Broadly neutralizing antibody responses in a large longitudinal sub-Saharan HIV primary infection cohort

Author Summary Understanding how HIV-1-broadly neutralizing antibodies (bnAbs) develop during natural infection is essential to the design of an efficient HIV vaccine. We studied kinetics and correlates of neutralization breadth in a large sub-Saharan African longitudinal cohort of 439 participants with primary HIV-1 infection. Broadly nAb responses developed in 15% of individuals, on average three years after infection. Broad neutralization was associated with high viral load, low CD4+ T cell counts, virus subtype C infection and HLA*A3(-) genotype. A correlation with high overall plasma IgG levels and anti-Env binding titers was also found. Specificity mapping of the bnAb responses showed that glycan-dependent epitopes, in particular the N332 region, were most commonly targeted, in contrast to other bnAb epitopes, suggesting that the HIV Env N332-glycan epitope region may be a favorable target for vaccine design

Promiscuous Glycan Site Recognition by Antibodies to the High-Mannose Patch of gp120 Broadens Neutralization of HIV

Broadly neutralizing monoclonal antibodies (bnMAbs) that target the high-mannose patch centered around the glycan at position 332 on HIV Env are promising vaccine leads and therapeutic candidates as they effectively protect against mucosal SHIV challenge and strongly suppress SHIV viraemia in established infection in macaque models. However, these antibodies demonstrate varying degrees of dependency on the N332 glycan site and the origins of their neutralization breadth are not always obvious. By measuring neutralization on an extended range of glycan site viral variants, we found that some bnMAbs can utilize alternate N-linked glycans in the absence of the N332 glycan site and therefore neutralize a substantial number of viruses lacking the site. Furthermore, many of the antibodies can neutralize viruses in which the N332 glycan site is shifted to the 334 position. Finally, we found that a combination of three antibody families that target the high-mannose patch can lead to 99% neutralization coverage of a large panel of viruses containing the N332/334 glycan site and up to 66% coverage for viruses that lack the N332/334 glycan site. The results indicate that a diverse response against the high-mannose patch may provide near equivalent coverage as a combination of bnMAbs targeting multiple epitopes. Additionally, the ability of some bnMAbs to utilize other N-linked glycan sites can help counter neutralization escape mediated by shifting of glycosylation sites. Overall, this work highlights the importance of promiscuous glycan binding properties in bnMAbs to the high-mannose patch for optimal anti-viral activity either in protective or therapeutic modalities

Early Antibody Lineage Diversification and Independent Limb Maturation Lead to Broad HIV-1 Neutralization Targeting the Env High-Mannose Patch.

International audienceThe high-mannose patch on HIV Env is a preferred target for broadly neutralizing antibodies (bnAbs), but to date, no vaccination regimen has elicited bnAbs against this region. Here, we present the development of a bnAb lineage targeting the high-mannose patch in an HIV-1 subtype-C-infected donor from sub-Saharan Africa. The Abs first acquired autologous neutralization, then gradually matured to achieve breadth. One Ab neutralized >47% of HIV-1 strains with only ∼11% somatic hypermutation and no insertions or deletions. By sequencing autologous env, we determined key residues that triggered the lineage and participated in Ab-Env coevolution. Next-generation sequencing of the Ab repertoire showed an early expansive diversification of the lineage followed by independent maturation of individual limbs, several of them developing notable breadth and potency. Overall, the findings are encouraging from a vaccine standpoint and suggest immunization strategies mimicking the evolution of the entire high-mannose patch and promoting maturation of multiple diverse Ab pathways

Antigen pressure from two founder viruses induces multiple insertions at a single antibody position to generate broadly neutralizing HIV antibodies.

