A self-amplifying mRNA SARS-CoV-2 vaccine candidate induces safe and robust protective immunity in preclinical models

Giulietta Maruggi, Corey P Mallett, Jason W Westerbeck, Tiffany Chen, Giuseppe Lofano, Kristian Friedrich, Lin Qu, Jennifer Tong Sun, Josie McAuliffe, Amey Kanitkar, Kathryn T Arrildt, Kai-Fen Wang, Ian McBee, Deborah McCoy, Rebecca Terry, Alison Rowles, Maia Araujo Abrahim, Michael A Ringenberg, Malcolm J Gains, Catherine Spickler, Xuping Xie, Jing Zou, Pei-Yong Shi, Taru Dutt, Marcela Henao-Tamayo, Izabela Ragan, Richard A Bowen, Russell Johnson, Sandra Nuti, Kate Luisi, Jeffrey B Ulmer, Ann-Muriel Steff, Rashmi Jalah, Sylvie Bertholet, Alan H Stokes, Dong Yu, Giulietta Maruggi, Corey P Mallett, Jason W Westerbeck, Tiffany Chen, Giuseppe Lofano, Kristian Friedrich, Lin Qu, Jennifer Tong Sun, Josie McAuliffe, Amey Kanitkar, Kathryn T Arrildt, Kai-Fen Wang, Ian McBee, Deborah McCoy, Rebecca Terry, Alison Rowles, Maia Araujo Abrahim, Michael A Ringenberg, Malcolm J Gains, Catherine Spickler, Xuping Xie, Jing Zou, Pei-Yong Shi, Taru Dutt, Marcela Henao-Tamayo, Izabela Ragan, Richard A Bowen, Russell Johnson, Sandra Nuti, Kate Luisi, Jeffrey B Ulmer, Ann-Muriel Steff, Rashmi Jalah, Sylvie Bertholet, Alan H Stokes, Dong Yu

Abstract

RNA vaccines have demonstrated efficacy against SARS-CoV-2 in humans, and the technology is being leveraged for rapid emergency response. In this report, we assessed immunogenicity and, for the first time, toxicity, biodistribution, and protective efficacy in preclinical models of a two-dose self-amplifying messenger RNA (SAM) vaccine, encoding a prefusion-stabilized spike antigen of SARS-CoV-2 Wuhan-Hu-1 strain and delivered by lipid nanoparticles (LNPs). In mice, one immunization with the SAM vaccine elicited a robust spike-specific antibody response, which was further boosted by a second immunization, and effectively neutralized the matched SARS-CoV-2 Wuhan strain as well as B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta) variants. High frequencies of spike-specific germinal center B, Th0/Th1 CD4, and CD8 T cell responses were observed in mice. Local tolerance, potential systemic toxicity, and biodistribution of the vaccine were characterized in rats. In hamsters, the vaccine candidate was well-tolerated, markedly reduced viral load in the upper and lower airways, and protected animals against disease in a dose-dependent manner, with no evidence of disease enhancement following SARS-CoV-2 challenge. Therefore, the SARS-CoV-2 SAM (LNP) vaccine candidate has a favorable safety profile, elicits robust protective immune responses against multiple SARS-CoV-2 variants, and has been advanced to phase 1 clinical evaluation (NCT04758962).

Keywords: SARS-CoV-2 vaccine; biodistribution; efficacy; immunogenicity; self-amplifying mRNA; spike antigen; toxicity.

Conflict of interest statement

Declaration of interests G.M., C.P.M., J.W., T.C., G.L., K.F., L.Q., J.T.S., J.M., A.K., K.A., K-F.W., I.M., R.T., A.R., M.A.R., A-M.S., R.Jo., S.N., R.J., K.L., S.B., J.B.U., A.H.S., and D.Y. are current or former employees of the GSK group of companies and may own GSK shares and/or restricted GSK shares. G.M., J.W., L.Q., K.L., J.B.U., and D.Y. are inventors on a patent application claiming subject matter related to the SARS-CoV-2 SAM vaccine candidates described herein. P.Y.S. is a member of the Scientific Advisory Boards of AbImmune and is Founder of FlaviTech. X.X. and P.-Y.S. have filed a patent on the reverse genetic system of SARS-CoV-2. M.A.A., M.G., and C.S. received compensation from GSK to perform the rat toxicity and biodistribution assays. The other authors declare no other competing interests.

Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Graphical abstract
Graphical abstract
Figure 1
Figure 1
Vaccine design and antigen characterization (A) Schematic representation of the SAM vector encoding the prefusion-stabilized SARS-CoV-2 spike (spikeFL-2P) protein (i.e., SARS-CoV-2 SAM). S1, S2, transmembrane (TM), and cytoplasmic (CT) domains, K986P, V987P, and GSAS mutations are indicated. (B) Percentage of spike-positive cells measured by intracellular flow cytometry 18 h after green fluorescent protein (GFP), prefusion-stabilized (spikeFL-2P), or wild-type (spikeFL) spike sequence SAM RNA electroporation of BHK cells, and intracellular staining with an anti-S2-specific antibody. (C) SAM-transfected BHK cells were also treated with (+) or without (−) BFA, and 18 h later, cell lysates were collected and analyzed by immunoblotting using anti-S2 or anti-actin antibodies. A recombinant spike protein was loaded as control. In addition, mouse C2C12 cells were transfected with SAM, treated with (+) or without (−) BFA, collected 18 h later, and surface-expressed spike protein was detected by flow cytometry using either the anti-S2-antibody (D) or incubated with soluble hACE2 and stained with an anti-hACE2 antibody (E). nsPs, non-structural proteins; UTR, untranslated regions; arrow, subgenomic promoter; kDa, kilodalton; S0, furin uncleaved full-length spike protein; S2, furin cleaved domain. Error bars represent SD of duplicate samples. Data shown are representative of at least two experiments.
Figure 2
Figure 2
Humoral responses elicited by SARS-CoV-2 SAM (LNP) vaccine in mice Pseudovirus-based spike 50% neutralization titers (pVNT50) of immunized mice sera or human COVID-19 convalescent sera (HCS) against Wuhan (A), Alpha (B.1.1.7) (C), Beta (B.1.351) (D), or Delta (B.1.617.2) (E) spike sequences. A panel of 22 HCS was tested against Wuhan, Alpha, and Beta, whereas a panel of 21 HCS (same panel minus one subject) was tested against Delta. Mouse sera were collected 3 weeks after the first vaccination (3wp1) and 2 weeks after the second vaccination (2wp2). (B) SARS-CoV-2 50% virus neutralization titers (VNT50) measured with a mNeonGreen reporter virus-based assay in sera collected 2wp2 from immunized mice or in a panel of COVID-19 HCS (n = 22). The dotted line indicates the limit of detection. Geometric means of each group ±95% confidence interval are shown. Each dot represents individual samples. Bars under p value asterisks indicate significantly different groups comparisons with ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001.
Figure 3
Figure 3
Cellular responses elicited by SARS-CoV-2 SAM (LNP) vaccine in mice Splenocytes of BALB/c mice (n = 5) immunized i.m. with SAM vaccine or saline control (mock) were harvested at 2wp2, stimulated ex vivo with a peptide mix specific to spikeFL-2P, and analyzed for CD8+ (A and C) and CD4+ (B and D) T cell responses by flow cytometry. The stacked bars (A and B) indicate distribution of the Tc0/Tc1/Tc2/Tc17 cytotoxic cells within the total CD8+ and the Th0/Th1/Th2/Th17 T helper cells within the CD4+ spike-specific T cells (mean ± SEM). Boolean combinations for spike-specific CD8+ and CD4+ T cell cytokines from splenocytes of individual immunized mice were background subtracted using Pestle software, and pies were generated using SPICE software (C and D). Each pie slice represents a proportion of the total spike-specific CD8+ or CD4+ T cell responses for a unique sub-population comprising CD107a, IFN-γ, IL-2, or TNF-α, as indicated in the legend. Bars under p value asterisks indicate significantly different group comparisons with ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001.
Figure 4
Figure 4
Viral loads from oropharyngeal swabs and respiratory tract tissues in SARS-CoV-2 SAM (LNP) vaccinated hamsters after challenge with SARS-CoV-2 virus (A and B) Oropharyngeal swabs were taken from all hamsters on 1, 2, 3, and 7 dpi, and viral titers were determined by plaque assay. (C–E) Virus titers were determined in nasal turbinates (C), cranial right (D), and caudal right (E) lung lobes of four female and four male hamsters from each group at 3 dpi. Bars represent GMT+95% CI. Asterisks above bars indicate statistically significant difference in viral titers between mock and vaccine groups (∗∗∗∗, p 

