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. 2013 May;87(10):5424-36.
doi: 10.1128/JVI.00201-13. Epub 2013 Mar 6.

Conformational epitope consisting of the V3 and V4 loops as a target for potent and broad neutralization of simian immunodeficiency viruses

Takeo Kuwata  1 , Kaori Takaki, Kazuhisa Yoshimura, Ikumi Enomoto, Fan Wu, Ilnour Ourmanov, Vanessa M Hirsch, Masaru Yokoyama, Hironori Sato, Shuzo Matsushita
Affiliations

Conformational epitope consisting of the V3 and V4 loops as a target for potent and broad neutralization of simian immunodeficiency viruses

Takeo Kuwata et al. J Virol. 2013 May.

Abstract

Inducing neutralizing antibodies (NAb) is the key to developing a protective vaccine against human immunodeficiency virus type 1 (HIV-1). To clarify the neutralization mechanism of simian immunodeficiency virus (SIV), we analyzed NAb B404, which showed potent and broad neutralizing activity against various SIV strains. In 4 SIVsmH635FC-infected macaques, B404-like antibodies using the specific VH3 gene with a long complementarity-determining region 3 loop and λ light chain were the major NAbs in terms of the number and neutralizing potency. This biased NAb induction was observed in all 4 SIVsmH635FC-infected macaques but not in 2 macaques infected with a SIV mix, suggesting that induction of B404-like NAbs depended on the inoculated virus. Analysis using Env mutants revealed that the V3 and V4 loops were critical for B404 binding. The reactivity to the B404 epitope on trimeric, but not monomeric, Env was enhanced by CD4 ligation. The B404-resistant variant, which was induced by passages with increasing concentrations of B404, accumulated amino acid substitutions in the C2 region of gp120. Molecular dynamics simulations of the gp120 outer domains indicated that the C2 mutations could effectively alter the structural dynamics of the V3/V4 loops and their neighboring regions. These results suggest that a conformational epitope consisting of the V3 and V4 loops is the target for potent and broad neutralization of SIV. Identifying the new neutralizing epitope, as well as specifying the VH3 gene used for epitope recognition, will help to develop HIV-1 vaccines.

