Vaccination with SARS-CoV-2 Spike Protein and AS03 Adjuvant Induces Rapid
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Anamnestic Antibodies in the Lung and Protects Against Virus Challenge in
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Nonhuman Primates
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4
5
6
Joseph R. Francica
1
, Barbara J. Flynn
1
, Kathryn E. Foulds
1
, Amy T. Noe
1
, Anne P. Werner
1
, Ian
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N. Moore
1
, Matthew Gagne
1
, Timothy S. Johnston
1
, Courtney Tucker
1
, Rachel L. Davis
1
, Britta
8
Flach
1
, Sarah O’Connell
1
, Shayne F. Andrew
1
, Evan Lamb
1
, Dillon R. Flebbe
1
, Saule T.
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Nurmukhambetova
1
, Mitzi M. Donaldson
1
, John-Paul M. Todd
1
, Alex Lee Zhu
2,3
, Caroline
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Atyeo
2,4
, Stephanie Fischinger
2,3
, Matthew J Gorman
2
, Sally Shin
2
, Venkata Viswanadh Edara
5,6,7
,
11
Katharine Floyd
5,6,7
, Lilin Lai
5,6,7
, Alida Tylor
1
, Elizabeth McCarthy
1
, Valerie Lecouturier
8
,
12
Sophie Ruiz
8
, Catherine Berry
8
, Timothy Tibbitts
9
,
Hanne Andersen
10
, Anthony Cook
10
, Alan
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Dodson
10
, Laurent Pessaint
10
, Alex Van Ry
10
, Marguerite Koutsoukos
11
, Cindy Gutzeit
12
, I-Ting
14
Teng
1
, Tongqing Zhou
1
, Dapeng Li
13
, Barton F. Haynes
13
, Peter D. Kwong
1
, Adrian McDermott
1
,
15
Mark G. Lewis
10
, Tong Ming Fu
9
, Roman Chicz
9
, Robbert van der Most
12
, Kizzmekia S. Corbett
1
,
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Mehul S. Suthar
5,6,7
, Galit Alter
2
, Mario Roederer
1
, Nancy J. Sullivan
1
, Daniel C. Douek
1
, Barney
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S. Graham
1
, Danilo Casimiro
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, and Robert A. Seder
1*
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21
22
1
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of
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Health, Bethesda, MD, USA
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2
Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA 02139, USA
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3
PhD program in Immunology and Virology, University of Duisburg-Essen, Essen, Germany
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4
PhD program in Virology, Division of Medical Sciences, Harvard University, Boston, MA, USA
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5
Centers for Childhood Infections and Vaccines; Children’s Healthcare of Atlanta and Emory University,
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Department of Pediatrics, Atlanta, GA, 30329, USA
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6
Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30329, USA
30
7
Yerkes National Primate Research Center, Atlanta, GA 30329, USA
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8
Sanofi Pasteur, Marcy l'Etoile, France
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9
Sanofi Pasteur, 38 Sidney Street, Cambridge, MA 02139, USA
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10
Bioqual, Inc., Rockville, MD, USA
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11
GSK, Wavre, Belgium
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12
GSK, Rixensart, Belgium
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13
Duke Human Vaccine Institute, Duke University, Durham, NC 27708, USA
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38
39
40
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42
* Corresponding author
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Cellular Immunology Section
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Vaccine Research Center
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National Institute of Allergy and Infectious Disease
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National Institutes of Health
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40 Convent Drive, MSC 3025, Building 40, Room 3512
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Bethesda, MD 20892
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E-mail address: rseder@mail.nih.gov
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105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC
The copyright holder for this preprintthis version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.433390doi: bioRxiv preprint
Abstract
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Adjuvanted soluble protein vaccines have been used extensively in humans for protection against various
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viral infections based on their robust induction of antibody responses. Here, soluble prefusion-stabilized
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spike trimers (preS dTM) from the severe acute respiratory syndrome coronavirus (SARS-CoV-2) were
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formulated with the adjuvant AS03 and administered twice to nonhuman primates (NHP). Binding and
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functional neutralization assays and systems serology revealed that NHP developed AS03-dependent
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multi-functional humoral responses that targeted multiple spike domains and bound to a variety of
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antibody F
C
receptors mediating effector functions in vitro. Pseudovirus and live virus neutralizing IC
50
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titers were on average greater than 1000 and significantly higher than a panel of human convalescent sera.
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NHP were challenged intranasally and intratracheally with a high dose (3x10
6
PFU) of SARS-CoV-2
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(USA-WA1/2020 isolate). Two days post-challenge, vaccinated NHP showed rapid control of viral
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replication in both the upper and lower airways. Notably, vaccinated NHP also had increased spike-
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specific IgG antibody responses in the lung as early as 2 days post challenge. Moreover, vaccine-induced
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IgG mediated protection from SARS-CoV-2 challenge following passive transfer to hamsters. These data
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show that antibodies induced by the AS03-adjuvanted preS dTM vaccine are sufficient to mediate
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protection against SARS-CoV-2 and support the evaluation of this vaccine in human clinical trials.
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105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC
The copyright holder for this preprintthis version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.433390doi: bioRxiv preprint
Introduction
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The 2019 outbreak of coronavirus disease (COVID-19) caused by the novel severe acute respiratory
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syndrome coronavirus (SARS-CoV-2) has become a global pandemic with 112,849,164 infections and
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2,503,390 deaths across 192 countries, as of February 25, 2021
1
. An effective prophylactic vaccine
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remains the most effective public health measure for controlling disease spread
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. To that end, two mRNA
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vaccines
3, 4
have received emergency use authorization from the FDA based on clinical efficacy of greater
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than 90% in the US. In addition, adenovirus-based vaccines have been approved for use in the EU, United
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Kingdom
5
and Russia
6
, and an inactivated virus vaccine is approved in China
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. Other candidates based on
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protein are currently in clinical testing
8
. With the exception of the inactivated virus vaccines
9, 10
, these
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approved and clinical-phase candidate vaccines use only the coronavirus spike (S) protein as their
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immunogen.
