mRNA-Based Vaccines and Mode of Action.

In the past 20 years, the mRNA vaccine technology has evolved from the first proof of concept to the first licensed vaccine against emerging pandemics such as SARS-CoV-2. Two mRNA vaccines targeting SARS-CoV-2 have received emergency use authorization by US FDA, conditional marketing authorization by EMA, as well as multiple additional national regulatory authorities. The simple composition of an mRNA encoding the antigen formulated in a lipid nanoparticle enables a fast adaptation to new emerging pathogens. This can speed up vaccine development in pandemics from antigen and sequence selection to clinical trial to only a few months. mRNA vaccines are well tolerated and efficacious in animal models for multiple pathogens and will further contribute to the development of vaccines for other unaddressed diseases. Here, we give an overview of the mRNA vaccine design and factors for further optimization of this new promising technology and discuss current knowledge on the mode of action of mRNA vaccines interacting with the innate and adaptive immune system.

Efficacy of an unmodified bivalent mRNA vaccine against SARS-CoV-2 variants in female small animal models.

Combining optimized spike (S) protein-encoding mRNA vaccines to target multiple SARS-CoV-2 variants could improve control of the COVID-19 pandemic. We compare monovalent and bivalent mRNA vaccines encoding B.1.351 (Beta) and/or B.1.617.2 (Delta) SARS-CoV-2 S-protein in a transgenic mouse and a Wistar rat model. The blended low-dose bivalent mRNA vaccine contains half the mRNA of each respective monovalent vaccine, but induces comparable neutralizing antibody titres, enrichment of lung-resident memory CD8+ T cells, antigen-specific CD4+ and CD8+ responses, and protects transgenic female mice from SARS-CoV-2 lethality. The bivalent mRNA vaccine significantly reduces viral replication in both Beta- and Delta-challenged mice. Sera from bivalent mRNA vaccine immunized female Wistar rats also contain neutralizing antibodies against the B.1.1.529 (Omicron BA.1 and BA.5) variants. These data suggest that low-dose and fit-for-purpose multivalent mRNA vaccines encoding distinct S-proteins are feasible approaches for extending the coverage of vaccines for emerging and co-circulating SARS-CoV-2 variants.

Assessment of Immunogenicity and Efficacy of CV0501 mRNA-Based Omicron COVID-19 Vaccination in Small Animal Models.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Omicron and its subvariants (BA.2, BA.4, BA.5) represented the most commonly circulating variants of concern (VOC) in the coronavirus disease 2019 (COVID-19) pandemic in 2022. Despite high vaccination rates with approved SARS-CoV-2 vaccines encoding the ancestral spike (S) protein, these Omicron subvariants have collectively resulted in increased viral transmission and disease incidence. This necessitates the development and characterization of vaccines incorporating later emerging S proteins to enhance protection against VOC. In this context, bivalent vaccine formulations may induce broad protection against VOC and potential future SARS-CoV-2 variants. Here, we report preclinical data for a lipid nanoparticle (LNP)-formulated RNActive® N1-methylpseudouridine (N1mΨ) modified mRNA vaccine (CV0501) based on our second-generation SARS-CoV-2 vaccine CV2CoV, encoding the S protein of Omicron BA.1. The immunogenicity of CV0501, alone or in combination with a corresponding vaccine encoding the ancestral S protein (ancestral N1mΨ), was first measured in dose-response and booster immunization studies performed in Wistar rats. Both monovalent CV0501 and bivalent CV0501/ancestral N1mΨ immunization induced robust neutralizing antibody titers against the BA.1, BA.2 and BA.5 Omicron subvariants, in addition to other SARS-CoV-2 variants in a booster immunization study. The protective efficacy of monovalent CV0501 against live SARS-CoV-2 BA.2 infection was then assessed in hamsters. Monovalent CV0501 significantly reduced SARS-CoV-2 BA.2 viral loads in the airways, demonstrating protection induced by CV0501 vaccination. CV0501 has now advanced into human Phase 1 clinical trials (ClinicalTrials.gov Identifier: NCT05477186).

A third dose of the unmodified COVID-19 mRNA vaccine CVnCoV enhances quality and quantity of immune responses.

