Among all approaches, a messenger RNA (mRNA)-based vaccine has emerged as a rapid and versatile platform to quickly respond to this challenge. Here, we developed a lipid nanoparticle-encapsulated mRNA (mRNA-LNP) encoding the receptor binding domain (RBD) of SARS-CoV-2 as a vaccine candidate (called ARCoV). Intramuscular immunization of ARCoV mRNA-LNP elicited robust neutralizing antibodies against SARS-CoV-2 as well as a Th1-biased cellular response in mice and non-human primates. Two doses of ARCoV immunization in mice conferred complete protection against the challenge of a SARS-CoV-2 mouse-adapted strain. Additionally, ARCoV is manufactured as a liquid formulation and can be stored at room temperature for at least 1 week. ARCoV is currently being evaluated in phase 1 clinical trials.
Lipid nanoparticles (LNPs) are one of the most appealing and commonly used mRNA delivery tools. Here we developed a vaccine platform based on modified mRNA encapsulated in LNPs for in vivo delivery. The RBD of SARS-CoV-2 (amino acids [aa] 319–541) was chosen as the target antigen for the mRNA coding sequence. Transfection of the RBD-encoding mRNA in multiple cell lines (HeLa, Huh7, HEK293T, and Vero) resulted in high expression of recombinant RBD in culture supernatants, with up to 917.4 ng/mL of RBD in mRNA-transfected HEK293F cells. RBD protein expressed from mRNA retained high affinity for recombinant human ACE2, as demonstrated by kinetics analysis using ForteBio Octet, and functionally inhibited entry of a vesicular stomatitis virus (VSV)-based pseudovirus expressing the SARS-CoV-2 S protein in Huh7 cells (Figure 1D). Immunostaining further demonstrated that this RBD protein can be recognized by a panel of monoclonal antibodies (mAbs) against SARS-CoV-2 RBD as well as convalescent sera from three COVID-19 patients
In the present study, we report the immunogenicity and efficacy of a novel COVID-19 mRNA vaccine candidate in various animal models. A single dose or two doses of immunization with ARCoV elicited robust antibody and T cell responses in mice and non-human primates against multiple epidemic SARS-CoV-2 strains. The NT50 in sera from non-human primates receiving low-dose (100 μg) ARCoV immunization was comparable with those from convalescent sera from 20 COVID-19 patients (Figure S9), whereas high-dose (1,000 μg) ARCoV immunization induced much higher titers of neutralizing antibodies compared with convalescent serum.It has been reported that two or three doses of inactivated SARS-CoV-2 virus vaccine could induce neutralizing antibodies at levels of ∼1/50, which provides full protection against SARS-CoV-2 in rhesus macaques. A recent report showed that two doses of immunization with a DNA vaccine candidate elicits mean neutralization titers between ∼1/70 and ∼1/170 in rhesus macaques. In the aforementioned studies, all animals exhibited anamnestic antibody responses following challenge, suggesting that vaccine protection was probably not sterilizing, which was consistent with relative lower neutralizing antibody titers. In our study, a single dose of ARCoV immunization induced an anamnestic antibody response, whereas animals receiving two doses of ARCoV immunization did not show enhancement of neutralizing antibody titers upon challenge, suggesting that sterilizing immunity may have been induced in mice Comparison of Neutralizing Antibody Titers in ARCoV-Immunized Cynomolgus Monkeys and Convalescent Sera from COVID-19 Patients, Related to
Further challenge experiments with a SARS-CoV-2 mouse-adapted strain, MASCp6 showed that two doses of immunization of ARCoV completely blocked viral replication in the lungs and trachea and prevented pulmonary pathology in mice. Although it still needs to be validated further in clinical settings, our results revealed that neutralizing antibody titer levels in mice correlate well with protection against SARS-CoV-2 challenge. Regression analysis revealed a cutoff value of ∼1:1,009 neutralizing antibody titers (PRNT50) as full protection from SARS-CoV-2 lung infection. To our knowledge, this is the first protection correlate identified in a mouse model. Considering the limited resource of non-human primates and strict requirement of biosafety facilities for SARS-CoV-2 challenge experiments, this protection correlate in a mouse model is a simple and useful benchmark for efficacy tests that will greatly facilitate and accelerate COVID-19 vaccine development. Because of biosafety facility limitations, at present we are not able to obtain protection efficacy data in non-human primates. However, based on the comparison of neutralizing antibody levels in macaques vaccinated with inactivated or DNA COVID-19 vaccine candidates, protective immunity can be expected in most macaques immunized with two doses of ARCoV. ARCoV is highly immunogenic in male and female macaques. Of particular note is that, although 1 in 10 vaccinated macaques in each group failed to produce detectable (1:30) neutralizing antibodies, an IFN-γ ELISPOT assay showed virus-specific IFN-γ secretion in all vaccinated macaques.