Figure 5

Histopathology following SARS-CoV-2 challenge of…

Figure 5

Histopathology following SARS-CoV-2 challenge of SARS-CoV-2 SAM (LNP) and mock vaccinated hamsters (A)…

Figure 5
Histopathology following SARS-CoV-2 challenge of SARS-CoV-2 SAM (LNP) and mock vaccinated hamsters (A) Lung pathology grading, with respect to inflammation (A) and centriacinar/bronchioloalveolar epithelial hyperplasia (B) in male and female hamsters necropsied at 3, 7, and 21 dpi with SARS-CoV-2 virus. (C) Representative lung sections from a naive male hamster showing the background level of atelectasis that can occur during necropsy. (D) Representative lung sections from mock vaccinated, SAM low dose (0.03μg), and SAM high dose (3μg) vaccinated male animals infected with SARS-CoV-2 virus and necropsied at 3, 7, and 21 dpi (histopathological features in females are consistent with those in males, females not shown). Sections are stained with H&E, and whole slide images were obtained using P250 scanner (3D Histech). Representative low-power (bottom left, scale bar 5 mm), medium-power (top, scale bar 200 μm), and higher power (bottom right, scale bar 50 μm) photomicrographs are shown from each group. “#,” hyperplasia of type II pneumocytes and bronchiolization of alveoli; “G,” hyperplasia of goblet cells within bronchial epithelium; “H,” areas of alveolar wall necrosis and hemorrhage within regions of inflammation. Further description of the histopathology at each time point is provided in the main text.

Figure 6

Repeated-dose toxicology assessment in SARS-CoV-2…

Figure 6

Repeated-dose toxicology assessment in SARS-CoV-2 SAM (LNP) vaccinated rats (A) Body temperature. Male…

Figure 6
Repeated-dose toxicology assessment in SARS-CoV-2 SAM (LNP) vaccinated rats (A) Body temperature. Male and female rats were immunized on three occasions (day 1, 15, and 29) with 12 μg of SARS-CoV-2 SAM (LNP) or saline. Rectal temperatures were measured 1 h before (Pre) and 2, 4, 6, 12, 18, 24, and 48 h post immunization. Results are shown as mean with SD. Statistical analysis: ∗, p 
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Figure 5
Figure 5
Histopathology following SARS-CoV-2 challenge of SARS-CoV-2 SAM (LNP) and mock vaccinated hamsters (A) Lung pathology grading, with respect to inflammation (A) and centriacinar/bronchioloalveolar epithelial hyperplasia (B) in male and female hamsters necropsied at 3, 7, and 21 dpi with SARS-CoV-2 virus. (C) Representative lung sections from a naive male hamster showing the background level of atelectasis that can occur during necropsy. (D) Representative lung sections from mock vaccinated, SAM low dose (0.03μg), and SAM high dose (3μg) vaccinated male animals infected with SARS-CoV-2 virus and necropsied at 3, 7, and 21 dpi (histopathological features in females are consistent with those in males, females not shown). Sections are stained with H&E, and whole slide images were obtained using P250 scanner (3D Histech). Representative low-power (bottom left, scale bar 5 mm), medium-power (top, scale bar 200 μm), and higher power (bottom right, scale bar 50 μm) photomicrographs are shown from each group. “#,” hyperplasia of type II pneumocytes and bronchiolization of alveoli; “G,” hyperplasia of goblet cells within bronchial epithelium; “H,” areas of alveolar wall necrosis and hemorrhage within regions of inflammation. Further description of the histopathology at each time point is provided in the main text.
Figure 6
Figure 6
Repeated-dose toxicology assessment in SARS-CoV-2 SAM (LNP) vaccinated rats (A) Body temperature. Male and female rats were immunized on three occasions (day 1, 15, and 29) with 12 μg of SARS-CoV-2 SAM (LNP) or saline. Rectal temperatures were measured 1 h before (Pre) and 2, 4, 6, 12, 18, 24, and 48 h post immunization. Results are shown as mean with SD. Statistical analysis: ∗, p 
All figures (7)

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