key: cord-1002319-1zg0q89n authors: Corleis, Björn; Hoffmann, Donata; Rauch, Susanne; Fricke, Charlie; Roth, Nicole; Gergen, Janina; Kovacikova, Kristina; Schlottau, Kore; Halwe, Nico Joel; Ulrich, Lorenz; Schön, Jacob; Wernike, Kerstin; Widera, Marek; Ciesek, Sandra; Mueller, Stefan O.; Mettenleiter, Thomas C.; Petsch, Benjamin; Beer, Martin; Dorhoi, Anca title: Low-dose bivalent mRNA vaccine is highly effective against different SARS-CoV-2 variants in a transgenic mouse model date: 2022-04-20 journal: bioRxiv DOI: 10.1101/2022.04.20.485440 sha: 193db15629a14f4dbf11104359b33c5a5fde01df doc_id: 1002319 cord_uid: 1zg0q89n Combining optimized spike (S) protein-encoding mRNA vaccines to target multiple SARS-CoV-2 variants could improve COVID-19 control. We compared monovalent and bivalent mRNA vaccines encoding B.1.351 (Beta) and/or B.1.617.2 (Delta) SARS-CoV-2 S-protein, primarily in a transgenic mouse model and a Wistar rat model. The low-dose bivalent mRNA vaccine contained half the mRNA of each respective monovalent vaccine, but induced comparable neutralizing antibody titres, enrichment of lung-resident memory CD8+ T cells, specific CD4+ and CD8+ responses, and fully protected transgenic mice from SARS-CoV-2 lethality. The bivalent mRNA vaccine significantly reduced viral replication in both Beta- and Delta-challenged mice. Sera from bivalent mRNA vaccine immunized Wistar rats also contained neutralizing antibodies against the B.1.1.529 (Omicron BA.1) variant. These data suggest that low-dose and fit-for-purpose multivalent mRNA vaccines encoding distinct S-proteins is a feasible approach for increasing the potency of vaccines against emerging and co-circulating SARS-CoV-2 variants. 3 Effective vaccines are critical for the control of the COVID-19 pandemic, especially as nations begin to scale back non-pharmaceutical interventions such as social distancing, travel restrictions, and isolation (https://www.ecdc.europa.eu/en/covid-19/prevention-andcontrol/vaccines). Since the beginning of the pandemic, new SARS-CoV-2 variants, including some classed as variants of concern (VOCs) have appeared, each characterized by different virulence, Several VOCs which have mutated extensively, such as Beta 1 and Omicron, 2 have been able to evade humoral responses elicited by vaccines based on ancestral S-protein sequences. 3 As a result, Omicron has quickly become globally prevalent, despite high immunization rates. Unfortunately, while Omicron appears to cause less severe disease than other variants, 4 it does not induce relevant cross-protective neutralizing antibody (nAb) titres in SARS-CoV-2 naïve populations, meaning they may be less protected against future infection compared with those previously exposed to other variants or vaccinated. 5 The evolution of further VOCs is unpredictable; however, it is likely that new escape variants will emerge. Therefore, developing effective vaccines and vaccine strategies will remain essential. 6 Table S3 ). Recent studies have revealed that repeated immunization extends neutralization to non-homologous variants, possibly through affinity maturation of memory B cell populations. 9,13 Following the effective virus clearance in conchae, and the subsequent impact on Delta transmission, it will be important to evaluate the impact of bivalent, and higher valency, mRNA vaccines on nonneutralizing antibody functions, e.g., antibody-dependent natural killer cell activation or cellular phagocytosis, which are elicited by mRNA vaccines and maintained despite reductions in nAbs over time. 14 These non-neutralizing antibodies may facilitate virus clearance like that observed in the URT of Delta-challenged mice. Multivalent influenza 15 and cytomegalovirus 16 mRNA vaccines in pre-clinical models have shown that integration of multiple antigens can lead to robust nAb responses. Furthermore, nAbs have emerged as correlates of immune protection and vaccine efficacy 17 to inform immunization schedules. 