Vaccination strategies aimed at maturing broadly neutralizing antibodies (bnAbs) from naïve precursors are hindered by unusual features that characterize these Abs, including insertions and deletions (indels). Longitudinal studies of natural HIV infection cases shed light on the complex processes underlying bnAb development and have suggested a role for superinfection as a potential enhancer of neutralization breadth. Here we describe the development of a potent bnAb lineage that was elicited by two founder viruses to inform vaccine design. The V3-glycan targeting bnAb lineage (PC39-1) was isolated from subtype C-infected IAVI Protocol C elite neutralizer, donor PC39, and is defined by the presence of multiple independent insertions in CDRH1 that range from 1-11 amino acids in length. Memory B cell members of this lineage are predominantly atypical in phenotype yet also span the class-switched and antibody-secreting cell compartments. Development of neutralization breadth occurred concomitantly with extensive recombination between founder viruses before each virus separated into two distinct population arms that evolved independently to escape the PC39-1 lineage. Ab crystal structures show an extended CDRH1 that can help stabilize the CDRH3. Overall, these findings suggest that early exposure of the humoral system to multiple related Env molecules could promote the induction of bnAbs by focusing Ab responses to conserved epitopes

HIV Envelope Glycoform Heterogeneity and Localized Diversity Govern the Initiation and Maturation of a V2 Apex Broadly Neutralizing Antibody Lineage

International audienceUnderstanding how broadly neutralizing antibodies (bnAbs) to HIV envelope (Env) develop during natural infection can help guide the rational design of an HIV vaccine. Here, we described a bnAb lineage targeting the Env V2 apex and the Ab-Env co-evolution that led to development of neutralization breadth. The lineage Abs bore an anionic heavy chain complementarity-determining region 3 (CDRH3) of 25 amino acids, among the shortest known for this class of Abs, and achieved breadth with only 10% nucleotide somatic hypermutation and no insertions or deletions. The data suggested a role for Env glycoform heterogeneity in the activation of the lineage germline B cell. Finally, we showed that localized diversity at key V2 epitope residues drove bnAb maturation toward breadth, mirroring the Env evolution pattern described for another donor who developed V2-apex targeting bnAbs. Overall, these findings suggest potential strategies for vaccine approaches based on germline-targeting and serial immunogen design

Antigen pressure from two founder viruses induces multiple insertions at a single antibody position to generate broadly neutralizing HIV antibodies.

Vaccination strategies aimed at maturing broadly neutralizing antibodies (bnAbs) from naïve precursors are hindered by unusual features that characterize these Abs, including insertions and deletions (indels). Longitudinal studies of natural HIV infection cases shed light on the complex processes underlying bnAb development and have suggested a role for superinfection as a potential enhancer of neutralization breadth. Here we describe the development of a potent bnAb lineage that was elicited by two founder viruses to inform vaccine design. The V3-glycan targeting bnAb lineage (PC39-1) was isolated from subtype C-infected IAVI Protocol C elite neutralizer, donor PC39, and is defined by the presence of multiple independent insertions in CDRH1 that range from 1-11 amino acids in length. Memory B cell members of this lineage are predominantly atypical in phenotype yet also span the class-switched and antibody-secreting cell compartments. Development of neutralization breadth occurred concomitantly with extensive recombination between founder viruses before each virus separated into two distinct population "arms" that evolved independently to escape the PC39-1 lineage. Ab crystal structures show an extended CDRH1 that can help stabilize the CDRH3. Overall, these findings suggest that early exposure of the humoral system to multiple related Env molecules could promote the induction of bnAbs by focusing Ab responses to conserved epitopes

Correlation between clinical parameters and development of broadly neutralizing antibody responses.

<p>(A) Bivariate and multivariable GLM correlation analyses between the listed variables, and the best neutralization score for the M48+ subset of Protocol C participants. Number of participants in each subgroup (N) is indicated. Estimated coefficients (EstCoef), p-values, q-values, odd ratios (ExpEst) upper (U95) and lower (L95) values of the 95% confidence interval are indicated. P-values are color coded as follows: 0.01< p-value < 0.05, in green; 0.001< p-value < 0.01, in yellow; 2E-16 < p-value <0.001, in red. Q-values below 0.1 are indicated in bold. (B) Kaplan Meier curves recording the time for Protocol C neutralizers (best neutralization score ≥ 0.5, N = 157) within the indicated subgroups to reach a neutralization score ≥ 0.5. Log-Rank test p-values are indicated. DC: Discordant couple, OHS: Other Heterosexual transmission, HSM: Women to Men Heterosexual transmission, HSW: Men to Women Heterosexual transmission, MSM: Men who have Sex with Men.</p

Evolution of broadly neutralizing antibody responses in plasma from Protocol C participants.

