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. 2011 Apr;85(7):3128-41.
doi: 10.1128/JVI.02658-10. Epub 2011 Jan 26.

Potent and broad neutralization of HIV-1 subtype C by plasma antibodies targeting a quaternary epitope including residues in the V2 loop

Penny L Moore  1 , Elin S Gray, Daniel Sheward, Maphuti Madiga, Nthabeleng Ranchobe, Zhong Lai, William J Honnen, Molati Nonyane, Nancy Tumba, Tandile Hermanus, Sengeziwe Sibeko, Koleka Mlisana, Salim S Abdool Karim, Carolyn Williamson, Abraham Pinter, Lynn Morris; CAPRISA 002 Study
Affiliations

Potent and broad neutralization of HIV-1 subtype C by plasma antibodies targeting a quaternary epitope including residues in the V2 loop

Penny L Moore et al. J Virol. 2011 Apr.

Abstract

The targets of broadly cross-neutralizing (BCN) antibodies are of great interest in the HIV vaccine field. We have identified a subtype C HIV-1-superinfected individual, CAP256, with high-level BCN activity, and characterized the antibody specificity mediating breadth. CAP256 developed potent BCN activity peaking at 3 years postinfection, neutralizing 32 (76%) of 42 heterologous viruses, with titers of antibodies against some viruses exceeding 1:10,000. CAP256 showed a subtype bias, preferentially neutralizing subtype C and A viruses over subtype B viruses. CAP256 BCN serum targeted a quaternary epitope which included the V1V2 region. Further mapping identified residues F159, N160, L165, R166, D167, K169, and K171 (forming the FN/LRD-K-K motif) in the V2 region as crucial to the CAP256 epitope. However, the fine specificity of the BCN response varied over time and, while consistently dependent on R166 and K169, became gradually less dependent on D167 and K171, possibly contributing to the incremental increase in breadth over 4 years. The presence of an intact FN/LRD-K-K motif in heterologous viruses was associated with sensitivity, although the length of the adjacent V1 loop modulated the degree of sensitivity, with a shorter V1 region significantly associated with higher titers. Repair of the FN/LRD-K-K motif in resistant heterologous viruses conferred sensitivity, with titers sometimes exceeding 1:10,000. Comparison of the CAP256 epitope with that of the PG9/PG16 monoclonal antibodies suggested that these epitopes overlapped, adding to the mounting evidence that this may represent a common neutralization target that should be further investigated as a potential vaccine candidate.

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Figures

FIG. 1.
FIG. 1.
CAP256 viral loads and CD4+ T cell counts over 4 years of infection. Key time points for neutralization assays at 1, 2, 3, and 4 years p.i. are indicated with dotted lines. The peak viral load (VL) at the point of superinfection and the time point at which CAP256 was started on ARV therapy are shown by arrows. v16, visit 16, etc.
FIG. 2.
FIG. 2.
Development of cross-nAbs against heterologous viruses of subtypes A, B, and C in CAP256 over 1 to 4 years of infection. Titers shown are ID50.
FIG. 3.
FIG. 3.
Effect of rgp120 adsorption on CAP256 neutralization. (A) Binding of CAP256 plasma and rgp120-adsorbed plasma on binding to ConC rgp120, JRFL rgp120, and IIIB viral lysate measured by ELISA (optical density [OD] at 405 nm). (B) Neutralization by CAP256 plasma and ConC rgp120-adsorbed CAP256 plasma of ConC, ZM53M, autologous virus CAP256.1mo.C7, and SF162.
FIG. 4.
FIG. 4.
CAP256 plasma neutralizes CAP45 via a V1V2 region-dependent antibody. Neutralization of chimeric viruses constructed of CAP45 (sensitive) and CAP84 (resistant) is shown. Only the chimera containing the V1V2 region of CAP45 was sensitive to neutralization.
FIG. 5.
FIG. 5.
V1V2 region-dependent antibodies mediate cross-neutralization of subtype C and A viruses. Chimeric viruses containing the V1V2 region of sensitive subtype C or A viruses were tested against longitudinal plasma samples spanning 4 years of infection. Sensitive parental viruses are shown by black lines, gray lines denote resistant (backbone) viruses, and gray squares with black outlines are V1V2 region chimeras. Asterisks indicate graphs with differing scales to illustrate lower titers.
FIG. 6.
FIG. 6.
Identification of the FN/LRD-K-K motif in V2 as a major target of CAP256 plasma neutralizing activity. (A) V2-L and V2-M domain swap chimeras consisting of the ZM53M (sensitive) and JRFL (resistant) viruses. Shown at the top are residues in the sequences of the V2L and V2M regions of ZM53M and JRFL and the chimeras. The graph at the bottom shows the neutralization of the ZM53M and chimeric viruses containing the V2L or V2M domain from the JRFL virus, confirming the importance of both regions, but especially the V2M region, in forming the CAP256 epitope. (B) Site-directed mutagenesis of residues in V2 was used to map critical residues in the ConC background. Black highlighting denotes >75-fold titer reductions, dark gray denotes mutations resulting in >8-fold titer reductions, and light gray denotes >3-fold titer reductions. The FN/LRD-K-K motif represents residues affecting the CAP256 titers by more than 8-fold. Two other residues (168 and 181, not included in the motif) had a lesser effect. wt, wild type.
FIG. 7.
FIG. 7.
Changes in the fine specificity of the V2-dependent nAb present in CAP256 plasma. Longitudinal assessment of the neutralization sensitivity of CAP45 (parent virus) compared to that of viruses with the F159A, N160A, L165A, R166A, D167N, K169E, and K171E mutations to CAP256 plasma from 1 to 4 years p.i.
FIG. 8.
FIG. 8.
The FN/LRD-K-K motif and neutralization sensitivity of subtype C, A, and B viruses. (A) Analysis of the FN/LRD-K-K motif in subtype C, A, and B viruses (ranked by neutralization sensitivity at 3 years p.i.). Amino acids in blue are positively charged, and those in red are negatively charged. A point denotes conservation compared to the FNLDRKK motif. (B) Comparison of the V1 loop lengths of subtype C viruses neutralized at titers exceeding 1:10,000 at 3 years p.i. compared to those neutralized at 1:1,000 to 1:10,000 at the same time point. (C) Analysis of the frequencies of each residue within the FN/LRD-K-K motif, comparing envelope sequences of subtype C (n = 1,375), subtype A (n = 746), and subtype B (n = 2,630). Sequences were downloaded from the Los Alamos sequence database.
FIG. 9.
FIG. 9.
Restoring the FN/LRD-K-K motif in neutralization-resistant viruses confers neutralization sensitivity. Resistant subtype C (A), subtype B (B), and subtype A (C) viruses were mutated to restore the FN/LRD-K-K motif to various degrees. Parental viruses (resistant) are shown in gray. Mutant viruses are shown in black. Asterisks indicate graphs with differing scales to illustrate lower titers. Sequence changes from the FN/LRD-K-K motif and mutations introduced are shown below the graphs.
FIG. 10.
FIG. 10.
Effects of V2 and V3 mutations on CAP256 plasma neutralization compared with that of MAbs PG9 and PG16.
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