18 In our study, anti-RBD total immunoglobulin levels were high in all vaccinated mice, with no notable differences between groups (Fig. S2e ,f). The bivalent mRNA vaccine induced similarly high nAb titres as the Beta and Delta monovalent vaccines with their respective homologous challenges (Fig. 2a,b) , despite the bivalent vaccine containing half the mRNA dose of each monovalent vaccine (0.25 µg vs. 0.5 µg). Irrespective of the challenge, the bivalent mRNA vaccine-induced nAb titres were statistically significantly higher than those induced by CV2CoV, whereas with the monovalent mRNA vaccines the nAB titres were statistically significantly higher with the respective homologous challenges only (Fig. 2a,b) . In separate experiments, serum from Wistar rats vaccinated with CV2CoV or CV2CoV.617.2 mRNA vaccines (monovalent; 8 µg), or vaccines combining CV2CoV617.2 with either CV2CoV or CV2CoV.351 (bivalent; 4 µg of each) on Day 0 and Day 21 (Fig. S5 ) contained high level nAb titres against Delta (Fig. 2c) . Neutralizing antibody titres were notably diminished against Omicron BA.1 in all but the bivalent vaccine group containing Beta and Delta S-protein sequences. Including the Beta S-protein sequence in the vaccine resulted in nAb titres that were 2× higher against Omicron BA.1 than those induced by Delta alone, whereas the nAb titres induced by vaccines without Beta S-protein were 3-9× lower against Omicron BA.1 than those induced by Delta (Fig. 2c) . The bivalent formulation contained a half dose of each mRNA compared with the monovalent vaccines with better antigen coverage; this strategy may be advantageous in case of the emergence of additional antigenic distant variants. Cellular immunity also contributes to protection against COVID-19 and recent evidence suggests that viral-vector vaccines and mRNA vaccines elicit long-lasting S-specific CD4 + and CD8 + T cell responses with broad cross-reactivity against VOCs. 19 While tissue resident memory T cells (T RM ) are induced by high concentration mRNA vaccines; 20 we investigated if lower concentrations of mono-or bivalent mRNA vaccines could induce lung parenchymal tissue resident T cells. We observed that both the monovalent and bivalent mRNA vaccines triggered potent S-specific CD4 + and CD8 + responses, with 2-3 log increases in lung CD45iv -CD8 + T cells, compared with naïve non-vaccinated animals ( Fig. 2d and Fig.S4a ). Further characterization of this T cell population, using markers of lung T cell residency, confirmed significantly higher proportions of T RM cells compared with the CD45iv + populations ( Fig. 2e and Fig.S4b-e) , while mRNA vaccination also induced an enrichment of IFNγ and granzyme-B producing CD8 + T cells in the lung parenchyma (Fig.S4f-i) . The accumulation of memory cells at mucous membrane sites is critical for the control of viral pneumonia and their presence has been reported to be associated with less severe COVID-19 symptoms. 21 CD8 + T cells may contribute to protection when antibodies titres are suboptimal in non-human primates. 22 The role of the durability and specificity of T RM CD8 + cells in protection from disease requires further investigation using antibody-mediated depletion. Depletion of CD8 + cells following immunization with mRNA vaccine, prior to SARS-CoV-2 challenge, led to different outcomes in a transgenic mouse model, possibly due to the vaccine dose used and variability in nAb concentrations. 23, 24 Interest in vaccine-elicited T cell responses has increased due to immune escape by various VOCs, including Omicron. 19 6 Preservation of the epitope repertoire may be critical for the defence against current and future VOCs and may bring substantial benefits by contributing to protection against severe disease. This feature along with tissue residency makes the bivalent mRNA vaccine, and more generally, multivalent vaccines, highly appropriate candidates for further development. In summary, SARS-CoV-2 evolution is a challenge for vaccine-based strategies for disease control. Our study demonstrates that a low-dose, bivalent, unmodified mRNA vaccine is highly efficacious in pre-clinical mouse and rat models and suggests that dose-sparing, multivalent vaccines combining mRNA encoding the S-protein from the variants with unrelated lineages may induce heterologous protection and thus increase the breadth of immune responses. Given their exceptional flexibility in antigen formulation, mRNA vaccine platforms offer advantages regarding adaptability to circulating VOCs and opportunities to