The COVID-19 pandemic has put global public health at high risk, rapidly spreading around the world. Although several COVID-19 vaccines are available for mass immunization, the world still urgently needs highly effective, reliable, cost-effective, and safe SARS-CoV-2 coronavirus vaccines, as well as antiviral and therapeutic drugs, to control the COVID-19 pandemic given the emerging variant strains of the virus. Recently, we successfully produced receptor-binding domain (RBD) variants in the Nicotiana benthamiana plant as promising vaccine candidates against COVID-19 and demonstrated that mice immunized with these antigens elicited a high titer of RBD-specific antibodies with potent neutralizing activity against SARS-CoV-2. In this study, we engineered the nucleocapsid (N) protein and co-expressed it with RBD of SARS-CoV-2 in Nicotiana benthamiana plant to produce an antigen cocktail. The purification yields were about 22 or 24 mg of pure protein/kg of plant biomass for N or N+RBD antigens, respectively. The purified plant produced N protein was recognized by N protein-specific monoclonal and polyclonal antibodies demonstrating specific reactivity of mAb to plant-produced N protein. In this study, for the first time, we report the co-expression of RBD with N protein to produce a cocktail antigen of SARS-CoV-2, which elicited high-titer antibodies with potent neutralizing activity against SARS-CoV-2. Thus, obtained data support that a plant-produced antigen cocktail, developed in this study, is a promising vaccine candidate against COVID-19.

The rapid spread of SARS-CoV-2 with its mutating strains has posed a global threat to safety during this COVID-19 pandemic. Thus far, there are 123 candidate vaccines in human clinical trials and more than 190 candidates in preclinical development worldwide as per the WHO on 1 October 2021. The various types of vaccines that are currently approved for emergency use include viral vectors (e.g., adenovirus, University of Oxford/AstraZeneca, Gamaleya Sputnik V, and Johnson & Johnson), mRNA (Moderna and Pfizer-BioNTech), and whole inactivated (Sinovac Biotech and Sinopharm) vaccines. Amidst the emerging cases and shortages of vaccines for global distribution, it is vital to develop a vaccine candidate that recapitulates the severe and fatal progression of COVID-19 and further helps to cope with the current outbreak. Hence, we present the preclinical immunogenicity, protective efficacy, and safety evaluation of a whole-virion inactivated SARS-CoV-2 vaccine candidate (ERUCoV-VAC) formulated in aluminium hydroxide, in three animal models, BALB/c mice, transgenic mice (K18-hACE2), and ferrets. The hCoV-19/Turkey/ERAGEM-001/2020 strain was used for the safety evaluation of ERUCoV-VAC. It was found that ERUCoV-VAC was highly immunogenic and elicited a strong immune response in BALB/c mice. The protective efficacy of the vaccine in K18-hACE2 showed that ERUCoV-VAC induced complete protection of the mice from a lethal SARS-CoV-2 challenge. Similar viral clearance rates with the safety evaluation of the vaccine in upper respiratory tracts were also positively appreciable in the ferret models. ERUCoV-VAC has been authorized by the Turkish Medicines and Medical Devices Agency and has now entered phase 3 clinical development (NCT04942405). The name of ERUCoV-VAC has been changed to TURKOVAC in the phase 3 clinical trial.

