2 glycan units bind with apparent nanomolar affinities to UCAs of V1V2 BnAbs PG9 and CH01 and with micromolar affinity to the UCA of a V2 strain-specific antibody. The higher-affinity binding of these V1V2 glycopeptides to BnAbs and their UCAs renders these glycopeptide constructs particularly attractive immunogens for targeting subdominant HIV-1 envelope V1V2-neutralizing antibody-producing B cells.

Chemical synthesis of highly congested gp120 V1V2 N-glycopeptide antigens for potential HIV-1-directed vaccines.

Critical to the search for an effective HIV-1 vaccine is the development of immunogens capable of inducing broadly neutralizing antibodies (BnAbs). A key first step in this process is to design immunogens that can be recognized by known BnAbs. The monoclonal antibody PG9 is a BnAb that neutralizes diverse strains of HIV-1 by targeting a conserved carbohydrate-protein epitope in the variable 1 and 2 (V1V2) region of the viral envelope. Important for recognition are two closely spaced N-glycans at Asn(160) and Asn(156). Glycopeptides containing this synthetically challenging bis-N-glycosylated motif were prepared by convergent assembly, and were shown to be antigenic for PG9. Synthetic glycopeptides such as these may be useful for the development of HIV-1 vaccines based on the envelope V1V2 BnAb epitope.

Postnatally-transmitted HIV-1 Envelope variants have similar neutralization-sensitivity and function to that of nontransmitted breast milk variants.

BACKGROUND: Breastfeeding is a leading cause of infant HIV-1 infection in the developing world, yet only a minority of infants exposed to HIV-1 via breastfeeding become infected. As a genetic bottleneck severely restricts the number of postnatally-transmitted variants, genetic or phenotypic properties of the virus Envelope (Env) could be important for the establishment of infant infection. We examined the efficiency of virologic functions required for initiation of infection in the gastrointestinal tract and the neutralization sensitivity of HIV-1 Env variants isolated from milk of three postnatally-transmitting mothers (n = 13 viruses), five clinically-matched nontransmitting mothers (n = 16 viruses), and seven postnatally-infected infants (n = 7 postnatally-transmitted/founder (T/F) viruses). RESULTS: There was no difference in the efficiency of epithelial cell interactions between Env virus variants from the breast milk of transmitting and nontransmitting mothers. Moreover, there was similar efficiency of DC-mediated trans-infection, CCR5-usage, target cell fusion, and infectivity between HIV-1 Env-pseudoviruses from nontransmitting mothers and postnatal T/F viruses. Milk Env-pseudoviruses were generally sensitive to neutralization by autologous maternal plasma and resistant to breast milk neutralization. Infant T/F Env-pseudoviruses were equally sensitive to neutralization by broadly-neutralizing monoclonal and polyclonal antibodies as compared to nontransmitted breast milk Env variants. CONCLUSION: Postnatally-T/F Env variants do not appear to possess a superior ability to interact with and cross a mucosal barrier or an exceptional resistance to neutralization that define their capability to initiate infection across the infant gastrointestinal tract in the setting of preexisting maternal antibodies.

Antigenicity and immunogenicity of RV144 vaccine AIDSVAX clade E envelope immunogen is enhanced by a gp120 N-terminal deletion.

An immune correlates analysis of the RV144 HIV-1 vaccine trial revealed that antibody responses to the gp120 V1/V2 region correlated inversely with infection risk. The RV144 protein immunogens (A244-rp120 and MN-rgp120) were modified by an N-terminal 11-amino-acid deletion (Δ11) and addition of a herpes simplex virus (HSV) gD protein-derived tag (gD). We investigated the effects of these modifications on gp120 expression, antigenicity, and immunogenicity by comparing unmodified A244 gp120 with both Δ11 deletion and gD tag and with Δ11 only. Analysis of A244 gp120, with or without Δ11 or gD, demonstrated that the Δ11 deletion, without the addition of gD, was sufficient for enhanced antigenicity to gp120 C1 region, conformational V2, and V1/V2 gp120 conformational epitopes. RV144 vaccinee serum IgGs bound more avidly to A244 gp120 Δ11 than to the unmodified gp120, and their binding was blocked by C1, V2, and V1/V2 antibodies. Rhesus macaques immunized with the three different forms of A244 gp120 proteins gave similar levels of gp120 antibody titers, although higher antibody titers developed earlier in A244 Δ11 gp120-immunized animals. Conformational V1/V2 monoclonal antibodies (MAbs) gave significantly higher levels of blocking of plasma IgG from A244 Δ11 gp120-immunized animals than IgG from animals immunized with unmodified A244 gp120, thus indicating a qualitative difference in the V1/V2 antibodies induced by A244 Δ11 gp120. These results demonstrate that deletion of N-terminal residues in the RV144 A244 gp120 immunogen improves both envelope antigenicity and immunogenicity.

Induction of antibodies in rhesus macaques that recognize a fusion-intermediate conformation of HIV-1 gp41.

