Broad and potent neutralizing antibody responses elicited in natural HIV-2 infection

Abstract

Compared with human immunodeficiency virus type 1 (HIV-1), little is known about the susceptibility of HIV-2 to antibody neutralization. We characterized the potency and breadth of neutralizing antibody (NAb) responses in 64 subjects chronically infected with HIV-2 against three primary HIV-2 strains: HIV-2(7312A), HIV-2(ST), and HIV-2(UC1). Surprisingly, we observed in a single-cycle JC53bl-13/TZM-bl virus entry assay median reciprocal 50% inhibitory concentration (IC(50)) NAb titers of 1.7 × 10(5), 2.8 × 10(4), and 3.3 × 10(4), respectively. A subset of 5 patient plasma samples tested against a larger panel of 17 HIV-2 strains where the extracellular gp160 domain was substituted into the HIV-2(7312A) proviral backbone showed potent neutralization of all but 4 viruses. The specificity of antibody neutralization was confirmed using IgG purified from patient plasma, HIV-2 Envs cloned by single-genome amplification, viruses grown in human CD4(+) T cells and tested for neutralization sensitivity on human CD4(+) T target cells, and, as negative controls, env-minus viruses pseudotyped with HIV-1, vesicular stomatitis virus, or murine leukemia virus Env glycoproteins. Human monoclonal antibodies (MAbs) specific for HIV-2 V3 (6.10F), V4 (1.7A), CD4 binding site (CD4bs; 6.10B), CD4 induced (CD4i; 1.4H), and membrane-proximal external region (MPER; 4E10) epitopes potently neutralized the majority of 32 HIV-2 strains bearing Envs from 13 subjects. Patient antibodies competed with V3, V4, and CD4bs MAbs for binding to monomeric HIV-2 gp120 at titers that correlated significantly with NAb titers. HIV-2 MPER antibodies did not contribute to neutralization breadth or potency. These findings indicate that HIV-2 Env is highly immunogenic in natural infection, that high-titer broadly neutralizing antibodies are commonly elicited, and that unlike HIV-1, native HIV-2 Env trimers expose multiple broadly cross-reactive epitopes readily accessible to NAbs.

Fig 1

Neutralization of primary HIV-2 and HIV-1 strains by plasma or serum from patients chronically infected by HIV-2 or HIV-1. (A) Neutralization titers (reciprocal IC₅₀s) of HIV-2-infected patient plasma or serum (n = 64) against primary HIV-2 strains HIV-2_7312A (group A), HIV-2_ST (group A), and HIV-2_UC1 (group B) and HIV-1 strain HIV-1_BG1168.1. IC₅₀s not reached at a starting dilution of 1:100 or 1:20 are represented as <100 and <20, respectively. Neutralization titers of 10 clade B (n = 5) or clade C (n = 5) HIV-1-infected patient plasma samples against the primary HIV-1 strains HIV-1_CH40 (subtype B, tier 2), HIV-1_ZM249 (subtype C, tier 2), and HIV-1_TRJO (subtype B, tier 3) are shown. Virus stocks were grown in 293T cells. Median values are represented by a horizontal line. (B) Neutralization titers of five HIV-2-infected patient plasma samples (7312Apl2003, SLRHCpl1995, 10849pl1995, 8704Apl2007, and 7810Apl1993) against 17 p7/SNAG-HIV2env chimeric viruses.

Fig 2

IgG-mediated neutralization of HIV-2. Neutralization of 293T-grown HIV-2_7312A by plasma from HIV-2-infected patients 7312Apl2003, SLRHCpl1995, and 10849pl1995 and healthy donors B, D, and J. IgG purified from these plasma samples are indicated 7312A IgG, SLRHC IgG, 10849 IgG, Donor B IgG, Donor D IgG, and Donor J IgG in the TZM-bl assay. Purified IgG was reconstituted in buffer having the same volume as the original plasma, and similarity in IgG concentrations was confirmed. Error bars represent three independent experiments. Dashed line indicates 50% reduction in virus infectivity.

Fig 3

Comparison of HIV-2 neutralization titrations using different assay formats. (A) Neutralization of human CD4⁺ lymphocyte-grown HIV-2_7312A and HIV-2_ST stocks by four HIV-2-infected patient plasma samples in the human CD4⁺ T-cell multicycle infectivity assay. (B) Neutralization of human CD4⁺ T-cell-grown versus 293T-cell-grown HIV-2_7312A and HIV-2_ST by five HIV-2-infected patient plasma samples in the TZM-bl assay. (C) Summary analysis of reciprocal IC₅₀ neutralization shown in panels A and B. One-tailed paired t tests were performed, and P values are shown. Error bars were generated on the basis of at least three independent experiments. Dashed line indicates 50% reduction in virus infectivity.

