Introduction of two prolines and removal of the polybasic cleavage site leads to optimal efficacy of a recombinant spike based SARS-CoV-2 vaccine in the mouse model

TL;DR: The data presented here validates the choice of antigens that contain the PP mutation and suggests that deletion of the polybasic cleavage site could lead to a further optimized design of vaccine design.

Abstract: The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the prime target for vaccine development. The spike protein mediates both binding to host cells and membrane fusion and is also so far the only known viral target of neutralizing antibodies. Coronavirus spike proteins are large trimers that are relatively instable, a feature that might be enhanced by the presence of a polybasic cleavage site in the SARS-CoV-2 spike. Exchange of K986 and V987 to prolines has been shown to stabilize the trimers of SARS-CoV-1 and the Middle Eastern respiratory syndrome coronavirus spikes. Here, we test multiple versions of a soluble spike protein for their immunogenicity and protective effect against SARS-CoV-2 challenge in a mouse model that transiently expresses human angiotensin converting enzyme 2 via adenovirus transduction. Variants tested include spike protein with a deleted polybasic cleavage site, the proline mutations, a combination thereof, as well as the wild type protein. While all versions of the protein were able to induce neutralizing antibodies, only the antigen with both a deleted cleavage site and the PP mutations completely protected from challenge in this mouse model.

Summary

Construct design and recombinant protein expression

• The sequence based on the S gene of SARS-CoV-2 strain Wuhan-1 was initially codon optimized for mammalian cell expression.
• This construct was termed wild type (WT).
• The proteins were then expressed in a baculovirus expression system and purified.
• For WT, the most prominent detected smaller band ran at 80 kDa, was visualized with an antibody recognizing the C-terminal hexa-histidine tag and likely represents S2 (Figure 1F ).
• The two constructs containing the PP mutations also produced an additional band at approximately 40 kDa (Figure 1E ), potentially representing a fragment downstream of S2'.

All versions of the recombinant spike protein induce robust immune responses in mice

• To test the immunogenicity of the four spike constructs, all proteins were used in a simple prime-boost study in mice (Figure 2A ).
• A control group received an irrelevant immunogen, recombinant influenza virus hemagglutinin (HA), also expressed in insect cells, with AddaVax.
• To determine antibody levels to the RBD, the authors performed enzyme-linked immunosorbent assays against recombinant, mammalian cell expressed RBD (18, 19) .
• In addition, the authors also performed cell-based ELISAs with Vero cells infected with SARS-CoV-2 as target.
• Here, the WT, PP and ΔCS groups showed similar levels of neutralization while the ΔCS-PP group animals had higher serum neutralization titers (Figure . 2F ).

Vaccination with recombinant S protein variants protects mice from challenge with SARS-CoV-2

• In order to perform challenge studies, mice were sensitized to infection with SARS-CoV-2 by intranasal (i.n.) transduction with an adenovirus expressing hACE2 (AdV-hACE2), using a treatment regimen described previously (Figure 2A ) (15, 21, 22) .
• Additional animals were euthanized on day 2 and day 4 to harvest lungs for histopathological assessment and immunohistochemistry, and on day 2 and day 5 to measure virus titers in the lung.
• After challenge, all groups lost weight trending with the negative control group (irrelevant HA protein vaccination), except for the ΔCS-PP group which displayed minimal weight loss (Figure 3A ).
• Only on days 4-6 the WT, PP and ΔCS groups showed a trend towards less weight loss then the control group.
• All animals recovered and by day 14 and no mortality was observed.

Lung immunohistochemistry and pathology

• Lungs were harvested on days 2 and 4 post challenge.
• The ΔCS-PP group showed very few positive cells, especially on day 4 while antigen was detected more widespread in all other groups.
• These results correlate well with the viral lung titers shown above.
• Alveolar congestion and edema were also more pronounced in S vaccinated groups as compared with the irrelevant control HA immunogen.
• Scores were slightly higher in vaccinated than control animals which may reflect the infiltration of CoV-2 antigen-specific immune cells into the lung, which would be absent in the irrelevant HA immunized control mice (Figure 4C and D ).

