Development of Quantitative Real-Time PCR and Loop-Mediated Isothermal Amplification Assays for the Surveillance and Diagnosis of Herpes B Virus Infection.

Herpes B virus (BV) is a zoonotic virus which can be transmitted from macaques to humans, which is often associated with high mortality rates. Because macaques often exhibit asymptomatic infections, individuals who come into contact with these animals face unexpected risks of BV infections. A serological test is widely performed to investigate BV infections. However, the assay's sensitivity and specificity appeared to be inadequate, and it does not necessarily indicate ongoing viral shedding. Here, we developed LAMP and qPCR assays aiming to detect BVs with a high sensitivity and specificity in various macaque species and validated them using oral swab samples collected from 97 wild cynomolgus macaques living in Thailand. Our LAMP and qPCR assays detected more than 50 and 10 copies of the target sequences per reaction, respectively. The LAMP assay could detect BV within 25 min, indicating its advantages for the rapid detection of BV. Collectively, our findings indicated that both assays developed in this study exhibit advantages and usefulness for BV surveillance and the diagnosis of BV infections in macaques. Furthermore, for the first time, we determined the partial genome sequences of BVs detected in cynomolgus macaques in Thailand. Phylogenetic analysis revealed the species-specific evolution of BV within macaques.

A recombinant subunit vaccine candidate produced in plants elicits neutralizing antibodies against SARS-CoV-2 variants in macaques.

Since the outbreak of the coronavirus disease (COVID) pandemic in 2019, the development of effective vaccines to combat the infection has been accelerated. With the recent emergence of highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC), there are concerns regarding the immune escape from vaccine-induced immunity. Hence an effective vaccine against VOC with a potent immune response is required. Our previous study confirmed that the two doses of the plant-produced receptor-binding domain (RBD) of SARS-CoV-2 fused with the Fc region of human IgG1, namely Baiya SARS-CoV-2 Vax 1, showed high immunogenicity in mice and monkeys. Here, we aimed to evaluate the immunogenicity of a three-dose intramuscular injection of Baiya SARS-CoV-2 Vax 1 on days 0, 21, and 133 in cynomolgus monkeys. At 14 days after immunization, blood samples were collected to determine RBD-specific antibody titer, neutralizing antibody, and pseudovirus neutralizing antibody titers. Immunized monkeys developed significantly high levels of antigen-specific antibodies against SARS-CoV-2 compared to the control group. Interestingly, the sera collected from immunized monkeys also showed a neutralizing antibody response against the SARS-CoV-2 VOCs; Alpha, Beta, Gamma, Delta, and Omicron. These findings demonstrate that a three-dose regimen of Baiya SARS-CoV-2 Vax 1 vaccine elicits neutralizing immune response against SARS-CoV-2 variants.

Preclinical evaluation of a plant-derived SARS-CoV-2 subunit vaccine: Protective efficacy, immunogenicity, safety, and toxicity.

Coronavirus disease 2019 (COVID-19) is an acute respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The prevention of SARS-CoV-2 transmission has become a global priority. Previously, we showed that a protein subunit vaccine that was developed based on the fusion of the SARS-CoV-2 receptor-binding domain (RBD) to the Fc portion of human IgG1 (RBD-Fc), produced in Nicotiana benthamiana, and adjuvanted with alum, namely, Baiya SARS-CoV-2 Vax 1, induced potent immunological responses in both mice and cynomolgus monkeys. Hence, this study evaluated the protective efficacy, safety, and toxicity of Baiya SARS-CoV-2 Vax 1 in K18-hACE2 mice, monkeys and Wistar rats. Two doses of vaccine were administered three weeks apart on Days 0 and 21. The administration of the vaccine to K18-hACE2 mice reduced viral loads in the lungs and brains of the vaccinated animals and protected the mice against challenge with SARS-CoV-2. In monkeys, the results of safety pharmacology tests, general clinical observations, and a core battery of studies of three vital systems, namely, the central nervous, cardiovascular, and respiratory systems, did not reveal any safety concerns. The toxicology study of the vaccine in rats showed no vaccine-related pathological changes, and all the animals remained healthy under the conditions of this study. Furthermore, the vaccine did not cause any abnormal toxicity in rats and was clinically tolerated even at the highest tested concentration. In addition, general health status, body temperature, local toxicity at the administration site, hematology, and blood chemistry parameters were also monitored. Overall, this work presents the results of the first systematic study of the safety profile of a plant-derived vaccine, Baiya SARS-CoV-2 Vax 1; this approach can be considered a viable strategy for the development of vaccines against COVID-19.

Receptor binding domain proteins of SARS-CoV-2 variants produced in Nicotiana benthamiana elicit neutralizing antibodies against variants of concern.

The constantly emerging severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) variants of concerns (VOCs) with mutations in the receptor-binding domain (RBD) spread rapidly and has become a severe public health problem worldwide. Effective vaccines and optimized booster vaccination strategies are thus highly required. Here, the gene encoding six different RBD (Alpha, Beta, Gamma, Kappa, Delta, and Epsilon variants) along with the Fc fragment of human IgG1 (RBD-Fc) was cloned into plant expression vector and produced in Nicotiana benthamiana by transient expression. Further, the immunogenicity of plant-produced variant RBD-Fc fusion proteins were tested in cynomolgus monkeys. Each group of cynomolgus monkeys was immunized three times intramuscularly with variant RBD-Fc vaccines at Day 0, 21, 42, and neutralizing antibody responses were evaluated against ancestral (Wuhan), Alpha, Beta, Gamma, and Delta variants. The results showed that three doses of the RBD-Fc vaccine significantly enhanced the immune response against all tested SARS-CoV-2 variants. In particular, the vaccines based on Delta and Epsilon mutant RBD elicit broadly neutralizing antibodies against ancestral (Wuhan), Alpha, and Delta SARS-CoV-2 variants whereas Beta and Gamma RBD-Fc vaccines elicit neutralizing antibodies against their respective SARS-CoV-2 strains. The Delta and Epsilon RBD-Fc based vaccines displayed cross-reactive immunogenicity and might be applied as a booster vaccine to induce broadly neutralizing antibodies. These proof-of-concept results will be helpful for the development of plant-derived RBD-Fc-based vaccines against SARS-CoV-2 and its variants.

Plant-Produced Receptor-Binding Domain of SARS-CoV-2 Elicits Potent Neutralizing Responses in Mice and Non-human Primates.

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.