Broadly neutralizing antibodies to SARS-related viruses can be readily induced in rhesus macaques

TLDR: This paper showed that immunization of rhesus macaques with SARS-CoV-2 spike (S) protein generates potent receptor binding domain cross-neutralizing antibody (nAb) responses to both SARS and CoV-1.

Abstract: To prepare for future coronavirus (CoV) pandemics, it is desirable to generate vaccines capable of eliciting neutralizing antibody responses against multiple CoVs. Because of the phylogenetic similarity to humans, rhesus macaques are an animal model of choice for many virus-challenge and vaccine-evaluation studies, including SARS-CoV-2. Here, we show that immunization of macaques with SARS-CoV-2 spike (S) protein generates potent receptor binding domain cross- neutralizing antibody (nAb) responses to both SARS-CoV-2 and SARS-CoV-1, in contrast to human infection or vaccination where responses are typically SARS-CoV-2-specific. Furthermore, the macaque nAbs are equally effective against SARS-CoV-2 variants of concern. Structural studies show that different immunodominant sites are targeted by the two primate species. Human antibodies generally target epitopes strongly overlapping the ACE2 receptor binding site (RBS), whereas the macaque antibodies recognize a relatively conserved region proximal to the RBS that represents another potential pan-SARS-related virus site rarely targeted by human antibodies. B cell repertoire differences between the two primates appear to significantly influence the vaccine response and suggest care in the use of rhesus macaques in evaluation of vaccines to SARS-related viruses intended for human use. ONE SENTENCE SUMMARY Broadly neutralizing antibodies to an unappreciated site of conservation in the RBD in SARS- related viruses can be readily induced in rhesus macaques because of distinct properties of the naive macaque B cell repertoire that suggest prudence in the use of the macaque model in SARS vaccine evaluation and design.

Figures

Fig. 4. Immunogenetic and epitope properties of sarbecovirus cross-neutralizing macaque antibodies. (A) Phylogenetic trees derived from heavy chain (VH)-gene sequences of S-protein specific antibodies isolated from K398 and K288 macaques. Gene assignments used the rhesus macaque (Macaca mulatta) germline database described in (62). The IGHV gene usage for each mAb depicted in the tree is shown by different colors indicated in the lower panel of the color scheme. Neutralizing and non-neutralizing mAbs are shown in red and blue, respectively. (B) IGHV gene usage for neutralizing and non-neutralizing mAbs in K288 and K398 macaques. (C) CDRH3 length and number of SHMs for neutralizing and non-neutralizing mAbs. (*p <0.1; ****p < 0.0001). AA, amino acids. (D) Heatmap showing BLI competition-based epitope binning of macaque cross-neutralizing mAbs with human RBD-specific nAbs CC12.3, CC12.19, CR3022, and S309.The gene usage, geomean neutralization potency and breadth (calculated from neutralization with 5 viruses in figure 3B-C) for each nAb are indicated. The BLI competition

Fig. 2. SARS-CoV-2 S-protein immunized macaque sera show neutralization with sarbecovirus spike RBD-swapped chimeric SARS-CoV-2 pseudotyped viruses and SARSCoV-2 variants circulating in humans. (A) Phylogenetic tree of 12 spike glycoproteins representing major sarbecovirus lineages. The five taxa highlighted in beige are SARS-like viruses, related by a common ancestor, that circulate in humans, bats and pangolins. Highlighted strains that produced infectious viruses in an hACE2-expressing reporter assay were selected for the characterization of cross-reactive neutralizing antibody responses. (B) Heatmap showing ID50 neutralizing antibody titers of SARS-CoV-2 S-protein-immunized wk14 macaque immune sera (left), COVID-19 convalescent sera (middle), and spike-mRNA human vaccinee sera with SARSCoV-2, SARS-CoV-1, and RBD-swapped WIV1, RaTG13, and pang17 chimeric SARS-CoV-2 pseudoviruses. ID50 neutralization titers less than 60 are shown in gray. (C) SARS-CoV-2 spike variants of concern (VOCs), B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma) and B.1.617 (delta),

