Neutralization properties of simian immunodeficiency viruses infecting chimpanzees and gorillas

Abstract

Broadly cross-reactive neutralizing antibodies (bNabs) represent powerful tools to combat human immunodeficiency virus type 1 (HIV-1) infection. Here, we examined whether HIV-1-specific bNabs are capable of cross-neutralizing distantly related simian immunodeficiency viruses (SIVs) infecting central (Pan troglodytes troglodytes) (SIVcpzPtt) and eastern (Pan troglodytes schweinfurthii) (SIVcpzPts) chimpanzees (n = 11) as well as western gorillas (Gorilla gorilla gorilla) (SIVgor) (n = 1). We found that bNabs directed against the CD4 binding site (n = 10), peptidoglycans at the base of variable loop 3 (V3) (n = 5), and epitopes at the interface of surface (gp120) and membrane-bound (gp41) envelope glycoproteins (n = 5) failed to neutralize SIVcpz and SIVgor strains. In addition, apex V2-directed bNabs (n = 3) as well as llama-derived (heavy chain only) antibodies (n = 6) recognizing both the CD4 binding site and gp41 epitopes were either completely inactive or neutralized only a fraction of SIVcpzPtt strains. In contrast, one antibody targeting the membrane-proximal external region (MPER) of gp41 (10E8), functional CD4 and CCR5 receptor mimetics (eCD4-Ig, eCD4-Ig(mim2), CD4-218.3-E51, and CD4-218.3-E51-mim2), as well as mono- and bispecific anti-human CD4 (iMab and LM52) and CCR5 (PRO140, PRO140-10E8) receptor antibodies neutralized >90% of SIVcpz and SIVgor strains with low-nanomolar (0.13 to 8.4 nM) potency. Importantly, the latter antibodies blocked virus entry not only in TZM-bl cells but also in Cf2Th cells expressing chimpanzee CD4 and CCR5 and neutralized SIVcpz in chimpanzee CD4(+) T cells, with 50% inhibitory concentrations (IC50s) ranging from 3.6 to 40.5 nM. These findings provide new insight into the protective capacity of anti-HIV-1 bNabs and identify candidates for further development to combat SIVcpz infection.

Importance: SIVcpz is widespread in wild-living chimpanzees and can cause AIDS-like immunopathology and clinical disease. HIV-1 infection of humans can be controlled by antiretroviral therapy; however, treatment of wild-living African apes with current drug regimens is not feasible. Nonetheless, it may be possible to curb the spread of SIVcpz in select ape communities using vectored immunoprophylaxis and/or therapy. Here, we show that antibodies and antibody-like inhibitors developed to combat HIV-1 infection in humans are capable of neutralizing genetically diverse SIVcpz and SIVgor strains with considerable breadth and potency, including in primary chimpanzee CD4(+) T cells. These reagents provide an important first step toward translating intervention strategies currently developed to treat and prevent AIDS in humans to SIV-infected apes.

FIG 1

Neutralizing antibody responses in long-term HIV-1- and SIVcpz-infected chimpanzees. (A) Phylogenetic relationship of HIV-1, SIVcpz, and SIVgor infectious molecular clones (IMCs). A maximum likelihood phylogenetic tree of Env (gp160) protein sequences is depicted, with sequences color coded to differentiate HIV-1 (black), SIVgor (green), SIVcpzPtt (red), and SIVcpzPts (blue) strains. Bootstrap values of ≥70% are shown; the scale bar represents 0.1 amino acid replacement per site. (B) Plasma samples from eight long-term-infected chimpanzees (listed on the right and shown in Table 1) were tested against HIV-1 (n = 3), SIVcpzPtt (n = 6), SIVgor (n = 1), and SIVcpzPts (n = 5) strains (bottom) in the TZM-bl neutralization assay. (Collection dates are indicated for samples from the same individual.) IC₅₀s (expressed as plasma dilutions) averaged from three replicate experiments are shown, with a heat map indicating the relative neutralization potency.

