Broad and ultra-potent cross-clade neutralization of HIV-1 by a vaccine-induced CD4 binding site bovine antibody

Abstract

Human immunodeficiency virus type 1 (HIV-1) vaccination of cows has elicited broadly neutralizing antibodies (bNAbs). In this study, monoclonal antibodies (mAbs) are isolated from a clade A (KNH1144 and BG505) vaccinated cow using a heterologous clade B antigen (AD8). CD4 binding site (CD4bs) bNAb (MEL-1872) is more potent than a majority of CD4bs bNAbs isolated so far. MEL-1872 mAb with CDRH3 of 57 amino acids shows more potency (geometric mean half-maximal inhibitory concentration [IC₅₀]: 0.009 μg/mL; breadth: 66%) than VRC01 against clade B viruses (29-fold) and than CHO1-31 against tested clade A viruses (21-fold). It also shows more breadth and potency than NC-Cow1, the only other reported anti-HIV-1 bovine bNAb, which has 60% breadth with geometric mean IC₅₀ of 0.090 μg/mL in this study. Using successive different stable-structured SOSIP trimers in bovines can elicit bNAbs focusing on epitopes ubiquitous across subtypes. Furthermore, the cross-clade selection strategy also results in ultra-potent bNAbs.

Keywords: CDRH3; HIV-1; antibody; antiretroviral; cows; envelope glycoprotein; neutralization; vaccine.

Graphical abstract

Figure 1

HIV Env binding and neutralization of serum samples collected from immunized cows (A) Vaccination of cows during pregnancy. Cow no. 1 was vaccinated with KNH1144 SOSIP gp140 and BG505 gp140, while cows no. 2 and no. 3 were vaccinated with AD8 uncleaved gp140 followed by BG505 uncleaved gp140 and AD8 SOSIP gp140, respectively. (B) Env binding of bovine IgGs in sera of vaccinated cows. Binding was measured against Env vaccine (cow no. 1: BG505 SOSIP; cow no. 2 and cow no. 3: AD8 SOSIP) through direct ELISA. (C) Neutralization assays were performed against seven pseudoviruses from clades A, B, and C and tiers 1A, 1B, and 2; as negative control, MuLV pseudovirus was used. The values show ID₅₀. Heatmap scale shows no neutralization from a value of ID₅₀ = 10 (white values) to the highest neutralization achieved at ID₅₀ = 1,000 (red values). ELISA assays were performed in duplicate with two independent biological replicates.

Figure 2

Isolation of bNAbs from HIV-1 vaccinated cows (A) Cow PBMCs were sorted for IgG⁺ cells that bound to biotinylated AD8 SOSIP-AviTag conjugated to phycoerythrin (PE) and antigen-presenting cell (APC) fluorophores. FSC, forward scatter; SSC, side scatter. (B) Bovine bNAbs showed potent cross-clade neutralization against tier 1 and tier 2 viruses with low geometric mean IC₅₀. Neutralization assays were performed in duplicates with two independent biological replicates.

Figure 3

Neutralization profile of bNAb MEL-1842, MEL-1872, and MEL-2129 Comparison of IC₅₀ (A) and IC₈₀ (B) values of isolated mAbs with bNAb NC-COW1. Categorization and comparison of neutralization activity against different HIV clades using IC₅₀ (C) and IC₈₀ (D) values is shown. The black lines represent the geometric mean IC₅₀ and IC₈₀. Neutralization assays were performed in duplicates with two independent biological replicates.

Figure 4

Amino acid alignment of heavy- and light-chain sequences of MEL-1872 mAb with the germline genes Purple: IGLV1-47∗01 germline gene; gray: IGLJ4∗01 germline gene; blue: IGHV1-7∗02 germline gene; green: IGHD8-2∗01 germline gene; red: IGHJ2-4∗01 germline gene.

Figure 5

Epitope mapping of bovine monoclonal antibodies (A) Binding of bovine bNAbs to HIV Envs. Bovine bNAbs were tested in direct ELISA assays to evaluate their binding to different forms of Env (monomeric gp120, uncleaved gp140, and SOSIP gp140) as well as ConM SOSIP, which is an Env trimer based on a consensus sequence of all HIV-1 group M isolates. (B) Competition of bovine bNAbs with human bNAb for Env binding. The table shows the competition ELISA assay with values demonstrating Env binding inhibition (percentage) of human bNAbs by bovine antibodies. Competition ELISA between antibodies MEL-1842, MEL-1872, and MEL-2129 showed these antibodies bind to the same or proximate epitopes. Higher competition is shown in red, and lower inhibition values are in increasingly pale shades of orange. ELISA assays were performed in duplicates with two independent biological replicates. (C) The effect of alanine mutagenesis on binding of bovine antibodies to AD8 gp120 captured from lysed virions. ELISA assay was performed using a constant half-maximal effective concentration (EC₅₀) of each antibody to AD8 WT Env. Stars also show significant neutralization IC₅₀ increase of each antibody against mutated virus compared with WT virus (5-fold for all mAbs, except 198). PGT121 (V3-glycan epitope) and b12 and VRC01 (CD4bs epitope) were included for comparison. ELISA and neutralization assays were performed in duplicate with two independent biological replicates.

Figure 6

Binding of bovine bNAbs to CD4bs Highlighted on the BG505 SOSIP.664 trimer (top left) are residues from the D loop (275–283: SNFTDNAKN, pale pink), CD4 binding loop (362–375: NQSSGGDPEIVMHS, cyan), and V5 loop (458–469: GGNNHNNDTETFR, pale orange). Binding site of antibodies MEL-1842, MEL-1872, MEL-2129, and VRC01 to AD8 SOSIP V4.1 are shown with red color, indicating the residues that binding of bovine antibodies to them was reduced through mutagenesis. Homology model of AD8 SOSIP was obtained using Swiss Modeller.

Figure 7

Assessment of bovine bNAb polyreactivity (A) Polyreactivity test in Hep-2 cells. Bovine bNAbs MEL-1842, MEL-1872, MEL-2129, and MEL-198 did not show any polyreactivity against human Hep-2 cells. 2F5 is human anti-HIV bNAb with polyreactivity, while anti-HIV bNAb PGT121 is not polyreactive. (B) Assessment of antibody polyreactivity against human antigens. ELISA assay was performed against human antigens using constant amount of 100 μg/mL from tested mAbs. Black line indicates cutoff values as indicated by the manufacturer. Each assay was repeated twice. snRNP, small nuclear ribonucleoprotein particle.