design pan-sarbecovirus vaccines. Wistar rats were vaccinated on Day 0 (prime) and Day 21 (booster), as detailed in Fig. S5 . Vaccine-induced T cells were characterized by flow cytometry. Table S4 . Cells were stored in PBS at 4°C in the dark for no longer than 24 hours before acquisition on a BD Fortessa™ i n s t r u m e n t . Details of the T cell gating strategy are presented in Fig. S3 . Detection of a SARS-CoV-2 variant of concern in South Africa Structural basis of SARS-CoV-2 Omicron immune evasion and receptor engagement Considerable escape of SARS-CoV-2 Omicron to antibody neutralization Early assessment of the clinical severity of the SARS-CoV-2 omicron variant in South Africa: a data linkage study Neutralization profile of Omicron variant convalescent individuals The future of SARS-CoV-2 vaccination -Lessons from Influenza Elicitation of broadly protective sarbecovirus immunity by receptorbinding domain nanoparticle vaccines SARS-CoV-2 mRNA vaccines induce persistent human germinal centre responses SARS-CoV-2 Omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses Mechanisms of innate and adaptive immunity to the Pfizer-BioNTech BNT162b2 vaccine ACE2: The major cell entry receptor for SARS-CoV-2 SARS-CoV-2 infection of human ACE2-transgenic mice causes severe lung inflammation and impaired function mRNA booster immunization elicits potent neutralizing serum activity against the SARS-CoV-2 Omicron variant mRNA-1273 vaccine-induced antibodies maintain Fc effector functions across SARS-CoV-2 variants of concern Development of multivalent mRNA vaccine candidates for seasonal or pandemic influenza Multi-antigenic human cytomegalovirus mRNA vaccines that elicit potent humoral and cell-mediated immunity Neutralizing antibody levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infection Homologous and heterologous COVID-19 booster vaccinations SARS-CoV-2 vaccination induces immunological T cell memory able to cross-recognize variants from Alpha to Omicron Protective activity of mRNA vaccines against ancestral and variant SARS-CoV-2 strains Single-cell landscape of bronchoalveolar immune cells in patients with COVID-19 Correlates of protection against SARS-CoV-2 in rhesus macaques Adaptive immune determinants of viral clearance and protection in mouse models of SARS-CoV-2 A single dose of self-transcribing and replicating RNA-based SARS-CoV-2 vaccine produces protective adaptive immunity in mice Reference Sequence NC_045512.2, GenBank accession number YP_009724390.1 and encodes for full length S featuring K986P and V987P mutations SARS-CoV-2 B.1.1.529 sublineage BA.1 "Omicron" FFM-ZAF0396/2021; GenBank accession: OM617939.1, GISAID accession EPI_ISL_6959868 27 was used for virus neutralization assay. Virus stocks were propagated (three passages for Delta, two passages for Beta and Omicron) on Vero E6 cells (Collection of Cell Lines in Veterinary Medicine CCLV-RIE 0929) using a mixture of equal volumes of Eagle MEM (Hanks' balanced salts solution) and Eagle MEM (Earle's balanced salts solution 120 mg/L sodium pyruvate, 10% fetal bovine serum (FBS), pH 7.2. The virus was harvested after 72 hours, titrated on Vero E6 cells and stored at -80 °C until further use Antibodies reactive against the receptor binding domain (RBD) of the ancestral SARS-CoV-2 were measured using the established ELISA protocol as previously described To evaluate specifically the presence of virus-neutralizing antibodies in serum samples the virus neutralization test was performed with use of the viruses introduced for the challenge infection and Omicron in concentrations as described References (methods) Protocol: Real-time RT-PCR assays for the detection of SARS-CoV-2 Institut Pasteur CVnCoV and CV2CoV protect human ACE2 transgenic mice from ancestral B BavPat1 and emerging B.1.351 SARS-CoV-2 Reduced neutralization of SARS-CoV-2 omicron variant by vaccine sera and monoclonal antibodies Multi-species ELISA for the detection of antibodies against SARS-CoV-2 in animals The authors would like to thank Mareen Lange, Patrick Zitzow, and Laura Timm for their excellent technical assistance, Andrea Aebischer for protein preparations, Claudia Wylezich The mRNA vaccine is based on the RNActive ® platform (claimed and described in e.g.