Fig. 6. Crystal structures of two rhesus macaque IGHV3-73-encoded neutralizing antibodies bound to SARS-CoV-2 RBD. The SARS-CoV-2 RBD is shown in white throughout the figure, while human ACE2 (hACE2) is in pale green, heavy and light chains of K288.2 are in cyan and light cyan, and those of K398.22 in orange and yellow. For clarity, only variable domains of the antibodies are shown. Structures of SARS-CoV-2 RBD in complex with K288.2 antibody (A-D) and in complex with K398.22 antibody (E-H). Germline genes encoding K288.2 and K398.22 macaque antibodies are labeled in (A, E). Structures of SARS-CoV-2 RBD in complex with K288.2 or K398.22 superimposed onto an RBD/hACE2 complex structure (PDB 6M0J, (74)) show that the K288.2/K398.22 antibody would clash with the hACE2 receptor (indicated with a red circle). (B, F) The epitope of K288.2 or K398.22 is shown in purple. CDR loops that interact

Fig. 3. Isolation and characterization of cross-neutralizing monoclonal antibodies from two SARS-CoV-2 S-protein-immunized rhesus macaques. (A) Flow cytometry plots showing the sorting strategy used to isolate single isotype-switched B cells that specifically bind to both SARSCoV-1 and SARS-CoV-2 S-protein fluorescently labelled probes. The class-switched memory B cells were gated as SSL, CD3−, CD4−, CD8−, CD14−, CD19+/CD20+, IgM−, IgG+. The antigen

Fig. 1. SARS-CoV-2 and SARS-CoV-1 cross-reactive binding and neutralizing antibody responses in SARS-CoV-2 S protein-immunized rhesus macaques. (A) SARS-CoV-2 S protein prime-boost immunization in rhesus macaques and sampling schedule. Animals were

Fig. 5. Electron microscopy structures of sarbecovirus cross-neutralizing macaque mAbs with SARS-CoV-2 and SARS-CoV-1 S-proteins. (A) Electron microscopy 3D reconstructions of rhesus macaque nAb Fabs with SARS-CoV-2 and SARS-CoV-1 S-proteins. Fabs of six RM nAbs, K288.2 (blue), K398.22 (red), K398.8 (green), (K398.18 (yellow), K398.25 (purple) and K398.16 (orange) were complexed with SARS-CoV-2 and SARS-CoV-1 S-proteins and 3D reconstructions were generated from 2D class averages. The heavy chain germline gene usage is indicated for each macaque nAb. The spikes S1 and S2 subunits and the RBD sites are labelled. (B) Side and top views showing 3D reconstructions of all 6 RBD-directed macaque cross-nAbs bound to SARSCoV-2 (upper panel) and SARS-CoV-1 (lower panel) S-proteins. (C) S-protein binding angles of

Full text

1
Broadly neutralizing antibodies to SARS-related viruses
can be readily induced in rhesus macaques
Wan-ting He
1,3,4*
, Meng Yuan
2,*
, Sean Callaghan
1,3,4*
, Rami Musharrafieh
1,3,4
, Ge Song
1,3,4
, Murillo
Silva
4,5
, Nathan Beutler
1
, Wilma Lee
2
, Peter Yong
1,3,4
, Jonathan Torres
2
, Mariane Melo
4,5
, Panpan
Zhou
1,3,4
, Fangzhu Zhao
1,3,4,
, Xueyong Zhu
2
, Linghang Peng
1
, Deli Huang
1
, Fabio Anzanello
1,3,4
,
James Ricketts
1
, Mara Parren
1
, Elijah Garcia
1
, Melissa Ferguson
6
, William Rinaldi
6
, Stephen A.
Rawlings
7
, David Nemazee
1
, Davey M. Smith
7
, Bryan Briney
1,3,4
, Yana Safonova
8
, Thomas F.
Rogers
1,7
, Shane Crotty
4,7,9
, Darrell J. Irvine
4,5,10,11,12
, Andrew B. Ward
2,3,4
, Ian A. Wilson
2,3,4,13,†
,
Dennis R. Burton
1,3,4,12,†
, Raiees Andrabi
1,3,4,†
1
Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA
92037, USA.
2
Department of Integrative Structural and Computational Biology, The Scripps Research
Institute, La Jolla, CA 92037, USA.
3
IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA 92037, USA
4
Consortium for HIV/AIDS Vaccine Development (CHAVD), The Scripps Research Institute, La
Jolla, CA 92037, USA.
5
Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology,
Cambridge, MA 02139, USA
6
Alpha Genesis, Yemassee, SC 29945, USA
7
Division of Infectious Diseases and Global Public Health, Department of Medicine, University of
California, San Diego (UCSD), La Jolla, CA 92037, USA.
8
Computer Science and Engineering Department, University of California, San Diego (UCSD),
La Jolla, CA 92037, USA
.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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2
9
Center for Infectious Disease and Vaccine Research, La Jolla Institute for Immunology, La
Jolla, CA 92037, USA
10
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA
02139, USA
11
Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA
12
Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology,
and Harvard University, Cambridge, MA 02139, USA.
13
Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037,
USA
*
These authors contributed equally to this work.
†
Corresponding author. Email: wilson@scripps.edu (I.A.W); burton@scripps.edu (D.R.B.);
andrabi@scripps.edu (R.A.).
.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 6, 2021. ; https://doi.org/10.1101/2021.07.05.451222doi: bioRxiv preprint