FIG 2

Neutralizing capacity of CD4 binding site (CD4bs) antibodies. (A) The ability of CD4bs monoclonal antibodies (listed on the right) to neutralize HIV-1, SIVcpz, and SIVgor strains (listed on the bottom) is shown. Numbers indicate IC₅₀s (in micrograms per milliliter) in TZM-bl cells, averaged from three different experiments, with a heat map indicating the relative neutralization potency. The highest antibody concentration was 10 µg/ml. A herpesvirus antibody (anti-HSV-gD) was used as a negative control. (B) Conservation of HIV-1, SIVcpz, and SIVgor strains in the CD4 binding region. An alignment of Env protein sequences (left) in regions surrounding the CD4 binding site is shown. CD4 and VRC01 contact resides (indicated in the HXB2 reference strain) are highlighted in orange and green, respectively. A logo plot above the alignment denotes the conservation of each amino acid, with the height of each letter indicating the proportion of the sequences that contain the residue at that site. Dots indicate identity to the HXB2 reference sequence, and dashes represent gaps introduced to improve the alignment.

FIG 3

Neutralizing capacity of high-mannose-patch- and apex V2-directed antibodies. (A) The ability of peptidoglycan-associated monoclonal antibodies (listed on the right) to neutralize HIV-1, SIVcpz, and SIVgor strains (bottom) is shown. Numbers indicate IC₅₀s (in micrograms per milliliter) in TZM-bl cells, averaged from three different experiments, with a heat map indicating the relative neutralization potency. Colored circles to the right of each antibody indicate the N-linked glycans that are important for their neutralizing activity (orange, N156; purple, N160; pink, N301; green, N332; red, N386; yellow, N392) (27, 28, 52, 81). The highest antibody concentration used was 10 µg/ml. (B) Conservation of glycans associated with bNab activity. An alignment of HIV-1, SIVcpz, and SIVgor Env protein sequences is shown, with predicted N-linked glycans (NXS/T) highlighted in red. Four residues comprising a lysine-rich motif in the V1/V2 strand (C), which together with glycans N156 and N160 form the core epitope for PG9, PG16, and PGT145, are highlighted in purple. N-linked glycans known to influence bNab binding are highlighted above the alignment, with HXB2 numbering in black. The positions of variable loops (V1/V2, V3, and V4) are shown in gray below the alignment.

FIG 4

Neutralizing capacity of antibodies targeting the interface of HIV-1 gp120 and gp41 regions. (A) The ability of glycan-associated antibodies (right) to neutralize HIV-1, SIVcpz, and SIVgor strains (bottom) is shown. Numbers indicate IC₅₀s (in micrograms per milliliter) from TZM-bl cells, averaged from three different experiments, with a heat map indicating the relative neutralization potency. Colored shapes to the right of each antibody indicate the N-linked glycans that are associated with antibody neutralizing activity. Antibody 8ANC195 contacts N234 (green circle) and N276 (green square), 35O22 utilizes N88 (blue circle), N230 (blue square), N241 (blue triangle), and N625 (blue square), and PGT151 requires N611 (pink circle) and N637 (pink square) for optimal neutralization (53–56). The highest antibody concentration used was 10 µg/ml. (B) Conservation of glycans associated with antibody neutralizing activity. An alignment of HIV-1, SIVcpz, and SIVgor Env protein sequences is shown, with predicted N-linked glycans (NXS/T) highlighted in red. N-linked glycans known to influence bNab binding are highlighted above the alignment, with HXB2 numbering in black. The positions of various Env regions are shown in gray below the alignment.

FIG 5

Neutralizing capacity of camelid antibodies. (A) The ability of llama-derived (heavy-chain-only) antibodies (listed on the right) to neutralize HIV-1, SIVcpz, and SIVgor strains (bottom) is shown. Numbers indicate IC₅₀s (in micrograms per milliliter) from TZM-bl cells, averaged from three different experiments, with a heat map indicating the relative neutralization potency. The highest antibody concentration used was 10 µg/ml. (B to D) Conservation of antibody binding epitopes shown in panel A. Alignments of HIV-1, SIVcpz, and SIVgor Env protein sequences are depicted, with residues identical to the HXB2 reference shown in gray. Logo plots denote the conservation of individual amino acids within each epitope, with the height of each letter indicating the proportion of sequences that contain the residue at that site. The letter “X” indicates residues within the 2H10 epitope that do not impact neutralization. JM4 contact residues are indicated on top of the alignment. Sequences are numbered according to the HXB2 reference.

