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. 2018 Aug 16;92(17):e00878-18.
doi: 10.1128/JVI.00878-18. Print 2018 Sep 1.

HIV-1 Subtype C-Infected Children with Exceptional Neutralization Breadth Exhibit Polyclonal Responses Targeting Known Epitopes

Zanele Ditse  1   2 , Maximilian Muenchhoff  3   4   5   6 , Emily Adland  3 , Pieter Jooste  7 , Philip Goulder  3   4   7 , Penny L Moore  8   2   9 , Lynn Morris  8   2   9
Affiliations

HIV-1 Subtype C-Infected Children with Exceptional Neutralization Breadth Exhibit Polyclonal Responses Targeting Known Epitopes

Zanele Ditse et al. J Virol. .

Abstract

We have previously shown that HIV-1-infected children develop broader and more potent neutralizing antibody responses than adults. This study aimed to determine the antibody specificities in 16 HIV-1 subtype C-infected children who displayed exceptional neutralization breadth on a 22-multisubtype virus panel. All children were antiretroviral treatment (ART) naive with normal CD4 counts despite being infected for a median of 10.1 years with high viral loads. The specificity of broadly neutralizing antibodies (bNAbs) was determined using epitope-ablating mutants, chimeric constructs, and depletion or inhibition of activity with peptides and glycoproteins. We found that bNAbs in children largely targeted previously defined epitopes, including the V2-glycan, V3-glycan, CD4bs, and gp120-gp41 interface. Remarkably, 63% of children had antibodies targeting 2 or 3 and, in one case, 4 of these bNAb epitopes. Longitudinal analysis of plasma from a mother-child pair over 9 years showed that while they both had similar neutralization profiles, the antibody specificities differed. The mother developed antibodies targeting the V2-glycan and CD4bs, whereas bNAb specificities in the child could not be mapped until 6 years, when a minor V2-glycan response appeared. The child also developed high-titer membrane-proximal external region (MPER) binding antibodies not seen in the mother, although these were not a major bNAb specificity. Overall, exceptional neutralization breadth in this group of children may be the result of extended exposure to high antigenic load in the context of an intact immune system, which allowed for the activation of multiple B cell lineages and the generation of polyclonal responses targeting several bNAb epitopes.IMPORTANCE An HIV vaccine is likely to require bNAbs, which have been shown to prevent HIV acquisition in nonhuman primates. Recent evidence suggests that HIV-infected children are inherently better at generating bNAbs than adults. Here, we show that exceptional neutralization breadth in a group of viremic HIV-1 subtype C-infected children was due to the presence of polyclonal bNAb responses. These bNAbs targeted multiple epitopes on the HIV envelope glycoprotein previously defined in adult infection, suggesting that the immature immune system recognizes HIV antigens similarly. Since elicitation of a polyclonal bNAb response is the basis of next-generation HIV envelope vaccines, further studies of how bNAb lineages are stimulated in children is warranted. Furthermore, our findings suggest that children may respond particularly well to vaccines designed to elicit antibodies to multiple bNAb epitopes.

Keywords: HIV envelope targets; HIV vaccines; HIV-1 subtype C; bNAbs; epitope mapping; mother-child transmission; neutralization breadth; neutralizing antibodies; pediatric immunology.

