Pre-Clinical Safety and Immunogenicity Study of a Coronavirus Protein-Based Subunit Vaccine for COVID-19.

Creating an effective and safe vaccine is critical to fighting the coronavirus infection successfully. Several types of COVID-19 vaccines exist, including inactivated, live attenuated, recombinant, synthetic peptide, virus-like particle-based, DNA and mRNA-based, and sub-unit vaccines containing purified immunogenic viral proteins. However, the scale and speed at which COVID-19 is spreading demonstrate a global public demand for an effective prophylaxis that must be supplied more. The developed products promise a bright future for SARS-CoV-2 prevention; however, evidence of safety and immunogenicity is mandatory before any vaccine can be produced. In this paper, we report on the results of our work examining the safety, toxicity, immunizing dose choice, and immunogenicity of QazCoVac-P, a Kazakhstan-made sub-unit vaccine for COVID-19. First, we looked into the product's safety profile by assessing its pyrogenicity in vaccinated rabbit models and using the LAL (limulus amebocyte lysate) test. We examined the vaccine's acute and sub-chronic toxicity on BALB/c mice and rats. The vaccine did not cause clinically significant toxicity-related changes or symptoms in our toxicity experiments. Finally, we performed a double immunization of mice, ferrets, Syrian hamsters, and rhesus macaques (Macaca mulatta). We used ELISA to measure antibody titers with the maximum mean geometric titer of antibodies in the animals' blood sera totaling approximately 8 log2. The results of this and other studies warrant recommending the QazCoVac-P vaccine for clinical trials.

Safety and immunogenicity of the first Kazakh inactivated vaccine for COVID-19.

This article describes the results of a preclinical safety and immunogenicity study of QazCovid-in®, the first COVID-19 vaccine developed in Kazakhstan, on BALB/c mice, rats, ferrets, Syrian hamsters and rhesus macaques (Macaca mulatta). The study's safety data suggests that this immunobiological preparation can be technically considered a Class 5 nontoxic vaccine. The series of injections that were made did not produce any adverse effect or any change in the general condition of the model animals' health, while macroscopy and histology studies identified no changes in the internal organs of the BALB/c mice and rats. This study has demonstrated that a double immunization enhances the growth of antibody titers as assessed by the microneutralization assay (MNA) and the enzyme-linked immunosorbent assay (ELISA) in a pre-clinical immunogenicity test on animal models. The best GMT results were assessed in MNA and ELISA 7 days after re-vaccination; however, we noted that GMT antibody results in ELISA were lower than in MNA. A comparative GMT assessment after the first immunization and the re-immunization identified significant differences between model animal groups and a growth of GMT antibodies in all of them; also, differences between the gender groups were statistically significant. Moreover, the most marked MNA immune response to the QazCovid-in® vaccine was seen in the Syrian hamsters, while their SARS-CoV-2-specific antibody activity as assessed with ELISA was the lowest.

Efficacy and safety of an inactivated whole-virion vaccine against COVID-19, QazCovid-in®, in healthy adults: A multicentre, randomised, single-blind, placebo-controlled phase 3 clinical trial with a 6-month follow-up.

Background: Vaccination remains the primary measure to prevent the spread of the SARS-CoV-2 virus, further necessitating the use of effective licensed vaccines. Methods: From Dec 25, 2020, to July 11, 2021, we conducted a multicenter, randomised, single-blind, placebo-controlled phase 3 efficacy trial of the QazCovid-in® vaccine with a 180-day follow-up period in three clinical centres in Kazakhstan. A total of 3000 eligible participants aged 18 years or older were randomly assigned (4:1) to receive two doses of the vaccine (5 µg each, 21 days apart) or placebo administered intramuscularly. QazCovid-in® is a whole-virion formaldehyde-inactivated anti-COVID-19 vaccine, adjuvanted with aluminium hydroxide. The primary endpoint was the incidence of symptomatic cases of the SARS-CoV-2 infection confirmed by RT-PCR starting from day 14 after the first immunisation. The trial was registered with ClinicalTrials.gov NCT04691908. Findings: The QazCovid-in® vaccine was safe over the 6-month monitoring period after two intramuscular immunisations inducing only local short-lived adverse events. The concomitant diseases of participants did not affect the vaccine safety. Out of 2400 vaccinated participants, 31 were diagnosed with COVID-19; 43 COVID-19 cases were recorded in 600 placebo participants with onset of 14 days after the first dose within the 180-day observation period. Only one severe COVID-19 case was identified in a vaccine recipient with a comorbid chronic heart failure. The protective efficacy of the QazCovid-in® vaccine reached 82·0% (95% CI 71.1-88.5) within the 180-day observation period. Interpretation: Two immunisations with the inactivated QazCovid-in® vaccine achieved 82·0% (95% CI 71.1-88.5) protective efficacy against COVID-19 within a 180-day follow-up period. Funding: The work was funded by the Science Committee of the Ministry of Education and Science of Kazakhstan within the framework of the Scientific and Technical Program "Development of a vaccine against coronavirus infection COVID-19". State registration number 0.0927.