Discussion

• The spike protein of SARS-CoV-2 has been selected early on as a target for vaccine development, based on experience with SARS-CoV-1 and MERS CoV (6).
• While vaccination with all constructs induced neutralizing antibodies and led to control of virus replication in the lung, the authors observed notable differences.
• Current leading vaccine candidates in clinical trials include virus vectored and mRNA vaccines.
• The ChAdOx based vaccine candidate that is developed by AstraZeneca is using a wild type version of the spike protein (23), while Moderna's mRNA vaccine is based on a spike construct that includes the PP mutations but features a wild type cleavage site (24) .
• It is, therefore, not surprising that similar constructs for SARS-CoV-2 also provided protection.

Materials and methods

• Vero.E6 cells (ATCC CRL-1586-clone E6) were maintained in culture using Dulbecco's Modified Eagle Medium (DMEM; Gibco) which was supplemented with Antibiotic-Antimycotic (100 U/ml penicillin-100 μg/ml streptomycin-0.25 ug/ml Amphotericin B) (Gibco; 15240062) and 10% fetal bovine serum (FBS; Corning).
• Resources, NR-52281) was grown in Vero.E6 cells as previously described and was used for the in vivo challenge (20) .
• High titer Ad-hACE2 stocks were amplified in TRex™-293 cells, purified by CsCl ultracentrifugation and infectious titers determined by tissue-culture infectious dose-50 (TCID 50 ), adjusting for plaque forming unit (PFU) titers using the Kärber statistical method, as described previously (32) .
• All recombinant proteins were expressed and purified using the baculovirus expression system, as previously described (18, 33, 34) .
• ΔCS indicates that the cleavage site of the spike protein was removed by deletion of the arginine residues (RRAR to just A).

SDS-PAGE and Western blot.

• One ug of each respective protein was mixed at a 1:1 ratio with 2X Laemmli buffer (Bio-Rad) which was supplemented with 2% β-mercaptoethanol (Fisher Scientific).
• The membrane was blocked with phosphate buffered saline (PBS; Gibco) containing 3% non-fat milk (AmericanBio, catalog# AB10109-01000) for an hour at room temperature on an orbital shaker.
• Ninety-six well plates (Immulon 4 HBX; Thermo Fisher Scientific) were coated with recombinant RBD at a concentration of 2 ug/ml with 50µl/well overnight.
• Three weeks later, mice were again immunized, via intramuscular route, with 3 μg of each respective protein with adjuvant.
• The authors used a very detailed protocol that they published earlier for measuring neutralizing antibody in serum samples (18, 20) .

