key: cord-0821506-gnq8dz3x
authors: Wu, Kai; Choi, Angela; Koch, Matthew; Elbashir, Sayda; Ma, LingZhi; Lee, Diana; Woods, Angela; Henry, Carole; Palandjian, Charis; Hill, Anna; Jani, Hardik; Quinones, Julian; Nunna, Naveen; O'Connell, Sarah; McDermott, Adrian B; Falcone, Samantha; Narayanan, Elisabeth; Colpitts, Tonya; Bennett, Hamilton; Corbett, Kizzmekia S; Seder, Robert; Graham, Barney S; Stewart-Jones, Guillaume BE; Carfi, Andrea; Edwards, Darin K
title: Variant SARS-CoV-2 mRNA vaccines confer broad neutralization as primary or booster series in mice
date: 2021-11-08
journal: Vaccine
DOI: 10.1016/j.vaccine.2021.11.001
sha: fd92f8346c61d961f07fec55e1afdd6f3c9d6360
doc_id: 821506
cord_uid: gnq8dz3x

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of a global pandemic. Safe and effective COVID-19 vaccines are now available, including mRNA-1273, which has shown 94% efficacy in prevention of symptomatic COVID-19 disease. However, the emergence of SARS-CoV-2 variants has led to concerns of viral escape from vaccine-induced immunity. Several variants have shown decreased susceptibility to neutralization by vaccine-induced immunity, most notably B.1.351 (Beta), although the overall impact on vaccine efficacy remains to be determined. Here, we present the initial evaluation in mice of 2 updated mRNA vaccines designed to target SARS-CoV-2 variants: (1) monovalent mRNA-1273.351 encodes for the spike protein found in B.1.351 and (2) mRNA-1273.211 comprising a 1:1 mix of mRNA-1273 and mRNA-1273.351. Both vaccines were evaluated as a 2-dose primary series in mice; mRNA-1273.351 was also evaluated as a booster dose in animals previously vaccinated with mRNA-1273. The results demonstrated that a primary vaccination series of mRNA-1273.351 was effective at increasing neutralizing antibody titers against B.1.351, while mRNA-1273.211 was effective at providing broad cross-variant neutralization. A third (booster) dose of mRNA-1273.351 significantly increased both wild-type and B.1.351-specific neutralization titers. Both mRNA-1273.351 and mRNA-1273.211 are being evaluated in pre-clinical challenge and clinical studies.

Since the declaration of a global pandemic by the World Health Organization on March 11, 2020 , infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to approximately 4.7 million deaths worldwide [1, 2] . Shortly after the SARS-CoV-2 genetic sequence was determined, mRNA-1273-a novel lipid nanoparticle (LNP) encapsulated messenger RNA-based vaccine encoding for a prefusion-stabilized, full-length spike (S) glycoprotein of the Wuhan-Hu-1 isolate of SARS-CoV-2-was developed [3, 4] . Vaccination with two 100 µg doses of mRNA-1273 four weeks apart was 94% efficacious against symptomatic COVID-19 disease; mRNA-1273 was granted Emergency Use Authorization by the Food and Drug Administration in December 2020 [5, 6] .

The emergence of SARS-CoV-2 variants with substitutions in the receptor binding domain (RBD) and N-terminal domain (NTD) of the viral S protein has raised concerns among scientists and health officials [7] [8] [9] [10] . The entry of coronaviruses into host cells is mediated by interaction between the RBD of the viral S protein and the host receptor, angiotensin-converting enzyme 2 (ACE2) [3, [11] [12] [13] [14] . Several studies have shown that the RBD is the main target of neutralizing antibodies against SARS-CoV-2 [4, [15] [16] [17] . A neutralization "supersite" has also been identified in the NTD [18] . A decrease in vaccine-mediated viral neutralization has been correlated with amino acid substitutions in the RBD (eg, K417T/N, E484K, and N501Y) and NTD (eg, L18F, D80A, D215G, and Δ242-244) of the S protein. Some of the most recently circulating variants of concern (VOCs) and variants of interest (VOIs) with key mutations in the RBD and NTD- (Epsilon or CAL.20C) lineages-have shown reduced susceptibility to neutralization from convalescent serum and resistance to monoclonal antibodies [18] [19] [20] [21] [22] [23] [24] [25] . Note that mutations in the NTD domain, specifically the neutralization supersite, are extensive in the B.1.351 lineage virus [18] .