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Figures

FIG 1
FIG 1
Breadth of plasma neutralizing activity from 16 children and 1 transmitting mother. Plasma samples were tested against 22 heterologous viruses from subtypes A, B, and C using the TZM-bl neutralization assay. Neutralization titers for each virus-plasma combination are shown as the reciprocal of the plasma dilution required to inhibit 50% virus infection (ID50). Data are shown as a heat map, with titers of >1,000 in red, those between 100 and 999 in orange, and those of <100 in light yellow, as indicated by the key. Titers of 1:40 indicate no neutralization. MuLV was included as a negative control. These data were used to derive the GMT included in Table 1.
FIG 2
FIG 2
Differential effect of gp120 adsorption on neutralization suggests polyclonality. Anti-gp120 antibodies were depleted from plasma samples of all 16 children using tosyl-activated beads coated with ConC gp120 protein. Adsorbed plasma were tested for neutralization activity against ConC and heterologous viruses CAP45 and Q23.17. The percentage of gp120-directed neutralization was calculated as the reduction in ID50 of plasma incubated with gp120-coated beads relative to that of blank beads [((blank beads − gp120-coated beads)/blank beads) × 100]. Plasma samples where >50% of neutralizing activity was depleted by gp120 are colored red, and those with no depletion are colored blue. Those with some gp120-directed antibodies are colored yellow.
FIG 3
FIG 3
Majority of children develop neutralizing antibodies directed at V2-glycan and V3-glycan epitopes. Plasma samples from 16 children were assessed for V2-glycan antibodies using N160A/K and K169E mutants and for V3-glycan-directed responses using N332A mutants. Three heterologous viruses were assessed for each mutation and results are color coded with 3- to 5-fold reduction in neutralization activity shown in yellow, 5- to 10-fold reduction in orange, 10- to 100-fold reduction in red, and >100-fold reduction in dark red. V2/V3 specificity was assigned when plasma showed ≥3-fold reduction in at least 2 backbones. The V2 MAbs CAP256.08 and PG9 and the V3 MAb PGT128 were used as positive controls and showed the expected activity.
FIG 4
FIG 4
Mapping of CD4bs antibodies by binding and neutralization using the RSC3 protein. (A) Plasma samples were tested for binding to the RSC3 protein (solid line) and the CD4bs mutant protein RSC3Δ371I/P363N (dotted line) by ELISA. Shown are curves of 6 children with binding to the RSC3 protein but not the mutant protein, similar to VRC01 and suggestive of CD4bs antibodies. PID14 showed equivalent binding to both proteins and was used as a negative control. (B) The RSC3 protein was also used in a competition neutralization assay to detect CD4bs antibodies in the same 6 participants. Neutralization of Du156.12, CAP45, ConC, and Q23.17 was measured following incubation with RSC3. A reduction in neutralization of ≥30% (dotted line) was considered positive. Five of the 6 children showed activity against one virus, most commonly CAP45. VRC01 was used as a positive control, with RSC3 blocking neutralization of all 4 viruses as expected.
FIG 5
FIG 5
Mapping of anti-MPER neutralizing activity in four children. (A) Plasma samples from 4 children with high levels of neutralizing activity against the HIV-2/HIV-1 MPER C1 and C1C constructs were tested against 6 additional chimeric constructs containing point mutations to map known MPER antibodies. Also shown are the sequences carried by the MPER of each engrafted HIV-2/HIV-1 chimeric construct. Mutated amino acids are indicated in blue. Titers are shown as ID50 and are color coded with HIV-2 used as a negative control. 2F5, 4E10, and Z13e1 were used as positive controls. (B) Plasma from PID3 was incubated either with magnetic beads coated with MPR.03 peptide (left) or incubated directly with the MPR.03 peptide (right) to adsorb anti-MPER antibodies. MPER broad neutralization activity against untreated (solid lines) and adsorbed (dotted lines) samples was tested against heterologous viruses C1C and COT6. Adsorption of anti-MPER antibodies in PID3 reduced neutralization of heterologous viruses.
FIG 6
FIG 6
Children develop antibody responses to the gp120-gp41 interface, including the fusion peptide. (A) A reduction in neutralization of Du156.12, ConC, and BG505 was measured following incubation with the fusion peptide FP9. A cutoff of ≥30% (dotted line) was considered significant. Of the 16 children, 7 showed activity against 2 or 3 viruses that was FP directed. VRC34 was used as a positive control. (B) Plasma samples from 16 children were assessed for binding to FP9 using ELISA. Samples where FP9 competed for neutralization in panel A are shown in red, blue represents ELISA-positive samples where FP9 did not compete for neutralization, and black represents samples that were negative for both binding and neutralization. VRC34 was used as a positive control. (C) Mapping of responses using mutant viruses to the fusion peptide (THR514B), gp120-gp41 (residues 88, 234, 611, and 637), and the cleavage site (CAP45 CS Mut that contains 6 mutations known to expose the MPER). The fold change in neutralization breadth for the mutants relative to the wild-type strains is shown as a heat map for all 16 plasma samples. Loss of activity is shown in yellow (3- to 5-fold), orange (5- to 10-fold), and red (>10-fold), and enhancement is shown in blue. PGT151, 35O22, 4E10, VRC34, and CAP248-2B were used as positive controls. The overall specificity is shown in the last column, with those in brackets derived from data shown in panels A and B.
FIG 7
FIG 7
The majority of children develop antibodies targeting multiple epitopes. (A) Pie chart showing a summary of mapped neutralization epitopes targeted by plasma antibodies from children. (B) Breakdown of specificities detected in children with more than one bNAb. Shown in the last column is the total number of mapped specificities.
FIG 8
FIG 8
Mother-child pair develop antibodies that target distinct epitopes. (A) Longitudinal viral load (circles) and CD4 counts (triangles) for mother-child pair. Mother's data (PID1_M) are shown in red, and the child's data (PID1_C) are shown in blue. Stars indicate the time points used for plasma mapping. Absolute CD4 counts with the median and 10th and 90th percentiles for normal uninfected children over the first 10 years of life are shown in gray (53, 54). (B, top) Kinetics of development of neutralization breadth in the mother-child pair over a 9-year period. Shown is the percentage of viruses neutralized in a 22-virus panel with mother shown in red and child in blue. (Bottom) Longitudinal geometric mean ID50 titers (GMT) of all viruses in the 22-virus panel for mother and child. (C) Mapping of the specificities over time in mother and child. Longitudinal samples were assessed for V2-glycan using the K169E mutant in CAP45 relative to the wild-type strain. V3-glycan-directed responses were assessed using the N332A mutant in ConC relative to the wild-type strain. CD4bs activity was assessed by the RSC3 and the RSC3Δ371I/P363N CD4bs mutant in ELISA. MPER responses were determined using the MPER chimeric constructs C1 (black) and C1C (gray). Wild-type strains are shown in solid lines and closed circles, and mutants are shown in dotted lines. Mother's data (PID1_M) are shown on the left, and the child's data (PID1_C) are on the right. Mother's samples were missing at time points 3, 6, and 7 years of the child's age.
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