Full text

1
Introduction of two prolines and removal of the polybasic cleavage site leads to
1
optimal efficacy of a recombinant spike based SARS-CoV-2 vaccine in the mouse
2
model
3
4
Fatima Amanat
1,2
, Shirin Strohmeier
1
, Raveen Rathnasinghe
1,2,3
, Michael Schotsaert
1,3
, Lynda Coughlan
1
,
5
Adolfo García-Sastre
1,3,4,5
and Florian Krammer
1
*
6
7
1
Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
8
2
Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029,
9
USA
10
3
Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York,
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NY 10029, USA
12
4
Department of Medicine, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New
13
York, NY 10029, USA
14
5
The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
15
16
*To whom correspondence should be addressed: florian.krammer@mssm.edu
17
18
Abstract
19
The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified
20
as the prime target for vaccine development. The spike protein mediates both binding to host cells and
21
membrane fusion and is also so far the only known viral target of neutralizing antibodies. Coronavirus
22
spike proteins are large trimers that are relatively instable, a feature that might be enhanced by the
23
presence of a polybasic cleavage site in the SARS-CoV-2 spike. Exchange of K986 and V987 to prolines
24
has been shown to stabilize the trimers of SARS-CoV-1 and the Middle Eastern respiratory syndrome
25
coronavirus spikes. Here, we test multiple versions of a soluble spike protein for their immunogenicity
26
and protective effect against SARS-CoV-2 challenge in a mouse model that transiently expresses human
27
angiotensin converting enzyme 2 via adenovirus transduction. Variants tested include spike protein with
28
a deleted polybasic cleavage site, the proline mutations, a combination thereof, as well as the wild type
29
protein. While all versions of the protein were able to induce neutralizing antibodies, only the antigen
30
with both a deleted cleavage site and the PP mutations completely protected from challenge in this
31
mouse model.
32
33
Importance
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A vaccine for SARS-CoV-2 is urgently needed. A better understanding of antigen design and attributes
35
that vaccine candidates need to have to induce protective immunity is of high importance. The data
36
presented here validates the choice of antigens that contain the PP mutation and suggests that deletion
37
of the polybasic cleavage site could lead to a further optimized design.
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Introduction
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019 in China and has
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since then caused a coronavirus disease 2019 (COVID-19) pandemic (1-3). Vaccines are an urgently
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needed countermeasure to the virus. Vaccine candidates have been moved at unprecedented speed
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2
through the pipeline with first Phase III trials already taking place in summer 2020, only half a year after
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discovery of the virus sequence. From studies on SARS-CoV-1 and the Middle Eastern respiratory
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syndrome CoV (MERS-CoV), it was clear that the spike protein of the virus is the best target for vaccine
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development (4-6). Most coronaviruses (CoVs) only have one large surface glycoprotein (a minority also
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have a hemagglutinin-esterase) that is used by the virus to attach to the host cell and trigger fusion of
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viral and cellular membranes. The spike protein of SARS-CoV-2, like the one of SARS-CoV-1, binds to
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human angiotensin receptor 2 (ACE2) (7-9). In order to be able to trigger fusion, the spike protein has to
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be cleaved into the S1 and S2 subunit (10-12). Additionally, a site in S2 (S2’) that has to be cleaved to
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activate the fusion machinery has been reported as well (13). While the spike of SARS-CoV-1 contains a
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single basic amino acid at the cleavage site between S1 and S2, SARS-CoV-2 has a polybasic motif that
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can be activated by furin-like proteases (10-12), analogous to the hemagglutinin (HA) of highly
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pathogenic H5 and H7 avian influenza viruses. In addition, it has been reported that the activated spike
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protein of CoVs is relatively instable and multiple conformations might exist of which not all may
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present neutralizing epitopes to the immune system. For SARS-CoV-1 and MERS-CoV stabilizing
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mutations – a pair of prolines replacing K986 and V987 in S2 – have been described (14) and a beneficial
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effect on stability has also been shown for SARS-CoV-2 (9). Here, we set out to investigate if including
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these stabilizing mutations, removing the cleavage site between S1 and S2 or combining the two
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strategies to stabilize the spike would increase its immunogenicity and protective effect in a mouse
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model that transiently expressed hACE2 via adenovirus transduction (15). This information is important
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since it can help to optimize vaccine candidates, especially improved versions of vaccines that might be
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licensed at a later point in time.
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Results
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Construct design and recombinant protein expression
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The sequence based on the S gene of SARS-CoV-2 strain Wuhan-1 was initially codon optimized for
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mammalian cell expression. The wild type signal peptide and ectodomain (amino acid 1-1213) were
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fused to a T4 foldon trimerization domain followed by a hexa-histidine tag to facilitate purification. This
