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 (VH1-69) and variable kappa light chain (VK3-20) 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.

SUMMARY 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.

SUMMARY 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 pre-configured 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 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.

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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.

Significance Achieving an AIDS-free generation will require elimination of breast milk transmission of HIV-1, as breastfeeding is a cornerstone of infant survival in developing regions. Antiretroviral prophylaxis considerably reduces postnatal HIV-1 transmission, yet its efficacy is limited by access, adherence, toxicities, and resistance of maternal HIV-1 strains. Alternative, safe strategies of impeding postnatal HIV-1 transmission will be required to eliminate infant HIV-1 infection. In this paper, we identify an innate HIV-neutralizing protein in breast milk, Tenascin-C, which captures and neutralizes HIV-1 virions via binding to the chemokine coreceptor binding site on the HIV-1 Envelope. This protein has the potential to be developed as a prevention strategy for postnatal and other modes of HIV-1 transmission.

The human immunoglobulin (Ig) V H (heavy-chain variable region) genes are grouped into 7 families (V H 1 to V H 7) (4, 24, 30) that are not equally represented in the human B cell repertoire (7,8,39). While V H gene usage in cord blood lymphocytes reflects the relative frequency of V H family size, some V H subfamily genes are expressed at higher frequency (14,20,38). The control of V H expression by genetic factors has been described in studies on monozygotic twins (19, 41), and V H polymorphisms have been associated with autoimmune diseases (40,41,45,47). V H gene expression, shaped by both genetic and antigenic stimulation, can also regulate the ability of the host to induce an antibody response against a pathogen. The HIV-1 envelope protein has conserved regions in gp41 to which rare broadly neutralizing human antibodies like 2F5 and 4E10 have been isolated (5, 27), However, only ϳ10 to 20% of chronically infected subjects make broadly neutralizing antibodies (bNAbs) (32,43). Even when bNAbs are made, they are made not during the acute infection but rather only after months of chronic infection (15,32,34).Viral factors contributing to difficulty in inducing bNAbs include conformational masking of transient epitopes (3, 21) as well as glycan shielding (31, 44). HIV-1 gp41 bNAbs are more uncommon than those that target gp120, with one hypothesis being that lack of induction of gp41 neutralizing antibodies is due to viral mimicry of the gp41 membrane proximal external region (MPER), with host molecules leading to tolerance to gp41 bNAb induction (3,16,17,42). However, the nature of host factors regulating the ability to make HIV-1 Env bNAbs remains unknown. Xiao et al. have suggested the lack of antigen recognition by unmutated receptors on naïve B cells, therefore implying that there may be "holes" in the human germ line B cell repertoire for antigens that stimulate naïve B cells that can make broadly neutralizing antibodies, and in particular those that can make 2F5-like gp41 bNAbs (46).The 2F5 bNAb (25, 29) uses the V H 2-5 gene, for which 10 distinct alleles are known. Each uses either a D or an N residue at position H54 (22), and the relative frequencies of human V H 2-5-bearing antibodies that use N or D alleles are 54% and 46%, respectively (Table 1). The mature 2F5 bNAb usage of V H 2-5 includes the D H54 residue, which resides in the heavychain complementarity-determining region 2 (HCDR2) (27)

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. immunogen | broadly neutralizing antibodiesA major challenge for an effective HIV-1 vaccine is the inability of immunogens to induce broadly neutralizing antibodies (nAbs). One goal for antibody-based HIV-1 vaccine strategies is to elicit broadly neutralizing antibodies similar in breadth to the membrane-proximal external region (MPER) 2F5 and 4E10 monoclonal antibodies (mAbs) that neutralize a majority of transmitted viruses (1). MPER-specific broadly neutralizing antibodies are rarely made in HIV-1 infection (2-4), but recent studies from our group and others have shown that 2F5-like antibodies responsible for neutralization breadth can be found in ≈0.3% of HIV-1 infected subjects (5), whereas 4E10-like antibodies can be found in ∼3% of HIV-1 positive subjects (6). Vaccination with antigenic envelope constructs expressing 2F5 and 4E10 epitopes has not induced high-titered neutralizing antibodies (7-10). One hypothesis for the failure of such vaccines to elicit broadly neutralizing antibodies is that the Env epitopes presented to host B cells are not in the correct envelope conformation; for the MPER, this conformation may be the transient, prehairpin gp41 intermediate (11,12). Another hypothesis is that 2F5 and 4E10 mAbs, which are unusual in having long hydrophobic CDR3 loops, may be particularly effective in reaching epitopes near the virion lipid bilayer, but may be difficult to induce because of down-regulation of polyreactive B cell clones through B cell tolerance mechanisms (13-18).Previous work has identified two amino acid substitutions (T569A, I675V) (19) and L669S (5) in the MPER that increased neutralization sensitivity by MPER antibodies; however, the mechanism by which neutralization sensitivity was increased was not determined (5,19). We now report that the mechanism of enhanced neutralization sensitivity of a single amino acid substitution (leucine to serine substitution at position 669, L669S) in the heptad re...