Using 2 orthogonal pseudovirus neutralization (PsVN) assays based on vesicular stomatitis virus (VSV) and lentivirus expressing S variants, neutralizing capacity of sera from phase 1 participants and non-human primates (NHPs) that received 2 doses of mRNA-1273 was reported [26] . No significant impact on neutralization against the B. [31, 32] . Studies have demonstrated reduced neutralization titers against the full B.1.351 variant following mRNA-1273 vaccination, although levels are still significant and expected to be protective based on challenge studies in NHPs [26, 33, 34] . Despite this prediction of continued efficacy of mRNA-1273 against this key variant of concern (VOC), the magnitude and duration of vaccine-mediated protection is still unknown. Moreover, a key related question is 

No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.

A sequence-optimized mRNA encoding prefusion-stabilized Wuhan-Hu-1 or B.1.351-variant SARS-CoV-2 S-2P protein was synthesized in vitro using an optimized T7 RNA polymerasemediated transcription reaction with complete replacement of uridine by N1m-pseudouridine [35] .

The reaction included a DNA template containing the immunogen open-reading frame flanked by 5' untranslated region (UTR) and 3' UTR sequences and was terminated by an encoded polyA tail.

After transcription, the cap-1 structure was added to the 5' end using the Vaccinia capping enzyme (New England Biolabs) and Vaccinia 2'-O-methyltransferase (New England Biolabs). The mRNA was purified by oligo-dT affinity purification, buffer exchanged by tangential flow filtration into sodium acetate, pH 5.0, sterile filtered, and kept frozen at -20°C until further use.

The mRNA was encapsulated in an LNP through a modified ethanol-drop nanoprecipitation process described previously [36] . Ionizable, structural, helper, and polyethylene glycol lipids were briefly mixed with mRNA in an acetate buffer, pH 5.0, at a ratio of 2.5:1 (lipids:mRNA).

The mixture was neutralized with Tris-HCl, pH 7.5, sucrose was added as a cryoprotectant, and the final solution was sterile-filtered. Vials were filled with formulated LNP and stored frozen at -70°C until further use. The drug product underwent analytical characterization-which included the determination of particle size and polydispersity, encapsulation, mRNA purity, double-stranded RNA content, osmolality, pH, endotoxin, and bioburden-and the material was deemed acceptable for in vivo study.

Animal experiments were carried out in compliance with approval from the Animal Care and Use Committee of Moderna Inc. Female BALB/c mice (6 to 8 weeks old; Charles River Laboratories) were used. mRNA formulations were diluted in 50 µL of 1X phosphate-buffered saline (PBS), and mice were inoculated via intramuscular injection into the same hind leg for both prime, boost, and third dose. Control mice received PBS because prior studies have demonstrated that tested mRNA formulations do not create significant levels of non-specific immunity beyond a few days [37] [38] [39] . Sample size for animal experiments was determined on the basis of criteria set by the institutional Animal Care and Use Committee. Experiments were neither randomized nor blinded.

Microtiter plates (96-well; Thermo) were coated with 1 µg/mL S-2P protein (Genscript) corresponding to the S protein of the Wuhan-Hu-1 virus. After overnight incubation at 4°C, plates were washed four times with PBS/0.05% Tween-20 and blocked for 1.5 hours at 37°C (SuperBlock-Thermo). After washing, five-fold serial dilutions of mouse serum were added (assay diluent: 0.05% Tween-20 and 5% goat serum in PBS). Plates were incubated for 2 hours at 37°C, washed and horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG) (Southern Biotech) was added at a 1:20,000 dilution (S-2P) in assay diluent. Plates were incubated for 1 hour at 37°C, washed, and bound antibody was detected with a 3,3',5,5'-tetramethylbenzidine (TMB) substrate (SeraCare). After incubation for 10 minutes at room temperature, the reaction was stopped by adding a TMB stop solution (SeraCare) and the absorbance was measured at 450 nm. Titers were determined using a four-parameter logistic curve fit in Prism v.8 (GraphPad Software, Inc.) and defined as the reciprocal dilution at approximately optical density 450 = 1.0 (normalized to a mouse standard on each plate).