An ideal COVID-19 vaccine is supposed to avoid induction of non-neutralizing antibody and Th2-biased cellular immune responses because of safety concerns Unlike the mRNA-1273 vaccine from Moderna, our ARCoV vaccine chose the RBD as an antigen target. Compared with the full-length S protein, RBD antigen may induce fewer non-neutralizing antibodies, lowering the risk of potential ADE of SARS-CoV-2 infection; a similar phenomenon has been observed during other coronavirus infection experiments. A recent in vitro study also suggests that antibodies targeting the SARS-CoV-2 RBD at various concentrations did not induce ADE infection. S-specific IgG antibodies have been suggested to cause acute pulmonary injury in vaccine challenge animal models of SARS-CoV, although the exact S epitopes accounting for the lung pathology remain to be determined; use of the RBD may minimize this risk. Additionally, vaccine-associated enhanced respiratory disease has been linked to Th2-biased CD4+ T cell responses. As expected, our mRNA-based vaccine induced a Th1-prone T cell immune response to SARS-CoV-2 RBD in mice and macaques . Similar results have also been reported in DNA- and adenovirus-vectored COVID-19 vaccine candidates. In our mouse challenge experiments, we did not observe enhanced viral replication or clinical disease in vaccinated animals, even those receiving a single dose of ARCoV vaccination.
We also characterized the in vitro and in vivo expression pattern of our mRNA-LNP formulation. Upon i.m. injection, robust protein expression was readily detected in the muscle tissue at the injection site, and the most predominant expression was seen in the liver, which was similar to the results from other LNP formulations. Most importantly, a multiplex immune co-staining assay showed robust expression of SARS-CoV-2 RBD in multiple antigen-presenting cells, including monocytes, macrophages, and dendritic cells (DCs), in muscle and liver as well as lymph nodes from ARCoV-vaccinated mice. A recent study has shown that a yellow fever mRNA vaccine delivered by LNP was mainly expressed at the injection site as well as in draining lymph nodes in cynomolgus macaques. Further biodistribution profiles of ARCoV in cynomolgus monkeys are being tested in a good laboratory practice (GLP) lab.
To date, limited results have been reported regarding the safety and stability of LNP-based mRNA vaccines Our data from cynomolgus monkeys show that 100 μg of ARCoV is sufficient to induce high-level neutralizing antibodies and that 1,000 μg of ARCoV did not cause obvious adverse effects, highlighting the safety of our mRNA LNP formulation. Extrapolation of dose from animals to humans remains a huge challenge that requires careful consideration of safety and efficacy data. These preclinical data from mouse and non-human primate provide a critical reference for the starting dose of ARCoV in human trials. Last, accessibility and scalability of COVID-19 vaccines are major challenges to expediting delivery and massive immunization worldwide; therefore, a ready-to-use and thermostable vaccine is highly preferred. The final ARCoV mRNA-LNP vaccine is manufactured in a liquid formulation without the need of thawing or reconstitution before injection, and a single-dose vaccine is prepared in a prefilled syringe for quick self-administration. Stability test results showed that our formulation maintained in vivo delivery efficiency at 4°C and 25°C for at least 1 week; the long-term stability of the ARCoV vaccine is currently under evaluation. Additionally, ARCoV is administrated with the most commonly used i.m. vaccination route for human use. These unique features of ARCoV make it a promising COVID-19 vaccine candidate with universal availability and global accessibility.
In summary, we report a thermostable mRNA vaccine candidate for SARS-CoV-2 and provided first-line evidence of immunogenicity and efficacy in multiple animal models. Although two mRNA vaccine candidates from Moderna and BioNTech/Pfizer were tested in humans prior to our results, there is no report that an mRNA vaccine can protect animals from SARS-CoV-2 infection or the immune correlate of protection. During revision of our manuscript, immunogenicity and protection efficacy of mRNA-1273 in mice was also reported. The robust protection observed in both studies highlights the power of the mRNA vaccine platform and paves the path for a successful COVID-19 vaccine in the near future. Our ARCoV mRNA vaccine was approved for phase I clinical trials (ChiCTR2000034112) on June 19, 2020.
Limitations
The challenge experiments in our study were based on a mouse-adapted strain of SARS-CoV-2; further challenge experiments with a wild-type SARS-CoV-2 strain in transgenic ACE2 mice or non-human primates will provide more data regarding protective efficacy. Another limitation of our study is that the duration of neutralizing antibodies induced by ARCoV has yet to be determined. Experience from other human coronaviruses has indicated the possibility of re-infection because of a waning antibody response. Future studies are needed to evaluate the long-term immune response in animal models and the effectiveness of ARCoV in humans. Additionally, long-term stability assays with a clinical-grade ARCoV vaccine are under investigation
Reference & Source information: https://www.cell.com/
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