FIG 6

Neutralizing capacity of MPER antibodies. (A) The ability of two MPER antibodies (listed on the right) to neutralize a panel of HIV-1, SIVcpz, and SIVgor strains (bottom) is shown. Numbers indicate IC₅₀s (in micrograms per milliliter) from TZM-bl cells, averaged from three different experiments, with a heat map indicating the relative neutralization potency. The highest antibody concentration used was 10 µg/ml. (B) Conservation of 4E10 and 10E8 epitopes. An alignment of HIV-1, SIVcpz, and SIVgor Env protein sequences is depicted, with dots indicating identity to the HXB2 reference sequence. A logo plot denotes the conservation of individual amino acids, with the height of each letter indicating the proportion of sequences that contain the residue at that site. Contact residues of 4E10 (blue) and 10E8 (red) are highlighted above the alignment.

FIG 7

Neutralizing capacity of anti-host receptor antibodies (A) The ability of monospecific anti-human CD4 (iMab and LM52) and anti-CCR5 (PRO140) antibodies as well as their bispecific derivatives (PG9-iMab, PG16-iMab, LM52-PGT128, and PRO140-10E8) (listed on the right) to neutralize a panel of HIV-1, SIVcpz, and SIVgor strains (bottom) is shown. Numbers indicate IC₅₀s (in micrograms per milliliter) from TZM-bl cells, averaged from three different experiments, with a heat map indicating the relative neutralization potency. The geometric mean IC₅₀ is shown for each antibody. (Only values for SIVcpz and SIVgor strains that were below 10 µg/ml were included in the calculation.) (B) Correlation of TZM-bl- and Cf2Th-hu-derived neutralization data. Three bNabs (10E8, PG16, and eCD4-Ig^mim2) were used to neutralize a subset of HIV-1 and SIVcpz strains (YU-2, GAB1, EK505, MT145, and TAN13) in TZM-bl and Cf2Th cells expressing human CD4 and CCR5 receptors (Cf2Th-hu). IC₅₀s from TZM-bl (x axis) and Cf2Th-hu (y axis) cells were plotted and analyzed using the Spearman correlation test. (C) Correlation of Cf2Th-hu and Cf2Th-ch neutralization data. The same three bNabs shown in panel B were used to neutralize the same subset of HIV-1 and SIVcpz strains in Cf2Th cells expressing human (Cf2Th-hu) and chimpanzee (Cf2Th-ch) CD4 and CCR5 receptors. IC₅₀s from Cf2Th-hu (x axis) and Cf2Th-ch (y axis) cells were plotted and analyzed using the Spearman correlation. (D) The ability of antireceptor antibodies (listed on the right) to neutralize a subset of HIV-1 and SIVcpz strains (bottom) in Cf2Th cells expressing either human (left panel) or chimpanzee (right panel) CD4 and CCR5 receptors is shown. Numbers indicate IC₅₀s (in micrograms per milliliter) averaged from three different experiments, with heat maps indicating relative neutralizing potencies. The geometric mean IC₅₀ is shown for each antibody. (Only values from SIVcpz strains were included.)

FIG 8

Neutralizing capacity of human CD4 D1/D2 domain containing immunoadhesins. (A) Schematic representation of six constructs. Human CD4 D1 and D2 domains are shown in green, immunoglobulin (IgG) Fc and constant heavy- and light-chain (C_H/C_L) regions are shown in black, E51 variable heavy and light (V_H/V_L) regions are shown in blue, CCR5 mimetic peptides are shown in yellow, and the T-20 fusion inhibitor is shown in red. (B) The ability of human CD4 containing antibody-like constructs (listed on the right) to neutralize a panel of HIV-1, SIVcpz, and SIVgor strains (bottom) is shown. Numbers indicate IC₅₀s (in micrograms per milliliter) from TZM-bl cells, averaged from three different experiments, with a heat map indicating the relative neutralization potency. The highest antibody concentration used was 10 µg/ml. The geometric mean IC₅₀ is shown for each construct. (Only values for SIVcpz and SIVgor strains that were below 10 µg/ml were included in the calculation.)

FIG 9

Breadth and potency of antibodies and antibody-like constructs with anti-SIVcpz and anti-SIVgor neutralizing activity. For each bNab, the percentage of SIVcpz and SIVgor strains neutralized with IC₅₀s of <10 µg/ml (y axis) is plotted against the corresponding geometric mean IC₅₀ (nanomolar concentration) (x axis). Antibodies and antibody-like constructs are color coded, with those that exhibited no anti-SIVcpz and SIVgor activity listed in the left lower corner. Horizontal and vertical bars denote 90% breadth and 10 nM potency, respectively, and the most potent and cross-reactive reagents are circled.