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
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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
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18
Abstract
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The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified
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as the prime target for vaccine development. The spike protein mediates both binding to host cells and
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membrane fusion and is also so far the only known viral target of neutralizing antibodies. Coronavirus
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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
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mouse model.
32
33
Importance
34
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
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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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39
40
41
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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
47
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2
through the pipeline with first Phase III trials already taking place in summer 2020, only half a year after
48
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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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
5
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
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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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The spike protein of SARS-CoV-2 has been selected early on as a target for vaccine development, based
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on experience with SARS-CoV-1 and MERS CoV (6). The coronavirus spike protein is known to be
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relatively labile, and in addition to this inherent property the SARS-CoV-2 spike also contains a polybasic
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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
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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
205
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™-
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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
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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
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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.
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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
229
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