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Abstract Establishment of an mRNA vaccine platform in low- and middle-income countries (LMICs) is important to enhance vaccine accessibility and ensure future pandemic preparedness. Here, we describe the preclinical studies of “ChulaCov19”, a SARS-CoV-2 mRNA encoding prefusion-unstabilized ectodomain spike protein encapsulated in lipid nanoparticles (LNP). In female BALB/c mice, ChulaCov19 at 0.2, 1, 10, and 30 μg elicits robust neutralizing antibody (NAb) and T cell responses in a dose-dependent relationship. The geometric mean titers (GMTs) of NAb against wild-type (WT, Wuhan-Hu1) virus are 1,280, 11,762, 54,047, and 62,084, respectively. Higher doses induce better cross-NAb against Delta (B.1.617.2) and Omicron (BA.1 and BA.4/5) variants. This elicited immunogenicity is significantly higher than those induced by homologous CoronaVac or AZD1222 vaccination. In a heterologous prime-boost study, ChulaCov19 booster dose generates a 7-fold increase of NAb against Wuhan-Hu1 WT virus and also significantly increases NAb response against Omicron (BA.1 and BA.4/5) when compared to homologous CoronaVac or AZD1222 vaccination. Challenge studies show that ChulaCov19 protects human-ACE-2-expressing female mice from COVID-19 symptoms, prevents viremia and significantly reduces tissue viral load. Moreover, anamnestic NAb response is undetectable in challenge animals. ChulaCov19 is therefore a promising mRNA vaccine candidate either as a primary or boost vaccination and has entered clinical development.

The emergence of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected global public health and economy. Despite the substantial efforts, only few vaccines are currently approved and some are in the different stages of clinical trials. As the disease rapidly spreads, an affordable and effective vaccine is urgently needed. In this study, we investigated the immunogenicity of plant-produced receptor-binding domain (RBD) of SARS-CoV-2 in order to use as a subunit vaccine. In this regard, RBD of SARS-CoV-2 was fused with Fc fragment of human IgG1 and transiently expressed in Nicotiana benthamiana by agroinfiltration. The plant-produced RBD-Fc fusion protein was purified from the crude extract by using protein A affinity column chromatography. Two intramuscular administration of plant-produced RBD-Fc protein formulated with alum as an adjuvant have elicited high neutralization titers in immunized mice and cynomolgus monkeys. Further it has induced a mixed Th1/Th2 immune responses and vaccine-specific T-lymphocyte responses which was confirmed by interferon-gamma (IFN-γ) enzyme-linked immunospot assay. Altogether, our results demonstrated that the plant-produced SARS-CoV-2 RBD has the potential to be used as an effective vaccine candidate against SARS-CoV-2. To our knowledge, this is the first report demonstrating the immunogenicity of plant-produced SARS-CoV-2 RBD protein in mice and non-human primates.

Previous investigations conducted on a liposomal formulation for a SARS-CoV-2 DNA vaccine manufactured using the thin-film layer rehydration method showed promising immunogenicity results in mice. The adaptation of the liposomal formulation to a scalable and reproducible method of manufacture is necessary to continue the investigation of this vaccine candidate. Microfluidics manufacture shows high potential in method translation. The physicochemical characterization of the blank liposomes produced by thin-film layer rehydration or microfluidics were shown to be comparable. However, a difference in lipid nanostructure in the bilayer resulted in a significant difference in the hydration of the thin-film liposomes, ultimately altering their complexation behavior. A study on the complexation of liposomes with the DNA vaccine at various N/P ratios showed different sizes and Zeta-potential values between the two formulations. This difference in the complexation behavior resulted in distinct immunogenicity profiles in mice. The thin-film layer rehydration-manufactured liposomes induced a significantly higher response compared to the microfluidics-manufactured samples. The nanostructural analysis of the two samples revealed the critical importance of understanding the differences between the two formulations that resulted in the different immunogenicity in mice.