<p>(A-C) Plasma from HIV-1 infected participants collected at various time points post infection were assessed for neutralizing activity on a predictive 6v-panel [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005369#ppat.1005369.ref016" target="_blank">16</a>]. Neutralization score on the 6v-panel was calculated as indicated in Material and Methods (A) Best neutralization score across all time points tested for Protocol C participants. (B) Fraction of Protocol C individuals with the indicated plasma neutralization score at the indicated visits. Neutralization score is color-coded as indicated in (A). (C) Detailed evolution of neutralization score over time (months) for individual Protocol C best neutralizers (N = 46), organized by time to reach neutralization score ≥ 1 in months post infection (MPI). NT: Not Tested. ART: Participants was on Anti-Retroviral Therapy at this visit, OFF: Participant was off-study at this visit.</p

Specificities mediating neutralization breadth and potency in the top 42 Protocol C neutralizers.

<p>(A) Samples are ranked by their neutralization score on the 37-virus panel (37vP) (S4 Fig in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005369#ppat.1005369.s001" target="_blank">S1 Text</a> and S2 Table in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005369#ppat.1005369.s001" target="_blank">S1 Text</a>). VC: Visit Code (months post infection). Symbols recapitulate the strength of the phenotypes tested using the different approaches detailed in this manuscript (S7-S11 Figures in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005369#ppat.1005369.s001" target="_blank">S1 Text</a>) to determine the Env epitope region targeted by the plasma broadly neutralizing activity: gp120 absorption of bnAb activity, effect of b6 competition in gp120 absorption experiments, RSC3 binding and neutralization competition, HIV-2 chimera neutralization, viruses produced in presence of kifunensine or bearing mutations. Absent (-), very weak (+/-), weak (+), moderate (++), strong (+++), phenotype was attributed based on i) the median fold or average percent decrease in ID50 and ii) the fraction of viruses which neutralization ID50 was decreased <2 fold, <10 <50 fold or <20%, <40%, <60%, <80%. Blank = not tested, NB: not binding. A dominant specificity was attributed for each sample based on results from all these experiments. UD: Undefined. (B) Overall distribution of dominant nAb specificities mediating plasma neutralization breadth in the top 42 Protocol C neutralizers as detailed in (A).</p

Correlation between total and antigen-specific IgG responses and development of broadly neutralizing antibody responses.

<p>Correlations were assessed by Spearman analyses: p-values and r-values are indicated; (ns) not significant. Linear, semi-Log or Log-log regressions are also shown as dotted lines. Neutralization score corresponds to a participant’s best neutralization score on the 6-virus panel across all tested time points. (A,C) IgG binding activity to recombinant MN gp41 (Subtype B), BG505 gp120 (subtype A) and IAVIC22 gp120 (Subtype B) was assessed, by ELISA, in plasma samples of Protocol C participants (N = 61) from the M48+ subset, at visits matching development of bnAb responses (M24-72, mean = 36.6 mpi). (B) Avidity index for IAVIC22-gp120 IgG titers were calculated from high salt (1.5M or 3M NaSCN) ELISA experiments. (C) Total IgG titers were assessed by ELISA. (D) Total IgG titers in pre-infection (N = 27), ~4mpi (N = 56, M00, mean = 4.0 mpi) and ~36mpi (N = 61) M24-72) samples. (E) Total IgG titers in ~36mpi samples (M24-72). (F) ELISA binding ID50 and neutralization score from (A) were standardized to a reference concentration of 20mg/mL of total plasma IgG.</p