Codon-optimized full-length S protein of the original Wuhan-Hu-1 variant with D614G mutation (D614G) or the indicated S variants, listed in Table 1 , were cloned into a pCAGGS vector.

To make SARS-CoV-2 full-length S pseudotyped recombinant VSV-ΔG-firefly luciferase virus, BHK-21/WI-2 cells (Kerafast) were transfected with the S expression plasmid and subsequently infected with VSV∆G-firefly-luciferase as previously described [40] . For a neutralization assay, serially diluted serum samples were mixed with pseudovirus and incubated at 37°C for 45 minutes.

The virus-serum mix was subsequently used to infect A549-hACE2-TMPRSS2 cells [41] for 18 hours at 37°C before adding 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 four-parameter logistic curve (Prism v.8).

Animal studies were completed once with all in vitro testing completed in duplicate or triplicate with 1 replicate, unless otherwise stated. Two-sided Wilcoxon matched-pairs signed rank test was used to compare the same animals against different viruses or at different time points.

Statistical analyses were performed (Prism v.8). Geometric mean titers with 95% CIs and lower limits of detection are included, where applicable.

The immunogenicity of mRNA-1273.351 and mRNA-1273.211 vaccines against the original Wuhan-Hu-1 variant with the D614G mutation (referred to as wild-type) and the B.1.351 variant was evaluated in BALB/c mice 2 weeks after the first and second injection (Fig. 1 ). Animals were vaccinated with 1 or 10 µg of mRNA-1273, mRNA-1273.351, or mRNA-1273.211 on a 0, 21-day schedule (Fig. 2a) . S protein-binding antibody titers were assessed using a Wuhan-Hu-1 S-2P

ELISA. In addition, neutralizing antibody titers were assessed in a VSV-based PsVN assay against wild-type and variant viruses (Table 1) .

High levels of binding antibody were elicited by vaccination in all animals 2 weeks after the first and second injection, with 4.5 to 9.4-fold increased S-2P binding titers measured after the second dose (Fig. 2b) . Slightly lower antibody levels were observed for mRNA-1273.351 compared with mRNA-1273, potentially due to the coating S-2P protein used in the ELISA being homologous to mRNA-1273. These results demonstrate that both mRNA-1273.351 and mRNA-1273.211 are immunogenic in mice. mRNA-1273 elicited higher neutralization titers against the D614G than B.1.351 pseudovirus (Fig. 2c,d) , although the approximate 2-fold difference was smaller than previously measured with phase 1 clinical trial sera [26] . mRNA-1273.351 elicited higher neutralization titers against B.1.351 compared with the D614G pseudovirus (Fig. 2c,d) ,

with an approximate 4-fold difference in measured titers. When mRNA-1273.211 was used to vaccinate mice, similar neutralization titers were elicited against both the D614G and B.1.351 pseudoviruses (Fig. 2c,d) 

To evaluate the ability of the mRNA-1273.351 to boost pre-existing immunity and increase neutralization against both the wild-type and the B.1.351 virus, BALB/c mice were immunized with 1 or 0.1 µg mRNA-1273 on day 1 and 22, and the level and durability of the antibody responses were evaluated over the course of 7 months. Sera collection occurred on day 212, and a third dose of 1 or 0.1 µg mRNA-1273.351 was administered on day 213 (Fig. 3a) .

High levels of binding antibody were elicited by vaccination with mRNA-1273, with peak titers measured 2 weeks after the second dose (Fig. 3b) . After an initial drop in antibody levels, titers were stable over the 7-month monitoring period. Following the mRNA-1273.351 booster injection, antibody levels dramatically rose, exceeding the previously measured peak for both the 1 and 0.1 µg dose levels. (Fig. 3c,d) . The difference between the 2 assays narrowed to 2-fold following the booster dose (Fig. 3c,e) ; the GMT of 15,524 against B.1.351 was ~1.5 fold higher than the peak titer against D614G 2 weeks after the second dose (Fig. 3c) . Animals vaccinated at the 0.1 µg dose level had lower titers, but similar trends for binding antibody and neutralization titers were observed (data not shown).