More than 65 million people have been confirmed infection with SARS-CoV-2 and more than 1 million have died from COVID-19 and this pandemic remains critical worldwide. Effective vaccines are one of the most important strategies to limit the pandemic. Here, we report a construction strategy of DNA vaccine candidates expressing full length wild type SARS-CoV-2 spike (S) protein, S1 or S2 region and their immunogenicity in mice. All DNA vaccine constructs of pCMVkan-S, -S1 and -S2 induced high levels of specific binding IgG that showed a balance of IgG1/IgG2a response. However, only the sera from mice vaccinated with pCMKkan-S or -S1 DNA vaccines could inhibit viral RBD and ACE2 interaction. The highest neutralizing antibody (NAb) titer was found in pCMVkan-S group, followed by -S1, while -S2 showed the lowest PRNT50 titers. The geometric mean titers (GMTs) were 2,551, 1,005 and 291 for pCMVkan-S, -S1 and -S2, respectively. pCMVkan-S construct vaccine also induced the highest magnitude and breadth of T cells response. Analysis of IFN-γ positive cells after stimulation with SARS-CoV-2 spike peptide pools were 2,991, 1,376 and 1,885 SFC/106 splenocytes for pCMVkan-S, -S1 and -S2, respectively. Our findings highlighted that full-length S antigen is more potent than the truncated spike (S1 or S2) in inducing of neutralizing antibody and robust T cell responses.

In view of addressing the global necessity of an effective vaccine in the SARS-CoV-2 pandemic, a plasmid DNA vaccine, expressing for the spike (S) protein and formulated in lipoplexes, was manufactured and tested for in vitro transfection and in vivo immunogenicity. Blank cationic liposomes of 130.9 ± 5.8 nm in size and with a zeta potential of +48 ± 12 mV were formulated using the thin-film layer rehydration method. Liposomes were complexed with pCMVkan-S at different N/P ratios. Ratios of 0.25:1 and 1:1 were selected according to their complex stability and controlled size compared to other ratios and tested in vitro for transfection studies and in vivo for immunogenicity. Both selected formulations showed enhanced neutralizing antibody responses compared to pCMVkan-S injected alone, as well as an increased T cell response. The titers observed were similar to those of intramuscular electroporation (IM-EP), which was set as an efficacy goal.

Due to the rapid transmission of the coronavirus disease 2019 (COVID-19) causing serious public health problems and economic burden, the development of effective vaccines is a high priority for controlling the virus spread. Our group has previously demonstrated that the plant-produced receptor-binding domain (RBD) of SARS-CoV-2 fused with Fc of human IgG was capable of eliciting potent neutralizing antibody and cellular immune responses in animal studies, and the immunogenicity could be improved by the addition of an alum adjuvant. Here, we performed a head-to-head comparison of different commercially available adjuvants, including aluminum hydroxide gel (alum), AddaVax (MF59), monophosphoryl lipid A from Salmonella minnesota R595 (mPLA-SM), and polyinosinic-polycytidylic acid (poly(I:C)), in mice by combining them with plant-produced RBD-Fc, and the differences in the immunogenicity of RBD-Fc with different adjuvants were evaluated. The specific antibody responses in terms of total IgG, IgG1, and IgG2a subtypes and neutralizing antibodies, as well as vaccine-specific T-lymphocyte responses, induced by the different tested adjuvants were compared. We observed that all adjuvants tested here induced a high level of total IgG and neutralizing antibodies, but mPLA-SM and poly (I:C) showed the induction of a balanced IgG1 and IgG2a (Th2/Th1) immune response. Further, poly (I:C) significantly increased the frequency of IFN-γ-expressing cells compared with control, whereas no significant difference was observed between the adjuvanted groups. This data revealed the adjuvants’ role in enhancing the immune response of RBD-Fc vaccination and the immune profiles elicited by different adjuvants, which could prove helpful for the rational development of next-generation SARS-CoV-2 RBD-Fc subunit vaccines. However, additional research is essential to further investigate the efficacy and safety of this vaccine formulation before clinical trials.