key: cord-0685034-yppu5hp4
authors: Kuo, Tsun-Yung; Lien, Chia En; Lin, Yi-Jiun; Lin, Meei-Yun; Wu, Chung-Chin; Tang, Wei-Hsuan; Campbell, John D.; Traquina, Paula; Chuang, Ya-Shan; Liu, Luke Tzu-Chi; Cheng, Jinyi; Chen, Charles
title: Protection of Hamsters Challenged with SARS-CoV-2 Delta Variant after Two Doses of Adjuvanted SARS-CoV-2 Stabilized Prefusion Spike Protein (S-2P) and a Single Dose of Beta Variant S-2P
date: 2022-04-22
journal: J Infect Dis
DOI: 10.1093/infdis/jiac153
sha: a712bfbf2eb96137f12fac126ac65b04b454940f
doc_id: 685034
cord_uid: yppu5hp4

SARS-CoV-2 Variants of Concern (VoCs) negatively impact the effectiveness of vaccines. In this study, we challenge hamsters with the Delta variant after two- or three-dose inoculations with SARS-CoV-2 vaccines constructed from stabilized prefusion spike proteins (S-2P) of Wuhan (W) and Beta (B) variants. Compared to three doses of W S-2P, two doses of W S-2P followed by a third dose of B S-2P induced the highest neutralizing antibody titer against live SARS-CoV-2 virus and enhanced neutralization of Omicron variant pseudovirus. Reduced lung live virus titer and pathology suggested that all vaccination regimens protect hamsters from SARS-CoV-2 Delta variant challenge.

Despite mass vaccination programs against SARS-CoV-2, variants of concern (VoCs) such as the Delta and 2

Omicron variants have become dominant strains [1] . These VoCs have increased transmission rates, reduced in 3 vitro neutralizing capability and clinical effectiveness of currently available vaccines, and are more resistant to 4 neutralization by convalescent and vaccine-induced antibodies [2, 3] . The most current data point towards booster 5 vaccinations for enhancing immune response and improving effectiveness against the VoCs [4, 5] . 6

Medigen's MVC-COV1901 is a subunit vaccine based on a stabilized prefusion spike protein (S-2P) 7

adjuvanted with CpG 1018 and aluminum hydroxide that has been approved for emergency use in Taiwan [6, 7]. 8

We have previously shown that two doses of adjuvanted S-2P induced neutralizing antibodies against SARS-9

CoV-2 variants with a tendency of higher immunogenicity at higher dose levels [8] . A third dose of MVC-10 COV1901 in the phase 1 subjects was also found to improve neutralization response against the Omicron variant 11 [9] . The current study expands on our previous findings by investigating the immunogenicity of third dose 12 variant-specific booster against VoCs. 13

Female golden Syrian hamsters aged 8-10 weeks at study initiation were obtained from the National 16

Laboratory Animal Center (Taipei, Taiwan). Animal immunizations were conducted in the Testing Facility for 17

Biological Safety, TFBS Bioscience Inc., Taiwan. Seven weeks after the final immunization and after serum 18

immunizations with S-2P were adjuvanted with 150 μg of CpG 1018 and 75 μg of alum. Serum samples were 11 collected five weeks after the final immunization and immunogenicity was determined by neutralization assay 12 with SARS-CoV-2 virus and the variants. Approximately three weeks after the serum sampling (53 days after the 13 final immunization), hamsters were challenged with the SARS-CoV-2 Delta variant (TCDC#1144) and then 14 sacrificed at 3 d.p.i. (n = 5 per group) or 6 d.p.i. (n = 5 per group) for analyses of lung viral loads and lung 15 TCID 50 . Body weights of individual hamsters were tracked daily up to the time of sacrifice. After euthanization, 16 necropsy was performed and lungs of sacrificed hamsters were harvested, prepared, and sectioned and evaluated 17 with a lung histopathological scoring system as previously described [10] . 18 the Alpha, Beta, Gamma, and Delta variants showed 3.79-, 13.30-, 11.39-, and 2.97-fold reductions in 3 neutralizing titer levels, respectively, at five weeks after the second dose ( Figure 1A ). This demonstrated that two 4 doses of W S-2P were relatively effective in stimulating neutralizing antibody against the Alpha and Delta 5 variants but were less effective against the Beta and Gamma variants. GMT titers against the WT, Alpha, and Delta variants similar to those of the W+W group and induced higher 12 GMT titers against the Beta and Gamma variants than the W+W group ( Figure 1A ). 13