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Spike is a surface membrane-bound trimer that, by electron microscopy, gives viral particles a
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characteristic halo from which its family name “corona” is derived
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. It is a type-1 viral membrane fusion
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protein that exists in a metastable prefusion conformation and undergoes a dramatic structural
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rearrangement upon engagement of the receptor binding domain (RBD) with its receptor, angiotensin-
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converting enzyme 2 (ACE2)
12, 13, 14
, ultimately leading to membrane fusion. It has been shown that
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antibodies directed against the RBD can neutralize incoming virus by preventing receptor recognition and
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thus entry
15, 16, 17, 18, 19
. Because the RBD, as well as other regions such as the N-terminal domain (NTD)
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may contain neutralizing epitopes
20, 21
, the full-length spike is a preferred target antigen for vaccine
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development. Based upon successful structure-based immunogen designs for SARS-CoV and Middle
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Eastern Respiratory Virus (MERS) vaccines
22, 23
, mutations have been introduced to block cleavage of S
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into S1 and S2 subunits and stabilize a region between the central helix and heptad repeat 1, giving rise to
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homogeneous S protein trimers in the prefusion conformation
24
. This construct, referred to as S-2P, is the
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105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC
The copyright holder for this preprintthis version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.433390doi: bioRxiv preprint
basis for several SARS-CoV-2 vaccine candidates being delivered by adenoviral vectors
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, displayed on
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nanoparticles
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, or encoded by mRNA
27, 28, 29
.
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In contrast to vectored gene delivery vaccine platforms, adjuvanted soluble protein vaccine formulations
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have been approved for clinical use against several viral infections
30, 31, 32
and have a long history of being
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used across all age groups. Soluble protein subunit vaccines will likely require a potent adjuvant to elicit
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strong T and B cell responses
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. Here we have formulated a soluble S-2P-derived protein with the well-
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characterized adjuvant, AS03, an oil-in-water emulsion composed of squalene, polysorbate 80, and α-
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tocopherol. AS03 potently induces antibodies and has been shown to increase vaccine durability, promote
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heterologous strain cross-reactivity
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, and to have dose-sparing effects
35, 36, 37, 38, 39
. It was licensed for use
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in vaccines against pandemic influenza in Europe, with approximately 90 million doses administered
35, 39,
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40, 41
. Therefore, in this study, AS03-adjuvanted soluble S-2P trimers were evaluated for NHP
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immunogenicity and protection following SARS-CoV-2 challenge in advance of clinical trials. To date
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several advanced vaccine candidates have been characterized for the magnitude, quality and efficacy of
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the immune responses they elicit
25, 28, 42, 43
. Here we performed a thorough characterization humoral and
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cellular responses in the upper and lower respiratory tracts following vaccination and challenge. These
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studies establish that vaccine-induced antibody is sufficient for protection and highlight its role in rapid
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control of lower airway viral replication.
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(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC
The copyright holder for this preprintthis version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.433390doi: bioRxiv preprint
Results
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Soluble spike trimers are immunogenic when adjuvanted with AS03
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To create a SARS-CoV-2 protein vaccine, the S-2P stabilizing mutations were used as previously
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described
24
; the trimer was then expressed as a soluble protein by replacing the transmembrane domain
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with a T4 foldon domain, which has been shown to assist in trimerization of type-1 membrane fusion
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proteins
44, 45
(Fig. 1a). The resulting soluble trimeric protein immunogen is thus referred to as prefusion
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transmembrane-deleted spike, or preS dTM. PreS dTM trimers were then formulated by admixing with
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the oil-in-water emulsion, AS03. NHP were immunized intramuscularly three weeks apart with or without
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AS03 to confirm its role for improving antibody responses. AS03 was critical for the induction of high
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magnitude S-2P IgG binding and neutralization titers (Fig. S1a-b), and S-2P-specific IgA and IgG B cell
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responses (Fig. S1c-f). Systems serology was also performed to assess the quantitative and qualitative
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effector functions of vaccine responses induced by AS03. Antibodies bound to a broad array of human
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antibody F
C
receptors and enabled F
C
-mediated effector functions such as phagocytosis and complement
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activation (Fig. S1g,h). AS03 strongly enhanced all F
C
functions equally with no skewing to a particular
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receptor or function. Collectively these data establish the critical role of the AS03 adjuvant for improving
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the magnitude and quality of antibody responses.
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To study protective efficacy and perform a wider assessment of immunogenicity, rhesus macaques were
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immunized with 4 or 12 µg AS03-adjuvanted preS dTM; PBS was administered as a negative control
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(Fig. 1b). Animals did not experience any abnormal body weight or temperature changes in response to
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vaccination (Supplementary Table 1). Serum binding titers were detectible two weeks after the first
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immunization at levels that approximated those found in human convalescent donor sera (HCS) from two
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different benchmark cohorts; endpoint binding titers were significantly increased from 2.9x10
3
to 7.4x10
4
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following the second immunization in the high dose group (Fig. 2a). Notably there was no significant
135
105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC
The copyright holder for this preprintthis version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.433390doi: bioRxiv preprint