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Figures

Fig 1
Fig 1
Potent and broad neutralization by monoclonal antibody B404 from a SIVsmH635FC-infected macaque. (A) The neutralizing potencies of IgG, Fab, and scFv of B404 are shown by inhibition kinetics against SIVsmE660FL14, SIVsmH805-24w-3, and SIVmac239. (B) The neutralizing potencies of IgG, Fab and scFv of B404 are shown by IC50 and IC90 (μg/ml). Seven SIV strains, HIV-2GH123 and HIV-1NL432 were examined for their neutralizing sensitivities against B404 in TZM-bl cells. The IC50 and IC90 values are shown in dark gray (<1.0 × 10−2 μg/ml), medium gray (1.0 × 10−2 to 1.0 × 100 μg/ml), light gray (1 to 100 μg/ml) and white (>100 μg/ml). Percent amino acid differences were calculated by pairwise comparison with SIVsmH635FC.
Fig 2
Fig 2
B404-like NAbs formed a major group in anti-Env antibodies from 4 SIVsmH635FC-infected rhesus macaques. NAbs were separated into 4 groups in the phylogenetic tree, which was generated using MEGA5 (70) from heavy-chain genes of 98, 155, 102, and 53 Fab clones from H704, H709, H714, and H723, respectively.
Fig 3
Fig 3
Bias in the Ig gene usage and CDR3 length of neutralizing Fab clones from SIVsmH635FC-infected macaques. The proportions of IgH (A) and Igκ and Igλ (B) repertoires and heavy-chain CDR3 length (C) are shown as percentages of NAbs and non-NAbs. In addition to Fabs isolated from 4 SIVsmH635FC-infected macaques, 87 Fab clones from H711, which was infected with a combination of SIVsmE543-3 and SIVsmH635FC, and 45 Fab clones from H725, which was infected with plasma samples from 2 SIVsmH445-infected macaques, H631 and H635, were similarly analyzed. Usage of Ig genes was analyzed using V-QUEST in the International Immunogenetics Database (44).
Fig 4
Fig 4
The specificity and potency of Fab clones in the B404 group are similar to those of B404. (A) Competition ELISA was performed using serially diluted B404 IgG as a competitor. B404 IgG significantly inhibited the binding of the Fabs in the B404 group (L6, L22, O9, and O19). In contrast, B404 IgG did not compete with the Fabs in groups III (K8 and K10) and IV (K12, K40, and K47) and even enhanced the binding of these Fabs. (B) Neutralization potencies of Fabs in the B404 group (left) and groups III and IV (right) are shown by inhibition of infection to TZM-bl cells with neutralization-resistant SIVsmE543-3 and genetically divergent SIVmac316.
Fig 5
Fig 5
B404 recognizes a conformational epitope, including the V3 and V4 loops, and sCD4 enhances the exposure of the epitope in trimeric Env. (A) Reactivity of B404, K8, and H301 (anti-V1 Fab) to Env mutants was examined using 293T cells transfected with plasmids to express SIVsmE543-3 Env (wild-type), mutants with deletions in the V1 (ΔV1), V2 (ΔV2), V3 (ΔV3), and V4 (ΔV4) loops, an N306A/N316A/N349A mutant lacking glycosylation sites near the V3 loop (ΔGly), a D385R mutant interfering with CD4bs antibodies (D385R), and an I434R mutant interfering with CD4i antibodies (I434R). The transfected cells were stained with Fabs B404, K8, and H301, and the reactivity of Env mutants was analyzed using flow cytometry. The percentage of Fab+ cells is shown. (B) Reactivity of Fabs B404, K8, and H301, and murine anti-V3 MAb KK46 to sCD4-treated trimeric Env on the cell surface. Cells transfected with the plasmid to express SIVsmE543-3 Env were incubated with 2 μg/ml sCD4 for 15 min, and the reactivities of antibodies were similarly examined. The tinted histogram represents cells stained by antibody in the absence of sCD4. The dotted line shows the unstained control. (C) Reactivity of Fab clones B404 and K8 to sCD4-treated monomeric Env. The reactivity of serially diluted Fab to Env was examined by ELISA using SIVsmE543-3 as an antigen in the absence or presence of 0.5 or 2.0 μg/ml sCD4.
Fig 6
Fig 6
Isolation of variants resistant to B404 and amino acid substitutions in gp120. The B404-resistant variant was induced from SIVmac316 by passages of viruses in PM-1/CCR5 cells with increasing concentrations of B404 Fab. A B404-resistant variant, P26B404, was obtained from the supernatant of passage 26. P26C was obtained after 26 passages without B404. (A) The sensitivities of P26B404, P26C, and parental SIVmac316 to neutralization are shown by inhibition of infection of TZM-bl cells. B404 and K8 Fabs, murine MAb M318T, which recognizes the V2 of gp120, and plasma samples from SIVsmH635FC-infected macaque H704 and SIVmac239-infected macaque MM324 were used for the neutralization assay. (B) Amino acid sequences of the N-terminal and C2 regions of gp120 from P26C and P26B404 are aligned with those of parental SIVmac316 and SIV strains, SIVmac239, SIVsmE543-3 and SIVsmH635FC. The number of clones per total number of clones is given in parentheses. Identical amino acids are shown as dots, and potential glycosylation sites in SIVmac316 are indicated with underlining.
Fig 7
Fig 7
Effects of F277V and N295S mutations on the structural dynamics of the gp120 outer domain. (A) Distribution of RMSF in the gp120 outer domain. MD simulations of gp120 outer domains of SIVmac316, F277V, and N295S were carried out at 1 atm and 310 K for 50 ns as described in Materials and Methods. The RMSF values, which indicate the atomic fluctuations of the main chains of individual amino acids during MD simulations, were calculated using 90,000 snapshots from 9 to 50 ns of each MD simulation. The numbers on the horizontal axes indicate amino acid positions in gp120. The RMSF values of the mutants are significantly different from those of parental SIVmac316 at the V3 loop, the V3 flanking regions (indicated by asterisks), the V4 loop, the β20β21/LF loop, and the V5 loop regions. (B) A structure at 50 ns of MD simulation of the SIVmac316 gp120 outer domain is shown as a representative to indicate the steric location of mutation sites and various loops. The regions that are proximal to the V3/V4 loops and displayed fluctuations that differed from those of parental SIVmac316 (Fig. 7A) are highlighted in orange (*), blue (**), and purple (***). Green sticks indicate glycans.
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