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construct was termed wild type (WT). Additional constructs were generated including one in which the
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polybasic cleavage site (RRAR) was replaced by a single alanine (termed ΔCS), one in which K986 and
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V987 in the S2 subunit were mutated to prolines (PP) and one in which both modifications were
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combined (ΔCS-PP) (Figure 1A-C). The proteins were then expressed in a baculovirus expression system
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and purified. At first inspection by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
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PAGE) and Coomassie staining, all four constructs appeared similar with a major clean band at
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approximately 180kDa (Figure 1E). When a Western blot was performed, additional bands were
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detected in the lanes with the WT, PP and ΔCS-PP constructs, suggesting cleavage of a fraction of the
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protein. For WT, the most prominent detected smaller band ran at 80 kDa, was visualized with an
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antibody recognizing the C-terminal hexa-histidine tag and likely represents S2 (Figure 1F). The two
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constructs containing the PP mutations also produced an additional band at approximately 40 kDa
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(Figure 1E), potentially representing a fragment downstream of S2’. While in general these bands were
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invisible on an SDS PAGE and therefore are likely only representing a tiny fraction of the purified spike
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protein, they might indicate vulnerability to proteolytic digest of the antigen in vivo. All constructs were
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also recognized in a similar manner by mAb CR3022 (16, 17), an antibody that binds to the RBD (Figure
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1F).
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All versions of the recombinant spike protein induce robust immune responses in mice
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95
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(which 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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3
To test the immunogenicity of the four spike constructs, all proteins were used in a simple prime-boost
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study in mice (Figure 2A). Animals were injected intramuscularly (i.m.) with 3μg of spike protein
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adjuvanted with AddaVax (a generic version of the oil-in-water adjuvant MF59) twice in a 3 week
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interval. A control group received an irrelevant immunogen, recombinant influenza virus hemagglutinin
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(HA), also expressed in insect cells, with AddaVax. Mice were bled three weeks after the prime and four
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weeks after the boost to assess the immune response that they mounted to the vaccine (Figure 2B). To
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determine antibody levels to the RBD, we performed enzyme-linked immunosorbent assays against
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recombinant, mammalian cell expressed RBD (18, 19). All animals made anti-RBD responses after the
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prime but they were higher in the ΔCS and ΔCS-PP groups than in the WT or PP groups (Figure 2C). The
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booster dose increased antibodies to the RBD significantly but the same pattern persisted (Figure 2D).
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Interestingly, the ΔCS-PP group showed very homogenous responses compared to the other groups
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were there was more spread between the animals. In addition, we also performed cell-based ELISAs
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with Vero cells infected with SARS-CoV-2 as target. While all groups showed good reactivity, a similar
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pattern emerged in which ΔCS and ΔCS-PP groups showed higher reactivity than WT and PP groups
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(Figure 2E). Finally, we performed microneutralization assays with authentic SARS-CoV-2 (20). Here, the
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WT, PP and ΔCS groups showed similar levels of neutralization while the ΔCS-PP group animals had
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higher serum neutralization titers (Figure. 2F).
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Vaccination with recombinant S protein variants protects mice from challenge with SARS-CoV-2
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In order to perform challenge studies, mice were sensitized to infection with SARS-CoV-2 by intranasal
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(i.n.) transduction with an adenovirus expressing hACE2 (AdV-hACE2), using a treatment regimen
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described previously (Figure 2A) (15, 21, 22). They were then challenged with 10
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plaque forming units
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(PFU) of SARS-CoV-2 and monitored for weight loss and mortality for 14 days. Additional animals were
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euthanized on day 2 and day 4 to harvest lungs for histopathological assessment and
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immunohistochemistry, and on day 2 and day 5 to measure virus titers in the lung. After challenge, all
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groups lost weight trending with the negative control group (irrelevant HA protein vaccination), except
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for the ΔCS-PP group which displayed minimal weight loss (Figure 3A). Only on days 4-6 the WT, PP and
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ΔCS groups showed a trend towards less weight loss then the control group. However, all animals
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recovered and by day 14 and no mortality was observed. Lung titers on day 2 suggested low virus