3
ABSTRACT
To prepare for future coronavirus (CoV) pandemics, it is desirable to generate vaccines capable
of eliciting neutralizing antibody responses against multiple CoVs. Because of the phylogenetic
similarity to humans, rhesus macaques are an animal model of choice for many virus-challenge
and vaccine-evaluation studies, including SARS-CoV-2. Here, we show that immunization of
macaques with SARS-CoV-2 spike (S) protein generates potent receptor binding domain cross-
neutralizing antibody (nAb) responses to both SARS-CoV-2 and SARS-CoV-1, in contrast to
human infection or vaccination where responses are typically SARS-CoV-2-specific. Furthermore,
the macaque nAbs are equally effective against SARS-CoV-2 variants of concern. Structural
studies show that different immunodominant sites are targeted by the two primate species.
Human antibodies generally target epitopes strongly overlapping the ACE2 receptor binding site
(RBS), whereas the macaque antibodies recognize a relatively conserved region proximal to the
RBS that represents another potential pan-SARS-related virus site rarely targeted by human
antibodies. B cell repertoire differences between the two primates appear to significantly influence
the vaccine response and suggest care in the use of rhesus macaques in evaluation of vaccines
to SARS-related viruses intended for human use.
ONE SENTENCE SUMMARY
Broadly neutralizing antibodies to an unappreciated site of conservation in the RBD in SARS-
related viruses can be readily induced in rhesus macaques because of distinct properties of the
naïve macaque B cell repertoire that suggest prudence in the use of the macaque model in SARS
vaccine evaluation and design.
.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 6, 2021. ; https://doi.org/10.1101/2021.07.05.451222doi: bioRxiv preprint