A third vaccine dose is often required to achieve potent, long-lasting immune responses. We investigated the effect of three 8-µg doses of CVnCoV, CureVac's severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine candidate containing sequence-optimized unmodified mRNA encoding the spike (S) glycoprotein, administered at 0, 4, and 28 weeks, on immune responses in rhesus macaques. After the third dose, S-specific binding and neutralizing antibodies increased 50-fold compared with post-dose 2 levels, with increased responses also evident in the lower airways and against the SARS-CoV-2 B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), and B.1.617.2 (Delta) variants. Enhanced binding affinity of serum antibodies after the third dose correlated with higher somatic hypermutation in S-specific B cells, corresponding with improved binding properties of monoclonal antibodies expressed from isolated B cells. Administration of low-dose mRNA led to fewer cells expressing antigen in vivo at the injection site and in the draining lymph nodes compared with a 10-fold higher dose, possibly reducing engagement of precursor cells with the antigen and resulting in the suboptimal response observed after two-dose vaccination schedules in phase IIb/III clinical trials of CVnCoV. However, when immune memory is established, a third dose efficiently boosts the immunological responses and improves antibody affinity and breadth.

Optimised Non-Coding Regions of mRNA SARS-CoV-2 Vaccine CV2CoV Improves Homologous and Heterologous Neutralising Antibody Responses.

More than two years after the emergence of SARS-CoV-2, 33 COVID-19 vaccines, based on different platforms, have been approved in 197 countries. Novel variants that are less efficiently neutralised by antibodies raised against ancestral SARS-CoV-2 are circulating, highlighting the need to adapt vaccination strategies. Here, we compare the immunogenicity of a first-generation mRNA vaccine candidate, CVnCoV, with a second-generation mRNA vaccine candidate, CV2CoV, in rats. Higher levels of spike (S) protein expression were observed in cell culture with the CV2CoV mRNA than with the CVnCoV mRNA. Vaccination with CV2CoV also induced higher titres of virus neutralising antibodies with accelerated kinetics in rats compared with CVnCoV. Significant cross-neutralisation of the SARS-CoV-2 variants, Alpha (B.1.1.7), Beta (B.1.351), and the 'mink' variant (B1.1.298) that were circulating at the time in early 2021 were also demonstrated. In addition, CV2CoV induced higher levels of antibodies at lower doses than CVnCoV, suggesting that dose-sparing could be possible with the next-generation SARS-CoV-2 vaccine, which could improve worldwide vaccine supply.

mRNA vaccines induce rapid antibody responses in mice.

mRNA vaccines can be developed and produced quickly, making them prime candidates for immediate outbreak responses. Furthermore, clinical trials have demonstrated rapid protection following mRNA vaccination. Thus, we sought to investigate how quickly mRNA vaccines elicit antibody responses compared to other vaccine modalities. We first compared the immune kinetics of mRNA and DNA vaccines expressing SARS-CoV-2 spike in mice. We observed rapid induction of antigen-specific binding and neutralizing antibodies by day 5 following mRNA (4 µg/mouse), but not DNA (50 µg/mouse), immunization. Comparing innate responses hours post immunization, the mRNA vaccine induced increased levels of IL-5, IL-6, and MCP-1 cytokines which maybe promoting humoral responses downstream. We then evaluated the immune kinetics of an HIV-1 mRNA vaccine in comparison to DNA, protein, and rhesus adenovirus 52 (RhAd52) vaccines of the same HIV-1 envelope antigen in mice. Again, induction of envelope-specific antibodies was observed by day 5 following mRNA vaccination, whereas antibodies were detected by day 7-14 following DNA, protein, and RhAd52 vaccination. Thus, eliciting rapid humoral immunity may be a unique and advantageous property of mRNA vaccines for controlling infectious disease outbreaks.

mRNA Vaccine Protects against Zika Virus.

Zika virus (ZIKV), a mosquito-borne flavivirus, has recently triggered global concern due to severe health complications. In 2015, a large ZIKV outbreak occurred in the Americas and established a link between ZIKV and microcephaly in newborn babies, spontaneous abortion, persistent viremia, and Guillain-Barré syndrome. While antivirals are being developed and prevention strategies focus on vector control, a safe and effective Zika vaccine remains unavailable. Messenger RNA (mRNA) vaccine technology has arisen as a flexible, simplified, and fast vaccine production platform. Here, we report on an mRNA vaccine candidate that encodes the pre-membrane and envelope (prM-E) glycoproteins of ZIKV strain Brazil SPH2015 and is encapsulated in lipid nanoparticles (LNPs). Our ZIKV prM-E mRNA-LNP vaccine candidate induced antibody responses that protected in AG129 mice deficient in interferon (IFN) alpha/beta/gamma (IFN-α/ß/γ) receptors. Notably, a single administration of ZIKV prM-E mRNA-LNP protected against a lethal dose of ZIKV, while a two-dose strategy induced strong protective immunity. E-specific double-positive IFN-γ and TNF-α T-cells were induced in BALB/c mice after immunizations with a two-dose strategy. With the success of mRNA vaccine technology in facing the coronavirus (COVID-19) pandemic, our data support the development of prM-E RNActive® as a promising mRNA vaccine against Zika to counter future epidemics.