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replication in the WT, PP and ΔCS groups with some animals having no detectable virus and no presence
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of replication competent virus in the ΔCS-PP (Figure 3B). Two of the control animals showed high virus
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replication while virus could not be recovered from the third animal. No virus could be detected in any
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of the vaccinated groups on day 5 while all three controls still had detectable virus in the 10
4
to 10
5
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range (Figure 3C).
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Lung immunohistochemistry and pathology
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Lungs were harvested on days 2 and 4 post challenge. Samples from both days were used for
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immunohistochemistry to detect viral nucleoprotein antigen. Viral antigen was detectable in all groups
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on day 2 as well as day 4 post infection (Figure 3D). However, the ΔCS-PP group showed very few
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positive cells, especially on day 4 while antigen was detected more widespread in all other groups.
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These results correlate well with the viral lung titers shown above. The samples were also hematoxylin
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and eosin (H&E) stained and scored for lung pathology by a qualified veterinary pathologist using a
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composite score with a maximum value of 24 (Figure 4A and C). At D2 post-infection with SARS-CoV-2,
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all mice were determined to exhibit histopathological lesions typical of interstitial pneumonia, with
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more severe alveolar inflammation in the WT group. Alveolar congestion and edema were also more
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pronounced in S vaccinated groups as compared with the irrelevant control HA immunogen. At this
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4
time-point, the overall pathology score was lowest for the irrelevant HA control group, followed by ΔCS-
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PP<PP<ΔCS<wild type (Figure 4A). On day 4 all groups showed mild to moderate pathology scores,
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reduced in severity as compared with D2. Observations included perivascular, bronchial and alveolar
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inflammation, as well as mild to moderate congestion or edema. Scores were slightly higher in
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vaccinated than control animals which may reflect the infiltration of CoV-2 antigen-specific immune cells
148
into the lung, which would be absent in the irrelevant HA immunized control mice (Figure 4C and D).
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Discussion
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152
The spike protein of SARS-CoV-2 has been selected early on as a target for vaccine development, based
153
on experience with SARS-CoV-1 and MERS CoV (6). The coronavirus spike protein is known to be
154
relatively labile, and in addition to this inherent property the SARS-CoV-2 spike also contains a polybasic
155
cleavage site between S1 and S2. Work on SARS-CoV-1 and MERS CoV had shown that introducing two
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prolines in positions 986 and 987 (SARS-CoV-2 numbering) improves stability and expression (14). In
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addition, removal of polybasic cleavage sites has been shown to stabilize hemagglutinin (HA) proteins of
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highly pathogenic influenza viruses. In this study, we tested different versions of the protein either
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lacking the polybasic cleavage site or including the stabilizing PP mutations or both. While vaccination
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with all constructs induced neutralizing antibodies and led to control of virus replication in the lung, we
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observed notable differences. Removing the polybasic cleavage side did increase the humoral immune
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response in ELISAs. Since we did not observe cleavage of the majority of protein when purified (although
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some cleavage could be observed), even with the polybasic cleavage site present, we speculate that
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removal of the site might make the protein more stable in vivo post vaccination. Longer stability could
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lead to stronger and potentially more uniform immune responses. The combination of deleting the
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polybasic cleavage site plus introducing the PP mutations performed best, also in terms of protection of
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mice from weight loss. It is important to note that all versions of the protein tested had a third
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stabilizing element present, which is a trimerization domain. This trimerization domain might have also
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increased stability and immunogenicity.
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Current leading vaccine candidates in clinical trials include virus vectored and mRNA vaccines. The
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ChAdOx based vaccine candidate that is developed by AstraZeneca is using a wild type version of the
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spike protein (23), while Moderna’s mRNA vaccine is based on a spike construct that includes the PP
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mutations but features a wild type cleavage site (24). It is currently unclear, if addition of the
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modifications shown here to enhance immunogenicity of recombinant protein spike antigens would also
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enhance immunogenicity of these constructs. However, it might be worth testing if these vaccine
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candidates can be improved by our strategy as well. Of note, ones study in non-human primates with