4
MAIN
The rapid development of multiple vaccines to counter the COVID-19 pandemic caused by SARS-
CoV-2 has been a triumph for science and medicine. The reason for these successes is likely
due, in part, to the relative ease of inducing protective neutralizing antibodies (nAbs) in humans
by immunization with the spike (S) protein, the key component of most vaccines (1-4). Different
presentations of S in the vaccines induce relatively high serum nAb responses in most individuals
that appear to strongly target the ACE2 receptor binding site (RBS) in the receptor binding domain
(RBD) (1, 5-9). Natural infection with SARS-CoV-2 targets the RBD similarly (1, 10-22). A key
contributor is the human VH3-53 antibody germline gene that encodes a major subset (RBS-A,
Class 1) of highly potent immunodominant nAbs that require minimal somatic hypermutation
(SHM) and are specific for SARS-CoV-2 (22-25). These and other nAbs to the RBS are likely
responsible for driving some of the mutations found in Variants of Concern (VOCs) (23, 26-30).
Here, we immunized rhesus macaques with SARS-CoV-2 S-protein to compare nAb responses
between macaques and humans with a particular emphasis on the breadth of elicited neutralizing
responses to sarbecoviruses and the effects of differences between the naive antibody repertoires
of the two primate species on responses.
SARS-CoV-2 S-protein prime-boost immunization leads to robust sarbecovirus broadly
neutralizing antibody (bnAb) responses in rhesus macaques
A recombinant prefusion-stabilized soluble S-protein in a saponin adjuvant (SMNP) was
administered through subcutaneous injection (s.c.) in naïve rhesus macaques (RMs) (n = 8).
Since the manner of immunogen administration may shape immune responses (31-33), two
protocols were evaluated: a conventional prime-boost consisting of bolus injections (100µg) at
weeks 0 and 10 for Group 1 animals (n = 4), and an escalating dose (ED) prime and bolus boost
for Group 2 (n=4) (Fig. 1A). Serum Ab responses to SARS-CoV-2 S-protein, evaluated by
enzyme-linked immunosorbent assay (ELISA) (figs. S1 and S2), were detected in both groups at
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was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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5
week 2 and, although responses at week 4 were stronger for the ED group, no major differences
were found subsequently. Boost immunization at week 10 increased EC
50
binding titers to the
10
3
-10
4
range for all animals, indicating a strong antibody recall response. ID
50
neutralization titers
showed a similar pattern where both immunization groups developed specific neutralizing
antibody responses by week 4, which were enhanced by week 12, post-boost (figs. S1 and S2).
Next, we examined serum responses for cross-reactivity with SARS-CoV-1 (Fig. 1B, fig. S2).
Strong cross-reactive binding responses to SARS-CoV-1 S protein were observed (Fig. 1B, fig.
S2), as well as strong cross-neutralizing antibody responses against SARS-CoV-1 (Fig. 1C, fig.
S2). ID
50
neutralization titers showed a modest correlation with EC
50
binding titers for both SARS-
CoV-2 and SARS-CoV-1 (Fig. 1D). The elicitation of potent cross-neutralizing responses in rhesus
macaques by S-protein immunization is in stark contrast to human SARS-CoV-2 natural infection,
which typically results in autologous nAb responses where cross-neutralizing activity against
SARS-CoV-1 is rare (Fig. 1E, fig. S3) (34-36). In humans vaccinated with mRNA S-protein, we
similarly found SARS-CoV-2 autologous but not SARS-CoV-1 cross-reactive nAb responses (Fig.
1E, fig. S3) as also described by others (37).
To examine the role of species-specific B cell immunogenetic differences, we vaccinated a group
of 5 mice twice (wk0-prime, wk3-boost) with SARS-CoV-2 S-protein and SMNP adjuvant (fig. S4).
Consistent with earlier studies (38-40), SARS-CoV-2 S protein immunization of mice elicited high
titers of autologous SARS-CoV-2 nAbs; however, the SARS-CoV-1 cross-neutralizing antibody
titers were significantly less than the macaque cross-nAb responses (Fig. 1E, fig. S4), indicating
a strong species-dependent contribution to development of bnAbs to SARS-CoV-2 S protein.
Specificities and breadth of neutralization of the polyclonal macaque nAbs
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was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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Frequently asked questions

Q1. What have the authors contributed in "Broadly neutralizing antibodies to sars-related viruses can be readily induced in rhesus macaques" ?

Here, the authors show that immunization of macaques with SARS-CoV-2 spike ( S ) protein generates potent receptor binding domain crossneutralizing antibody ( nAb ) responses to both SARS-CoV-2 and SARS-CoV-1, in contrast to human infection or vaccination where responses are typically SARS-CoV-2-specific. CC-BY-NC-ND 4. 0 International license available under a was not certified by peer review ) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Furthermore, the macaque nAbs are equally effective against SARS-CoV-2 variants of concern. Human antibodies generally target epitopes strongly overlapping the ACE2 receptor binding site ( RBS ), whereas the macaque antibodies recognize a relatively conserved region proximal to the RBS that represents another potential pan-SARS-related virus site rarely targeted by human antibodies. B cell repertoire differences between the two primates appear to significantly influence the vaccine response and suggest care in the use of rhesus macaques in evaluation of vaccines to SARS-related viruses intended for human use. Broadly neutralizing antibodies to an unappreciated site of conservation in the RBD in SARSrelated viruses can be readily induced in rhesus macaques because of distinct properties of the naïve macaque B cell repertoire that suggest prudence in the use of the macaque model in SARS vaccine evaluation and design.