Serum Neutralizing Activity of mRNA-1273 against SARS-CoV-2 Variants

ABSTRACT The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has led to growing concerns over increased transmissibility and the ability of some variants to partially escape immunity. Sera from participants immunized on a prime-boost schedule with the mRNA-1273 COVID-19 vaccine were tested for neutralizing activity against several SARS-CoV-2 variants, including variants of concern (VOCs) and variants of interest (VOIs), compared to neutralization of the wild-type SARS-CoV-2 virus (designated D614G). Results showed minimal, statistically nonsignificant effects on neutralization titers against the B.1.1.7 (Alpha) variant (1.2-fold reduction compared with D614G); other VOCs, such as B.1.351 (Beta, including B.1.351-v1, B.1.351-v2, and B.1.351-v3), P.1 (Gamma), and B.1.617.2 (Delta), showed significantly decreased neutralization titers ranging from 2.1-fold to 8.4-fold reductions compared with D614G, although all remained susceptible to mRNA-1273-elicited serum neutralization. IMPORTANCE In light of multiple variants of SARS-CoV-2 that have been documented globally during the COVID-19 pandemic, it remains important to continually assess the ability of currently available vaccines to confer protection against newly emerging variants. Data presented herein indicate that immunization with the mRNA-1273 COVID-19 vaccine produces neutralizing antibodies against key emerging variants tested, including variants of concern and variants of interest. While the serum neutralization elicited by mRNA-1273 against most variants tested was reduced compared with that against the wild-type virus, the level of neutralization is still expected to be protective. Such data are crucial to inform ongoing and future vaccination strategies to combat COVID-19.

IMPORTANCE In light of multiple variants of SARS-CoV-2 that have been documented globally during the COVID-19 pandemic, it remains important to continually assess the ability of currently available vaccines to confer protection against newly emerging variants. Data presented herein indicate that immunization with the mRNA-1273 COVID-19 vaccine produces neutralizing antibodies against key emerging variants tested, including variants of concern and variants of interest. While the serum neutralization elicited by mRNA-1273 against most variants tested was reduced compared with that against the wild-type virus, the level of neutralization is still expected to be protective. Such data are crucial to inform ongoing and future vaccination strategies to combat COVID-19. KEYWORDS COVID-19, SARS-CoV-2 variants of concern, mRNA-1273, neutralization A s the coronavirus disease 2019 (COVID-19) pandemic continues to escalate in various parts of the world, several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of interest (VOIs) and variants of concern (VOCs) have emerged, including in the United States (B.  (1). There is growing concern over these variants based on increased transmissibility and the ability of some variants to partially escape both natural and vaccine-induced immunity. Notably, the B.1.617.2 lineage has been classified as a VOC by the World Health Organization due to evidence of an increased rate of transmission, reduced effectiveness of monoclonal antibody treatment, and reduced susceptibility to neutralizing antibodies (1).

RESULTS
We assessed neutralization activity of sera against D614G pseudovirus (predominant variant in 2020), B.  (2). Results showed minimal, statistically nonsignificant effects on neutralization titers against B.1.1.7 and A.23.1-v1 compared to D614G (P = 0.64 and 0.46, respectively) ( Fig. 1). In contrast, all other variants examined showed significantly decreased neutralization titers compared with D614G (P , 0.01) (Fig. 1), although all remained susceptible to mRNA-1273-elicited serum neutralization. Reductions in neutralization titers for these variants ranged from a factor of 2.1 to 8.4 compared with that for D614G (Fig. 1A). Across the 3 versions of the B.1.351 variant tested, 6.9-fold to 8.4-fold reductions in neutralization were observed compared with that for D614G (Fig. 1A). Among all variants tested, the greatest effect on neutralization was observed for A.VOI.V2 and B.

DISCUSSION
Among VOCs tested, serum-elicited neutralization of the B.1.1.7 (Alpha) variant was comparable to that of D614G; a range of significantly reduced neutralization titers compared to D614G were observed for other VOCs, including the B.1.351 (Beta), P.1 (Gamma), and B.1.617.2 (Delta) variants, with reductions ranging from 2.1-fold to 8.4-fold. Results presented here are generally consistent with previous studies examining neutralization activity of mRNA-1273-induced immune sera against VOIs/VOCs (reviewed in reference 6), with similar overall trends using both live-virus and pseudovirus neutralization assays (6,7). Similar trends in neutralizing activity of VOIs/VOCs by sera from individuals immunized with BNT162b2 were also observed (6,(8)(9)(10). A limitation of this study is that differential variant spike incorporation into the various pseudoviruses might impact neutralization results. Nevertheless, these data emphasize the need to continually assess the ability of mRNA-1273 to confer protection against prevalent and emergent VOIs/VOCs. Such preclinical analyses in conjunction with epidemiological monitoring of the incidence and spread   of VOCs directly inform strategies around vaccines targeting SARS-CoV-2 variants. As new variants emerge, including those that lead to greater vaccine breakthrough cases, similar analyses could be designed to test vaccine-induced immunity against variants in either animal or clinical studies. Such data are crucial to inform necessary modifications to COVID-19 mRNA vaccines going forward, which may help to mitigate the ongoing spread of SARS-CoV-2 and the emergence of new variants.

MATERIALS AND METHODS
Clinical trial. Healthy adult participants (n = 8; age [mean 6 standard deviation], 34.8 6 9.7 years; male, 37.5%) were immunized with mRNA-1273 (100 mg) on a prime-boost schedule, and serum was collected 7 days after the booster (day 36). Study protocols and results have been reported previously (2).
Recombinant VSV-based pseudovirus assay. Codon-optimized full-length spike (S) protein of the original Wuhan-Hu-1 isolate with D614G mutation (D614G) was cloned into a pCAGGS vector. This codon-optimized D614G vector was used as a template for site-directed mutagenesis to incorporate the S variants, listed in Table 1. To make SARS-CoV-2 full-length S-pseudotyped recombinant vesicular stomatitis virus DG (VSVDG)-firefly luciferase virus, BHK-21/WI-2 cells (Kerafast) were transfected with the S expression plasmid and subsequently infected with VSVDG-firefly luciferase as previously described (11). For the neutralization assay, serially diluted serum samples were mixed with pseudovirus and incubated at 37°C for 45 min. The virus-serum mix was subsequently used to infect A549-hACE2-TMPRSS2 cells (12) for 18 h at 37°C before addition of ONE-Glo reagent (Promega) for measurement of the luciferase signal by relative luminescence units (RLUs). The percentage of neutralization was calculated based on the RLUs of the virus-only control and subsequently analyzed using the four-parameter logistic curve in Prism v.8 (GraphPad Software, Inc.).
Statistical analysis. A two-sided Wilcoxon matched-pairs signed-rank test was used to compare the same patients against different viruses. Statistical analyses were performed (Prism v.8). Geometric mean titers, lower limit of quantification, and fold change relative to D614G were included.

ACKNOWLEDGMENTS
This research was supported by Moderna, Inc., and Biomedical Advanced Research and Development Authority, Department of Health and Human Services (contract 75A50120C00034). This work used samples from the phase 1 mRNA-1273 study (NCT04283461). The mRNA-1273 phase 1 study was sponsored and primarily funded by the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, in part with federal funds from the NIAID under grant awards UM1AI148373 to Kaiser Washington, UM1AI148576, UM1AI148684, and NIH P51 OD011132 to Emory University, and NIH AID AI149644 and contract award HHSN272201500002C to Emmes. Funding for the manufacture of mRNA-1273 phase 1 material was provided by the Coalition for Epidemic Preparedness Innovation. A