The COVID-19 pandemic, caused by SARS-CoV-2, has rapidly spread to more than 222 countries and has put global public health at high risk. The world urgently needs cost-effective and safe SARS-CoV-2 vaccines, antiviral, and therapeutic drugs to control it. In this study, we engineered the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein and produced it in the plant Nicotiana benthamiana in a glycosylated and deglycosylated form. Expression levels of both glycosylated (gRBD) and deglycosylated (dRBD) RBD were greater than 45 mg/kg fresh weight. The purification yields were 22 mg of pure protein/kg of plant biomass for gRBD and 20 mg for dRBD, which would be sufficient for commercialization of these vaccine candidates. The purified plant-produced RBD protein was recognized by an S protein-specific monoclonal antibody, demonstrating specific reactivity of the antibody to the plant-produced RBD proteins. The SARS-CoV-2 RBD showed specific binding to angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 receptor. In mice, the plant-produced RBD antigens elicited high titers of antibodies with a potent virus-neutralizing activity. To our knowledge, this is the first report demonstrating that mice immunized with plant-produced deglycosylated RBD form elicited high titer of RBD-specific antibodies with potent neutralizing activity against SARS-CoV-2 infection. Thus, obtained data support that plant-produced glycosylated and in vivo deglycosylated RBD antigens, developed in this study, are promising vaccine candidates for the prevention of COVID-19.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus in the Nairoviridae family within the Bunyavirales order of viruses. Crimean-Congo hemorrhagic fever (CCHF) is the most widespread among tick-borne human viral diseases. It is endemic in many areas of Africa, Asia, the Middle East, in the Balkans, Russia and countries of the former Soviet Union. The confirmed CCHF cases were seen in Spain in 2016 to signify expansion of the virus into new geographical areas. CCHFV causes a viral human disease characterized by sudden onset of fever, headache, abdominal pain, nausea, hypotension, hemorrhage, and hepatic dysfunction with fatality rates up to 30%. Currently, there are no spesific treatments or licensed vaccines available for CCHFV. The absence of a susceptible animal model for CCHFV infection was severely hindered work on the development of vaccines. However, several animal models of CCHFV infection have been recently developed and used to assess vaccine efficacy. In this study, we have used the transiently immune-suppressed (IS) mouse model that MAb-5A3 was used to block IFN-I signaling in immune intact, wild-type mice at the time of CCHFV infection to evaluate the immune response and efficacy of the cell culture based and the mouse brain derived inactivated vaccines against CCHFV. Both vaccine preparations have provided complete protection but the cell culture based vaccine more effectively induced to CCFHV spesific antibodies and T cell responses. This is the first comparison of the cell culture based and the mouse brain derived vaccines for assessing the protective efficacy and the immunogenicity in the IS mouse CCHFV model.

Vaccine-induced immunity wanes over time and warrants booster doses. We investigated the long-term (32 weeks) immunogenicity and safety of a third, homologous, open-label booster dose of TURKOVAC, administered 12 weeks after completion of the primary series in a randomized, controlled, double-blind, phase 2 study. Forty-two participants included in the analysis were evaluated for neutralizing antibodies (NAbs) (with microneutralization (MNT₅₀) and focus reduction (FRNT₅₀) tests), SARS-CoV-2 S1 RBD (Spike S1 Receptor Binding Domain), and whole SARS-CoV-2 (with ELISA) IgGs on the day of booster injection and at weeks 1, 2, 4, 8, 16, 24, and 32 thereafter. Antibody titers increased significantly from week 1 and remained higher than the pre-booster titers until at least week 4 (week 8 for whole SARS-CoV-2) (p < 0.05 for all). Seroconversion (titers ≥ 4-fold compared with pre-immune status) persisted 16 weeks (MNT₅₀: 6-fold; FRNT₅₀: 5.4-fold) for NAbs and 32 weeks for S1 RBD (7.9-fold) and whole SARS-CoV-2 (9.4-fold) IgGs. Nine participants (20.9%) tested positive for SARS-CoV-2 RT-PCR between weeks 8 and 32 of booster vaccination; none of them were hospitalized or died. These findings suggest that boosting with TURKOVAC can provide effective protection against COVID-19 for at least 8 weeks and reduce the severity of the disease.

Background/Objectives: The rapid evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the emergence of variants with enhanced transmissibility and immune evasion, challenging existing vaccines. This study aimed to evaluate the immunogenicity and protective efficacy of inactivated bivalent vaccine formulations incorporating the ancestral SARS-CoV-2 strain (ERAGEM) with either Delta or Omicron (BA.5) variants. Methods: Bivalent vaccine formulations were prepared using beta-propiolactone-inactivated SARS-CoV-2 antigens and administered to K18-hACE2 transgenic mice. Following prime and booster immunizations, neutralizing antibody titers and viral loads were assessed through ELISA, microneutralization assays, and quantitative PCR. Mice were challenged with the respective variants, and the survival rates, temperature, and body weight changes were monitored for 21 days. Results: Both vaccine formulations elicited significant increases in neutralizing antibody titers post-booster immunization. The ERAGEM + Delta group demonstrated geometric mean titers (GMTs) of 6938.1 and 4935.0 for the ancestral and Delta variants, respectively, while the ERAGEM + Omicron (BA.5) group achieved GMTs of 16,280.7 and 24,215.9 for the ancestral and Omicron (BA.5) variants. Complete survival (100%) was observed in all the vaccinated groups post-challenge, with no detectable viral titers in the lungs and substantial reductions in the nasal turbinate viral loads compared to the unvaccinated controls. Conclusions: The bivalent inactivated vaccines demonstrated strong immunogenicity and complete protection against severe disease in preclinical models. These findings indicate the potential of bivalent vaccine strategies in addressing antigenic diversity and preparing for future pandemics caused by rapidly evolving pathogens.