Optimization of non-coding regions for a non-modified mRNA COVID-19 vaccine.

The CVnCoV (CureVac) mRNA vaccine for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was recently evaluated in a phase 2b/3 efficacy trial in humans1. CV2CoV is a second-generation mRNA vaccine containing non-modified nucleosides but with optimized non-coding regions and enhanced antigen expression. Here we report the results of a head-to-head comparison of the immunogenicity and protective efficacy of CVnCoV and CV2CoV in non-human primates. We immunized 18 cynomolgus macaques with two doses of 12 µg lipid nanoparticle-formulated CVnCoV or CV2CoV or with sham (n = 6 per group). Compared with CVnCoV, CV2CoV induced substantially higher titres of binding and neutralizing antibodies, memory B cell responses and T cell responses as well as more potent neutralizing antibody responses against SARS-CoV-2 variants, including the Delta variant. Moreover, CV2CoV was found to be comparably immunogenic to the BNT162b2 (Pfizer) vaccine in macaques. Although CVnCoV provided partial protection against SARS-CoV-2 challenge, CV2CoV afforded more robust protection with markedly lower viral loads in the upper and lower respiratory tracts. Binding and neutralizing antibody titres were correlated with protective efficacy. These data demonstrate that optimization of non-coding regions can greatly improve the immunogenicity and protective efficacy of a non-modified mRNA SARS-CoV-2 vaccine in non-human primates.

Immunogenicity of Low-Dose Prime-Boost Vaccination of mRNA Vaccine CV07050101 in Non-Human Primates.

Many different vaccine candidates against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, are currently approved and under development. Vaccine platforms vary from mRNA vaccines to viral-vectored vaccines, and several candidates have been shown to produce humoral and cellular responses in small animal models, non-human primates, and human volunteers. In this study, six non-human primates received a prime-boost intramuscular vaccination with 4 µg of mRNA vaccine candidate CV07050101, which encodes a pre-fusion stabilized spike (S) protein of SARS-CoV-2. Boost vaccination was performed 28 days post prime vaccination. As a control, six animals were similarly injected with PBS. Humoral and cellular immune responses were investigated at time of vaccination, and two weeks afterwards. No antibodies could be detected at two and four weeks after prime vaccination. Two weeks after boost vaccination, binding but no neutralizing antibodies were detected in four out of six non-human primates. SARS-CoV-2 S protein-specific T cell responses were detected in these four animals. In conclusion, prime-boost vaccination with 4 µg of vaccine candidate CV07050101 resulted in limited immune responses in four out of six non-human primates.

CVnCoV and CV2CoV protect human ACE2 transgenic mice from ancestral B BavPat1 and emerging B.1.351 SARS-CoV-2.

The ongoing SARS-CoV-2 pandemic necessitates the fast development of vaccines. Recently, viral mutants termed variants of concern (VOC) which may escape host immunity have emerged. The efficacy of spike encoding mRNA vaccines (CVnCoV and CV2CoV) against the ancestral strain and the VOC B.1.351 was tested in a K18-hACE2 transgenic mouse model. Naive mice and mice immunized with a formalin-inactivated SARS-CoV-2 preparation were used as controls. mRNA-immunized mice develop elevated SARS-CoV-2 RBD-specific antibody and neutralization titers which are readily detectable, but significantly reduced against VOC B.1.351. The mRNA vaccines fully protect from disease and mortality caused by either viral strain. SARS-CoV-2 remains undetected in swabs, lung, or brain in these groups. Despite lower neutralizing antibody titers compared to the ancestral strain BavPat1, CVnCoV and CV2CoV show complete disease protection against the novel VOC B.1.351 in our studies.

mRNA-based SARS-CoV-2 vaccine candidate CVnCoV induces high levels of virus-neutralising antibodies and mediates protection in rodents.

mRNA technologies have recently proven clinical efficacy against coronavirus disease 2019 and are among the most promising technologies to address the current pandemic. Here, we show preclinical data for our clinical candidate CVnCoV, a lipid nanoparticle-encapsulated mRNA vaccine that encodes full-length, pre-fusion stabilised severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein. In contrast to previously published approaches, CVnCoV is exclusively composed of naturally occurring nucleotides. Immunisation with CVnCoV induced strong humoral responses with high titres of virus-neutralising antibodies and robust T-cell responses. CVnCoV vaccination protected hamsters from challenge with wild-type SARS-CoV-2, demonstrated by the absence of viral replication in the lungs. Hamsters vaccinated with a suboptimal dose of CVnCoV leading to breakthrough viral replication exhibited no evidence of vaccine-enhanced disease. Overall, data presented here provide evidence that CVnCoV represents a potent and safe vaccine candidate against SARS-CoV-2.