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adenovirus 26-vectored vaccine candidates expressing different versions of the spike protein also
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showed that a ΔCS-PP (although including the transmembrane domain) performed best and this
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candidate is now moving forward into clinical trials (25). Similarly, Novavax is using a recombinant spike
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construct that features ΔCS-PP and, when adjuvanted, induced high neutralization titers in humans in a
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Phase I clinical trial (26).
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While vaccination with all constructs led to various degrees of control of virus replication,
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histopathology scores, especially on day 2 after challenge were above those of the negative controls
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animals. We do not believe that this is a signal of enhanced disease as it has been observed in some
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studies for SARS-CoV-2 but the hallmark of an antigen-specific immune response. This is also evidenced
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by significantly reduced weight loss in the ΔCS-PP group as well as complete control of virus replication
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despite having increased lung histopathology scores. However, future studies with recombinant protein
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vaccines that are routed for clinical testing, as outlined below, will need to assess this increase in lung
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pathology in more detail.
190
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5
Recombinant protein vaccines including the spike ectodomain (27, 28), membrane extracted spike (29)
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as well as S1 (30) and RBD (31) have been tested for SARS-CoV-1 and several studies show good efficacy
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against challenge in animal models. It is, therefore, not surprising that similar constructs for SARS-CoV-2
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also provided protection. While our goal was not vaccine development but studying the effect of
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stabilizing elements on the immunogenicity of the spike protein, Sanofi Pasteur has announced the
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development of a recombinant protein based SARS-CoV-2 vaccine and a second recombinant protein
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candidate is currently being developed by Seqirus. Our data shows that this approach could be effective.
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Materials and methods
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Cells and viruses. Vero.E6 cells (ATCC CRL‐1586-clone E6) were maintained in culture using Dulbecco's
202
Modified Eagle Medium (DMEM; Gibco) which was supplemented with Antibiotic-Antimycotic (100 U/ml
203
penicillin- 100 μg/ml streptomycin- 0.25 ug/ml Amphotericin B) (Gibco; 15240062) and 10% fetal bovine
204
serum (FBS; Corning). SARS-CoV-2 (isolate USA‐WA1/2020 BEI Resources, NR‐52281) was grown in
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Vero.E6 cells as previously described and was used for the in vivo challenge (20). A viral seed stock for a
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non-replicating human adenovirus type-5 (HAdV-C5) vector expressing the human ACE2 receptor was
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obtained from the Iowa Viral Vector Core Facility. High titer Ad-hACE2 stocks were amplified in TRex™-
208
293 cells, purified by CsCl ultracentrifugation and infectious titers determined by tissue-culture
209
infectious dose-50 (TCID
50
), adjusting for plaque forming unit (PFU) titers using the Kärber statistical
210
method, as described previously (32).
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Recombinant proteins. All recombinant proteins were expressed and purified using the baculovirus
212
expression system, as previously described (18, 33, 34). Different versions of the spike protein of SARS-
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CoV-2 (GenBank: MN908947.3) were expressed to assess immunogenicity. PP indicates that two
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stabilizing prolines were induced at K986 and K987. ΔCS indicates that the cleavage site of the spike
215
protein was removed by deletion of the arginine residues (RRAR to just A). The HA was also produced in
216
the baculovirus expression system similar to the spike variants.
217
SDS-PAGE and Western blot. One ug of each respective protein was mixed at a 1:1 ratio with 2X
218
Laemmli buffer (Bio-Rad) which was supplemented with 2% β-mercaptoethanol (Fisher Scientific). The
219
samples were heated at 90°C for 10 minutes and loaded onto a 4-20% precast polyacrylamide gel
220
(BioRad). The gel was stained with SimplyBlue SafeStain (Invitrogen) for 1 hour and then de-stained with
221
water for a few hours. For Western blot, the same process was used as mentioned above. After the gel
222
was run, the gel was transferred onto a nitrocellulose membrane, as described previously (33). The
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membrane was blocked with phosphate buffered saline (PBS; Gibco) containing 3% non-fat milk
224
(AmericanBio, catalog# AB10109‐01000) for an hour at room temperature on an orbital shaker. Next,
225
primary antibody was prepared in PBS containing 1% non-fat milk using anti-hexahistidine antibody
226
(Takara Bio, catalog #631212) at a dilution of 1:3000. The membrane was stained with primary antibody
227
solution for 1 hour at room temperature. The membrane was washed thrice with PBS containing 0.1%
228
Tween-20 (PBS-T; Fisher Scientific). The secondary solution was prepared with 1% non-fat milk in PBS-T
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using anti-mouse IgG (whole molecule)–alkaline phosphatase (AP) antibody produced in goat (Sigma-
230
Aldrich) at a dilution of 1:3,000. The membrane was developed using an AP conjugate substrate kit,
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catalog no. 1706432 (Bio-Rad).
232
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which 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 preprintthis version posted September 18, 2020. ; https://doi.org/10.1101/2020.09.16.300970doi: bioRxiv preprint