A component to the problem of inducing broad neutralizing HIV-1 gp41 membrane proximal external region (MPER) antibodies is the need to focus the antibody response to the transiently exposed MPER pre-hairpin intermediate neutralization epitope. Here we describe a HIV-1 envelope (Env) gp140 oligomer prime followed by MPER peptide-liposomes boost strategy for eliciting serum antibody responses in rhesus macaques that bind to a gp41 fusion intermediate protein. This Env-liposome immunization strategy induced antibodies to the 2F5 neutralizing epitope 664DKW residues, and these antibodies preferentially bound to a gp41 fusion intermediate construct as well as to MPER scaffolds stabilized in the 2F5-bound conformation. However, no serum lipid binding activity was observed nor was serum neutralizing activity for HIV-1 pseudoviruses present. Nonetheless, the Env-liposome prime-boost immunization strategy induced antibodies that recognized a gp41 fusion intermediate protein and was successful in focusing the antibody response to the desired epitope.

Isolation of a human anti-HIV gp41 membrane proximal region neutralizing antibody by antigen-specific single B cell sorting.

Broadly neutralizing antibodies are not commonly produced in HIV-1 infected individuals nor by experimental HIV-1 vaccines. When these antibodies do occur, it is important to be able to isolate and characterize them to provide clues for vaccine design. CAP206 is a South African subtype C HIV-1-infected individual previously shown to have broadly neutralizing plasma antibodies targeting the envelope gp41 distal membrane proximal external region (MPER). We have now used a fluoresceinated peptide tetramer antigen with specific cell sorting to isolate a human neutralizing monoclonal antibody (mAb) against the HIV-1 envelope gp41 MPER. The isolated recombinant mAb, CAP206-CH12, utilized a portion of the distal MPER (HXB2 amino acid residues, 673-680) and neutralized a subset of HIV-1 pseudoviruses sensitive to CAP206 plasma antibodies. Interestingly, this mAb was polyreactive and used the same germ-line variable heavy (V(H)1-69) and variable kappa light chain (V(K)3-20) gene families as the prototype broadly neutralizing anti-MPER mAb, 4E10 (residues 672-680). These data indicate that there are multiple immunogenic targets in the C-terminus of the MPER of HIV-1 gp41 envelope and suggests that gp41 neutralizing epitopes may interact with a restricted set of naive B cells during HIV-1 infection.

Differential reactivity of germ line allelic variants of a broadly neutralizing HIV-1 antibody to a gp41 fusion intermediate conformation.

Genetic factors, as well as antigenic stimuli, can influence antibody repertoire formation. Moreover, the affinity of antigen for unmutated naïve B cell receptors determines the threshold for activation of germinal center antibody responses. The gp41 2F5 broadly neutralizing antibody (bNAb) uses the V(H)2-5 gene, which has 10 distinct alleles that use either a heavy-chain complementarity-determining region 2 (HCDR2) aspartic acid (D(H54)) or an HCDR2 asparagine (N(H54)) residue. The 2F5 HCDR2 D(H54) residue has been shown to form a salt bridge with gp41 (665)K; the V(H)2-5 germ line allele variant containing N(H54) cannot do so and thus should bind less avidly to gp41. Thus, the induction of 2F5 bNAb is dependent on both genetic and structural factors that could affect antigen affinity of unmutated naïve B cell receptors. Here, we studied allelic variants of the V(H)2-5 inferred germ line forms of the HIV-1 gp41 bNAb 2F5 for their antigen binding affinities to gp41 linear peptide and conformational protein antigens. Both V(H)2-5 2F5 inferred germ line variants bound to gp41 peptides and protein, including the fusion intermediate protein mimic, although more weakly than the mature 2F5 antibody. As predicted, the affinity of the N(H54) variant for fusion-intermediate conformation was an order of magnitude lower than that of the D(H54) V(H)2-5 germ line antibody, demonstrating that allelic variants of 2F5 germ line antibodies differentially bind to gp41. Thus, these data demonstrate a genetically determined trait that may affect host responses to HIV-1 envelope epitopes recognized by broadly neutralizing antibodies and has implications for unmutated ancestor-based immunogen design.

Prolonged exposure of the HIV-1 gp41 membrane proximal region with L669S substitution.

The conserved membrane-proximal external region (MPER) of HIV-1 envelope is a target for the rare broadly neutralizing 2F5, Z13, and 4E10 monoclonal antibodies (mAbs). One strategy to elicit such antibodies is to design an immunogen with increased exposure of the 2F5 and 4E10 mAb epitopes. In this study we characterize a single leucine to serine substitution at position 669 (L669S) in the gp41 Env MPER that confers >250-fold more neutralization sensitivity to 2F5 and 4E10 mAbs than does the wild-type gp41 sequence. On synthetic liposomes, increased solvent exposure of MPER tryptophan residues and stable docking of 2F5 and 4E10 mAbs to mutant MPER peptide liposomes indicate more favorable membrane orientation of MPER neutralizing epitopes with L669S substitution. The time during which virus is sensitive to 2F5 mAb-mediated neutralization is approximately 3-fold longer when the mutation is present. These data suggest that a major contribution to the L669S mutant virus phenotype of enhanced susceptibility to MPER mAbs is prolonged exposure of the MPER neutralizing epitope during viral entry.