In this study, mRNA-1273.351 and mRNA-1273.211 were evaluated in mice as both a primary vaccination series and as a third booster dose in animals previously vaccinated with 2 injections of mRNA-1273. As a primary vaccination series, both vaccines were potently immunogenic after the first injection, with both S-2P binding and neutralizing antibody titers significantly increasing after the second injection. Neutralizing activity of mRNA-1273.351 against the D614G variant Several potential limitations to the current study should be highlighted. Sera from mRNA-1273 vaccinated NHP or human sera was shown to have 6-8 fold reduced neutralizing activity against the B.1.351 variant SARS-CoV-2 in several assessments, although the level of neutralization remained at levels that are predicted to be protective [26] . Results in this study, however, showed that after the primary series of mRNA-1273, only a 2-fold reduced neutralization against the B.1.351 virus was evident 2 weeks after the second dose. A 6.6-fold reduction was seen at day 

A pneumonia outbreak associated with a new coronavirus of probable bat origin

The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

An mRNA Vaccine against SARS-CoV-2 -Preliminary Report

Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine

FDA (2021) COVID-19 Vaccines

Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus

Spike mutation D614G alters SARS-CoV-2 fitness

Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity

Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein

Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses

Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2

Cell entry mechanisms of SARS-CoV-2

Phase 1-2 Trial of a SARS-CoV-2 Recombinant Spike Protein Nanoparticle Vaccine

Safety and Immunogenicity of Two RNA-Based Covid-19 Vaccine Candidates

Safety and Immunogenicity of SARS-CoV-2 mRNA-1273 Vaccine in Older Adults

N-terminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2

SARS-CoV-2 variants B.1.351 and P.1 escape from neutralizing antibodies

Decreased neutralization of SARS-CoV-2 global variants by therapeutic anti-spike protein monoclonal antibodies

Comprehensive mapping of mutations in the SARS-CoV-2 receptor-binding domain that affect recognition by polyclonal human plasma antibodies

Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7. Nature

1.526 SARS-CoV-2 Variants Identified in New York City are Neutralized by Vaccine-Elicited and Therapeutic Monoclonal Antibodies. mBio

Convalescent-Phase Sera and Vaccine-Elicited Antibodies Largely Maintain Neutralizing Titer against Global SARS-CoV-2 Variant Spikes

Transmission, infectivity, and neutralization of a spike L452R SARS-CoV-2 variant

Serum Neutralizing Activity Elicited by mRNA-1273 Vaccine

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK

Efficacy of ChAdOx1 nCoV-19 (AZD1222) vaccine against SARS-CoV-2 variant of concern

7): an exploratory analysis of a randomised controlled trial

Safety and Efficacy of Single-Dose Ad26.COV2.S Vaccine against Covid-19

Efficacy of NVX-CoV2373 Covid-19 Vaccine against the B.1.351 Variant

Evidence for increased breakthrough rates of SARS-CoV-2 variants of concern in BNT162b2-mRNA-vaccinated individuals

Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine through 6 Months

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

mRNA-1273 protects against SARS-CoV-2 beta infection in nonhuman primates

Impact of mRNA chemistry and manufacturing process on innate immune activation

Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines

Preclinical and Clinical Demonstration of Immunogenicity by mRNA Vaccines against H10N8 and H7N9 Influenza Viruses

Multi-antigenic human cytomegalovirus mRNA vaccines that elicit potent humoral and cell-mediated immunity

Self-Amplifying RNA Vaccines Give Equivalent Protection against Influenza to mRNA Vaccines but at Much Lower Doses

Generation of VSV pseudotypes using recombinant DeltaG-VSV for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines

SARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparedness

Safety and immunogenicity of SARS-CoV-2 variant mRNA vaccine boosters in healthy adults: an interim analysis

Innate transcriptional effects by adjuvants on the magnitude, quality, and durability of HIV envelope responses in NHPs

Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor

We thank Michael Brunner and Dr. Michael Whitt for kind support on recombinant VSV-based SARS-CoV-2 pseudovirus production. Medical writing and editorial assistance was provided by 

The authors declare that the data supporting the findings of this study are available within this article.

Employees of the study sponsor, Moderna, Inc., contributed to the study design, data collection, analysis and interpretation, and writing of the report.

This research was funded by Moderna Inc.