Fig 4

Autologous and heterologous neutralization of single-genome amplification (SGA)-derived HIV-2 Env-pseudotyped virus. (A) Neighbor-joining tree of HIV-2 gp160 env gene sequences amplified from 7312Apl1992 (blue) and 7312Apl2003 (red) by SGA. HIV-2 group A (HIV-2_7312A, HIV-2_ST, HIV-2_BEN, and HIV-2_ROD) and group B (HIV-2_D205 and HIV-2_UC1) reference sequences are included. The env genes tested for neutralization in panel B are labeled with asterisks. (B) 7312A-92 and 7312A-03 Envs were used to pseudotype the env-minus HIV-2_7312A backbone pJK7312AΔEnv in 293T cells. Autologous neutralization by plasma samples 7312Apl1992 (top) and 7312Apl2003 (bottom) in the TZM-bl assay is shown. (C) Neighbor-joining tree of HIV-2 gp160 env gene sequences amplified from 8704Apl2006 by SGA (orange). The env genes tested for neutralization in panel D are labeled with asterisks. (D) 8704A-06 Envs were used to pseudotype the env-minus HIV-2_7312A backbone pJK7312AΔEnv in 293T cells. Autologous neutralization by plasma samples 8704Apl2006 (top) and 8704Apl2007 (bottom) in the TZM-bl assay is shown. (E) Reciprocal IC₅₀ of autologous plasma (7312Apl1992 and 7312Apl2003) and heterologous plasma (SLRHCpl1995, 10849pl1995, and 8704Apl2007) against 7312A-92 variants and 7312A-03 variants. (F) Reciprocal IC₅₀ of autologous plasma (8704Apl2006 and 8704Apl2007) and heterologous plasma (7312Apl2003, SLRHCpl1995, and 10849pl1995) against 8704A-06 variants. Error bars reflect data from at least three independent experiments.

Fig 5

Absence of MPER neutralizing antibodies in serum from HIV-2-infected patients. (A) Construction of the HIV-1/HIV-2 chimeric virus BG1168.1-C1 containing the complete MPER from HIV-2_ST. (B) Neutralization of HIV-1_BG1168.1, HIV-2_ST, and BG1168.1-C1 by anti-MPER antibodies 4E10 and 2F5. (C) Neutralization of HIV-1_BG1168.1, HIV-2_ST, and BG1168.1-C1 by 53 HIV-2-infected patient serum samples. Positive neutralization was defined as a reciprocal IC₅₀ of at least 100. One HIV-2-infected serum sample was positive for neutralization of the BG1168.1-C1 chimera with a reciprocal IC₅₀ titer of 252. The median reciprocal IC₅₀ titer of the 53 serum specimens against HIV-2_ST was 2.8 × 10⁴.

Fig 6

Correlation between serum neutralization titers and MAb competition titers. (A) x axis, reciprocal serum dilutions yielding 50% inhibition of 1.7A binding to HIV-2_ST gp120 (reciprocal EC₅₀); y axis, virus neutralization titers (reciprocal IC₅₀) of serum against HIV-2_ST in the TZM-bl assay. Pearson correlation and linear regression analyses were performed. The correlation coefficient r, P value, and linear regression line are shown. Similar analyses are depicted for MAb 6.10F (B) and 6.10B (C) binding to HIV-2_ST gp120 and 1.7A binding to HIV-2_MVP15132 gp120 (D).

Fig 7

Potential N-linked glycosylation sites in the V4 loops of HIV-1 and HIV-2 strains. Mean values are shown as dashed lines. For HIV-1, the mean was 4.34, the median was 4, and the range was 2 to 7. For HIV-2, the mean was 2.48, the median was 2, and the range was 2 to 3. The HIV-1 V4 sequences exhibited a significantly higher level of glycosylation than the HIV-2 V4 sequences (P < 0.0001, Mann-Whitney test).

Fig 8

Structural models and relative neutralization sensitivities of primary and laboratory-adapted strains of HIV-1 compared with primary HIV-2 strains. Structural models of gp120s from HIV-2 primary (7312A), HIV-1 primary (YU2), and HIV-1 laboratory-adapted (HXBc2) strains are shown with modeled glycans and epitopes for V3, V4, CD4bs, and CD4i highlighted. Neutralization potencies of V3, V4, CD4bs, and CD4i antibodies are estimated schematically, with full bars corresponding to highly potent neutralization, medium bars to moderate neutralization, and small bars to very weak or nonneutralizing phenotypes. V4-directed antibodies are rarely observed in natural HIV-1 infection, but placement of antigenic tags into the V4 region reveals that antibodies directed here can neutralize HIV-1 potently (41). For V3-, CD4bs-, and CD4i-directed antibodies, epitopes are generally hidden on the assembled HIV-1 viral spike of primary HIV-1 strains by conformational masking. The increased flexibility of laboratory-adapted HIV-1 isolates allows exposure of these epitopes, which resembles the neutralization sensitivities observed with HIV-2 (Table 1).