Next, we immunized hamsters with a third dose of adjuvanted W S-2P, (Group D: W + W + W) and we 16 analyzed neutralizing titers five weeks later. Compared to the WT ( Figure 1A) , neutralizing titers against the 17 Alpha, Beta, Gamma, and Delta variants were reduced by 3.54-, 15.30-, 11.41-and 3.14-fold, respectively. 18

third dose. We also explored the possibility of using the adjuvanted Beta variant version of S-2P as the third dose 2

in Group E (W + W + B). Compared to WT, this resulted in reductions of neutralizing titers against the Alpha, 3

Beta, Gamma, and Delta variants of 3.52-, 6.42-, 5.09-and 1.85-fold, respectively. Compared to the other groups, 4 the W + W + B regimen resulted in the highest neutralization titers against the WT and all of the VoCs tested, 5 especially against the Delta variant. Overall, the neutralizing titers were lowest for the Beta and Gamma variants 6 and regardless of the treatment group ( Figure 1A) . 7

In the Omicron pseudovirus neutralization assay, the GMTs against Omicron were reduced dramatically in 8 all groups, but Group E (6.8-fold) showed less reduction than Group D (17.8-fold) ( Figure 1B) . Boosting with the 9

Beta variant S-2P (Group E) increased ID 50 GMT against WT and Omicron by 1.5 times and 3.8 times, 10 respectively, compared to Group D ( Figure 1B) . Thus, live virus and pseudovirus assays show that two doses of 11 W S-2P followed by a booster dose of B S-2P increase immunity against VoCs, including the Omicron variant, 12 better than three doses of W S-2P. 13

Hamsters in all treatment groups (Groups A to E) initially lost weight for up to 3 d.p.i. but recovered and did 16 not show significant weight loss by 6 d.p.i. (Figure 2A ). In contrast, the adjuvant control group showed a steady 17 decline in weight that coincided with high lung viral titer and RNA load in the adjuvant control group (Figures 18

2A, 2B, and 2C). At 3 d.p.i., lung viral RNA levels in the treatment groups were lower than in the adjuvant 1 control group, but the difference was only significant in Group E (p < 0.01) ( Figure 2B ). In contrast, by 6 d.p.i. 2 the viral RNA levels in all groups were significantly (p < 0.05) lower than in the adjuvant control group. TCID 50 3 levels were significantly lower (p < 0.05) at 3 d.p.i. in all treatment groups relative to the adjuvant control ( Figure  4 2C). There were no differences in histopathology scores at 3 d.p.i. between control and experimental groups 5 ( Figure 2D ). However, at 6 d.p.i, the adjuvant control group had significantly (p < 0.01) increased lung pathology 6 including extensive and severe immune cell infiltration, hemorrhage, and diffuse alveolar damage compared to 7 groups receiving three doses of S-2P (i.e. Groups D and E) (Figures 2D and S2 ). 8

Here we report live virus and pseudovirus neutralization titers elicited in hamsters by 5 combinations of 10 adjuvanted Wuhan and Beta S-2P vaccines given up to three times. We found that two doses of W S-2P followed 11 by a dose of B S-2P induced the highest neutralizing antibody titer and broadest spectrum against all VoCs tested. 12

