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Rapid elicitation of broadly neutralizing antibodies to HIV by immunization in cows

Abstract

No immunogen to date has reliably elicited broadly neutralizing antibodies to HIV in humans or animal models. Advances in the design of immunogens that antigenically mimic the HIV envelope glycoprotein (Env), such as the soluble cleaved trimer BG505 SOSIP1, have improved the elicitation of potent isolate-specific antibody responses in rabbits2 and macaques3, but so far failed to induce broadly neutralizing antibodies. One possible reason for this failure is that the relevant antibody repertoires are poorly suited to target the conserved epitope regions on Env, which are somewhat occluded relative to the exposed variable epitopes. Here, to test this hypothesis, we immunized four cows with BG505 SOSIP. The antibody repertoire of cows contains long third heavy chain complementary determining regions (HCDR3) with an ultralong subset that can reach more than 70 amino acids in length4,5,6,7,8,9. Remarkably, BG505 SOSIP immunization resulted in rapid elicitation of broad and potent serum antibody responses in all four cows. Longitudinal serum analysis for one cow showed the development of neutralization breadth (20%, n = 117 cross-clade isolates) in 42 days and 96% breadth (n = 117) at 381 days. A monoclonal antibody isolated from this cow harboured an ultralong HCDR3 of 60 amino acids and neutralized 72% of cross-clade isolates (n = 117) with a potent median IC50 of 0.028 μg ml−1. Breadth was elicited with a single trimer immunogen and did not require additional envelope diversity. Immunization of cows may provide an avenue to rapidly generate antibody prophylactics and therapeutics to address disease agents that have evolved to avoid human antibody responses.

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Figure 1: Cows develop broadly neutralizing serum antibodies when immunized with the HIV envelope trimer mimic BG505 SOSIP.
Figure 2: Rapid development of neutralization breadth and potency in cows.
Figure 3: Isolation and characterization of a broadly neutralizing cow antibody.

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NCBI Reference Sequence

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Acknowledgements

This work was supported by the International AIDS Vaccine Initiative Neutralizing Antibody Consortium through the Collaboration for AIDS Vaccine Discovery grant OPP1084519 (D.R.B., I.A.W., A.B.W.), NIH grants R21 AI120791 (V.V.S.), R01 GM105826 (V.V.S.), Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery Grant UM1AI100663 (D.R.B., I.A.W., A.B.W.), IOS 1257829 (M.F.C.), and USDA-NIFA grant number CSREES 2008-35204 (W.M.). D.R.B. acknowledges the support of the James and Jessie Minor Chair in Immunology. We thank B. Schief and S. Menis for providing MD39 for competition experiments. This work was funded in part by IAVI and made possible by the support of many donors, including: the Bill & Melinda Gates Foundation, the Ministry of Foreign Affairs of Denmark, Irish Aid, the Ministry of Finance of Japan in partnership with The World Bank, the Ministry of Foreign Affairs of the Netherlands, the Norwegian Agency for Development Cooperation (NORAD), the United Kingdom Department for International Development (DFID), and the United States Agency for International Development (USAID). The full list of IAVI donors is available at http://www.iavi.org. The contents of this manuscript do not necessarily reflect the views of USAID or the US Government.

Author information

Authors and Affiliations

Authors

Contributions

D.S., K.M.L., J.R. and A.R. performed antigen B cell sorts. K.M.L., J.R. and A.R. performed PCR and antibody cloning, expression, and purification. K.L.S.-F., K.M.L. and J.R. performed neutralization and mutagenesis experiments. M.L.V. and D.S. designed and validated VH and VL gene-specific primers. D.S., K.M.L., M.L.V. and V.V.S. analysed V-region sequences. L.K. and I.A.W. provided protein for immunization experiments. J.L.T., Z.T.B., R.S., A.B.W. and I.A.W. performed structural analysis. J.G.J. performed pH experiments. C.-H.L. performed polyreactivity experiments. P.L.C., M.F.C. and W.M. performed cow immunization and serum ELISA experiments. P.L.C., M.F.C., W.M. and M.L.V. processed lymphocytes and produced mRNA and cDNA. D.S., V.V.S. and D.R.B. helped design and oversaw experiments. D.S., V.V.S. and D.R.B. wrote the manuscript.

Corresponding authors

Correspondence to Vaughn V. Smider or Dennis R. Burton.

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Competing interests

D.S., D.R.B. and V.V.S. are inventors on a patent describing the NC-Cow antibodies: US provisional application filed on 14 July 2017.

Additional information

Reviewer Information Nature thanks J. Mascola and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 Sera collected at different time points over the course of immunization for cows 26 and 27 were evaluated for neutralization breath and potency on the 12-virus global indicator panel.

Values represent serum ID50 against the indicated virus.

Extended Data Figure 2 Serum tested at given time points for neutralization on BG505 pseudovirus.

Neutralization against autologous virus emerged at the same time as breadth (day 42) and increased in potency over time. Values represent serum ID50.

Extended Data Figure 3 Development of neutralization breadth at 35 days post immunization from cows in experiment 1.

Serum collected at day 35 from experiment 1 was tested for neutralization breadth and potency on the 12-virus global indicator panel. Values represent serum ID50.

