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.

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.

Background: Continuous antigenic evolution of SARS-CoV-2 has reduced the effectiveness of vaccines based on a single ancestral strain, highlighting the need for multivalent vaccine strategies. We developed a trivalent whole-virion inactivated vaccine containing ancestral (ERAGEM), Delta, and Omicron BA.5 antigens and evaluated its immunogenicity and protective efficacy in K18-hACE2 transgenic mice. Methods: β-propiolactone-inactivated ERAGEM, Delta, and Omicron BA.5 antigens were combined at 1 µg each (3 µg total) with aluminium hydroxide and administered intraperitoneally on days 0 and 21. Variant-specific binding and neutralizing antibody responses were assessed by enzyme-linked immunosorbent assay (ELISA) and microneutralization assays. On day 42, mice were challenged with ERAGEM, Delta, or Omicron BA.5. Survival, body weight, body temperature, infectious viral titers, and viral RNA loads were evaluated. Results: The trivalent vaccine induced binding and neutralizing antibodies against all three vaccine components, and titers rose after the booster dose. Neutralizing responses were highest against the BA.5 component. Vaccinated mice showed 100% survival following challenge with each vaccine-matched variant and maintained stable body weight and temperature throughout the observation period. Unvaccinated controls challenged with Omicron BA.5 showed no mortality, so protection against this component could be assessed only virologically. No infectious virus was detected in the lungs of vaccinated animals, while viral titers and RNA loads in the nasal turbinates were significantly reduced compared with unvaccinated controls. Conclusions: A trivalent whole-virion inactivated SARS-CoV-2 vaccine containing ancestral, Delta, and Omicron BA.5 antigens elicited variant-specific humoral immunity and protected mice against challenge with each vaccine-matched component: against lethal challenge for the ancestral and Delta components, and against viral burden for Omicron BA.5, which was not lethal in unvaccinated controls. These outcomes were obtained at a lower per-antigen dose than in our previously reported monovalent and bivalent formulations; because those were independent studies rather than contemporaneous comparators, this suggests a dose-sparing potential that warrants formal evaluation rather than demonstrating one. Cross-protection against variants not included in the formulation was not assessed.