The same vaccination regime also significantly increased neutralizing antibody titer against Omicron variant 13 pseudovirus ( Figure 1B ). All five vaccination regimens protected hamsters from weight loss and reduced viral 14 load after infection with Delta variant (Figure 2) . Interestingly, while group B had a relatively poor antibody 15 response against the Delta variant, the protection offered by this regimen against weight loss was comparable to 16 other groups in which the hamsters did not experience any weight loss or increase in lung pathology (Figure 2) . 17

This suggests that apart from neutralizing antibodies, protection from disease could also be attributed to innate 1 and cellular immunity as previously demonstrated in a ChAdOx1 nCoV-19 clinical study [11] . 2

As vaccines induce polyclonal neutralizing antibodies, they could be cross-reactive to different SARS-CoV-3 2 variants. SARS-CoV-2 vaccine could induce broadly neutralizing antibodies targeting the N-terminal domain 4 (NTD) and residues in the RBD that are conserved across SARS-CoV-2 variants [12]. Mechanistically, this could 5 be because a booster of Beta variant S-2P after two doses of W S-2P selects for B-cells that produce antibodies 6 against conserved epitopes between variants, and elicits a broadly reactive T-cell immune response as shown in a 7 study with recipients receiving a variety of vaccines [13] . The inability of RNA amplification assay to distinguish 8 between replicating virus and inactivated virus may explain for the discrepancy between detectable levels of viral 9

RNA and undetectable TCID 50 levels at 3 d.p.i., as observed in our previous hamster study [10] . 10

One limitation of this study is that we have not tested in vivo protection by our vaccine with VoCs other 11 than the Delta variant. Second, the natural course of infection in hamsters results in eventual convalescence, and 12 so the model does not permit evaluation of mortality or severe disease endpoints, and are inadequate as models for 13

Omicron infection due to limited weight loss and lower viral load [14] . The lung histopathology scoring system 14 we used in our animal model also did not distinguish between different levels of lung damage caused by different 15 degrees of viral replication in the lung, and no immunohistochemistry was done to visualize the presence of viral 16 antigens in order to extend on our viral RNA detection and TCID 50 assays. Finally, hamster T-cell responses were 17 not evaluated in this study, but a non-human primate challenge study at the US National Institutes of Health has 18

shown that adjuvanted W S-2P induced Th1-biased response with no detectable CD8 T cell response (Robert 1 Seder, personal communication). We would like to thank the team members at TFBS Bioscience Incorporation for their assistance with 13 hamster housing and the immunization process. We also thank Academia Sinica for the use of their Biosafety 

CoVariants: SARS-CoV-2 Mutations and Variants of Interest

Effectiveness of mRNA-1273 against SARS-CoV-2 omicron and 6 delta variants

Immunogenicity and Safety Following a Homologous Booster Dose 9 of a SARS-CoV-2 recombinant spike protein vaccine

Effectiveness of a third dose of BNT162b2 mRNA COVID-19

A retrospective cohort study

Development of CpG-adjuvanted stable prefusion SARS-CoV-2 spike 16 antigen as a subunit vaccine against COVID-19

Lin 18 YL. Safety and immunogenicity of CpG 1018 and aluminium hydroxide-adjuvanted SARS-CoV-2 S-2P 19 protein vaccine MVC-COV1901: interim results of a large-scale, double-blind, randomised, placebo-20 controlled phase 2 trial in Taiwan

Evaluating the Neutralizing Ability of a CpG-Adjuvanted S-2P Subunit 22 second dose of a protein subunit vaccine MVC-COV1901: An extension to an open-label, dose-escalation, 4 and non-randomized phase 1 study

CpG-adjuvanted stable prefusion SARS-CoV-2 spike protein protected 7 hamsters from SARS-CoV-2 challenge

AZD1222) vaccine in a phase 1/2 clinical trial

Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron 11 antigenic shift

Divergent SARS CoV-2 Omicron-reactive T-and B cell 13 responses in COVID-19 vaccine recipients

SARS-CoV-2 Omicron virus causes attenuated disease 16 in mice and hamsters

Preliminary Analysis of Safety and Immunogenicity of a SARS

Variant Vaccine Booster

 Figure 1