Extended Data Figure 4 Sorting strategy for isolating antigen-specific IgG+ B cells.

a, Cow PBMCs were sorted for IgG+ cells that bound to biotinylated BG505 SOSIP-AviTag conjugated on PE and APC fluorophores. b, To isolate epitope-specific antibodies, unliganded BG505 SOSIP (blue) and BG505 SOSIP liganded with NC-Cow1 was used to antigen-sort memory B cells. Epitope-specific B cells are defined as binding unliganded SOSIP and not binding to liganded SOSIP. This sort strategy yielded the broadly neutralizing antibodies NC-Cow7 to NC-Cow10.

Extended Data Figure 5 Functional screening and sequencing information for isolated antibodies.

a, Amplified heavy chains were paired with universal cow light chain or with NC-Cow1 light chain and tested for expression (anti-Fc ELISA), antigen binding (BG505 SOSIP), autologous neutralization (BG505 pseudovirus), and heterologous neutralization (Q23 pseudovirus). Sequence alignments of recovered heavy chains are listed underneath. As ultralong HCDR3 antibodies have been reported to pair with a single germline light chain (V30), amplified heavy chain genes were first paired with the universal cow light chain and screened for expression, binding to BG505 SOSIP, neutralization of BG505 virus, and neutralization of a clade A heterologous virus, Q23. From this dataset, three antibodies (NC-Cow1, NC-Cow2, and NC-Cow3) were selected that showed autologous neutralization (all from day 238) and corresponding native light chains were amplified, with success for only NC-Cow1. These three antibodies were expressed and purified at a larger scale for additional characterization by maintaining native pairing for NC-Cow1 and pairing with germline V30 for NC-Cow2 and NC-Cow3. For the day 70 time point, three heavy chains were selected that showed binding to BG505 SOSIP, but no neutralization, and these antibodies were produced with their native light chain pairs (NC-Cow4 to NC-Cow6). Finally, as NC-Cow1 could neutralize isolate Q23 in the neutralization screen, an additional sort with PBMCs from day 381 was performed, but used BG505 SOSIP liganded with and without NC-Cow1 to enrich for epitope-specific antibodies. The enrichment yielded an additional five hits by neutralization screen, and four out of these five antibodies were produced at larger scale with their native light chain pairs (NC-Cow7 to NC-Cow10). Small-scale screening was also performed with all heavy chains paired with NC-Cow1 light chain and no significant increase in neutralization breadth was observed, although there were slight improvements in BG505 SOSIP affinity and autologous potency. b, Nucleotide alignment of heavy chain sequences of isolated monoclonal antibodies36.

Extended Data Figure 6 NC-Cow1 is not polyreactive to human antigens.

a, NC-Cow1 was tested for antigen reactivity in a HEp-2 assay compared to the known polyreactive antibody 4E10, and negative and positive control sera supplied by the manufacturer. b, NC-Cow1 was also tested for reactivity with a range of typical human autoantigens by ELISA as well as for binding to solubilized membrane (SMP) and cytosolic preparations (SCP) from CHO cells. Values are optical density values (OD405) at a dose of 100 μg ml−1. Black lines indicate cut-off values as indicated by the manufacturer.

Extended Data Figure 7 Epitope mapping of NC-Cow1.

a, Representative 2D class averages of cow Fabs NC-Cow1 and NC-Cow2 bound to BG505 SOSIP trimers to demonstrate CD4bs specificity. The Fabs appear at slightly different angles relative to the trimer, perhaps owing to some flexibility between the Fab and HCDR3. In the 2D class averages for NC-Cow1, the HCDR3 appears as a faint density bridging between the Fab and CD4bs. b, Top and side views of 3D reconstruction of NC-Cow1 bound to BG505 SOSIP trimer with previously published Env trimer structure (green, PDB 5CEZ) and cow Fab (orange, PDB 5IJV) docked in. The body of NC-Cow1 Fab is approximately 20 Å from the CD4bs, which is the estimated length of the ultralong HCDR3. c, NC-Cow 1 was tested by ELISA for binding to BG505 or JRCSF gp120 captured from lysed virions with PGT121 (V3-glycan epitope) and VRC01 (CD4bs epitope) included for comparison. Values are EC50 in μg ml−1. NC-Cow1 was also tested for neutralization on BG505 or JR-CSF pseudoviruses and corresponding alanine mutants with PGT121 (V3-glycan epitope) and 12A12 (CD4bs epitope) included for comparison. Values are IC50 in μg ml−1. d, NC-Cow1 was tested for binding to wild-type and D368R protein. Antibodies VRC01 (CD4bs) and 14e (V3) were included for comparison.

Extended Data Figure 8 Effects of pH on binding of NC-Cow1 and CD4bs antibodies to gp120.

a, CD4bs antibodies PGV04, 12A12, 3BNC60 and b12 were tested for binding to gp120 (isolate 92BR020) by ELISA in buffers at different pHs (3.5, 4.0, 4.5, 5.0, 5.5 and 6.0). b, NC-Cow1 and VRC01 were tested for binding to BG505 gp120 in PBS buffer (pH 7.4) compared to simulated vaginal fluid (SVF) at pH 4.5.

Extended Data Table 1 Evaluation of purified recombinant antibody for neutralization breadth and potency
Extended Data Table 2 Mapping epitope specificity over time

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Sok, D., Le, K., Vadnais, M. et al. Rapid elicitation of broadly neutralizing antibodies to HIV by immunization in cows. Nature 548, 108–111 (2017). https://doi.org/10.1038/nature23301

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