Development of the Inactivated QazCovid-in Vaccine: Protective Efficacy of the Vaccine in Syrian Hamsters.

In March 2020, the first cases of the human coronavirus disease COVID-19 were registered in Kazakhstan. We isolated the SARS-CoV-2 virus from clinical materials from some of these patients. Subsequently, a whole virion inactivated candidate vaccine, QazCovid-in, was developed based on this virus. To develop the vaccine, a virus grown in Vero cell culture was used, which was inactivated with formaldehyde, purified, concentrated, sterilized by filtration, and then adsorbed on aluminum hydroxide gel particles. The formula virus and adjuvant in buffer saline solution were used as the vaccine. The safety and protective effectiveness of the developed vaccine were studied in Syrian hamsters. The results of the studies showed the absolute safety of the candidate vaccine in the Syrian hamsters. When studying the protective effectiveness, the developed vaccine with an immunizing dose of 5 µg/dose specific antigen protected animals from a wild homologous virus at a dose of 104.5 TCID50 /mL. The candidate vaccine induced the formation of virus-neutralizing antibodies in vaccinated hamsters at titers of 3.3 ± 1.45 log2 to 7.25 ± 0.78 log2, and these antibodies were retained for 6 months (observation period) for the indicated titers. No viral replication was detected in vaccinated hamsters, protected against the development of acute pneumonia, and ensured 100% survival of the animals. Further, no replicative virus was isolated from the lungs of vaccinated animals. However, a virulent virus was isolated from the lungs of unvaccinated animals at relatively high titers, reaching 4.5 ± 0.7 log TCID50/mL. After challenge infection, 100% of unvaccinated hamsters showed clinical symptoms (stress state, passivity, tousled coat, decreased body temperature, and body weight, and the development of acute pneumonia), with 25 ± 5% dying. These findings pave the way for testing the candidate vaccine in clinical human trials.

Registered Influenza Viral Vector Based Brucella abortus Vaccine for Cattle in Kazakhstan: Age-Wise Safety and Efficacy Studies.

A novel influenza viral vector based Brucella abortus vaccine (Flu-BA) was introduced for use in cattle in Kazakhstan in 2019. In this study, the safety and efficacy of the vaccine was evaluated in male and female cattle at different ages, and during pregnancy as a part of its registration process. Our data demonstrated that the Flu-BA vaccine was safe after prime or booster vaccination in calves (5-7 months old male and female), heifers (15-17 months old) and cows (6-7 years old) and was not abortogenic in pregnant animals. A mild, localized granuloma was observed at the Flu-BA injection site. Vaccinated animals did not show signs of influenza infection or reduced milk production in dairy cows, and the influenza viral vector (IVV) was not recovered from nasal swabs or milk. Vaccinated animals in all age groups demonstrated increased IgG antibody responses against Brucella Omp16 and L7/L12 proteins with calves demonstrating the greatest increase in humoral responses. Following experimental challenge with B. abortus 544, vaccinates demonstrated greater protection and no signs of clinical disease, including abortion, were observed. The vaccine effectiveness against B. abortus 544 infection was 75, 60 and 60%, respectively, in calves, heifers and adult cows. Brucella were not isolated from calves of vaccinated cattle that were experimentally challenged during pregnancy. Our data suggests that the Flu-BA vaccine is safe and efficacious in cattle, including pregnant animals; and can therefore be administered to cattle of any age.

A new candidate vaccine for human brucellosis based on influenza viral vectors: a preliminary investigation for the development of an immunization schedule in a guinea pig model.

BACKGROUND: A new candidate vector vaccine against human brucellosis based on recombinant influenza viral vectors (rIVV) subtypes H5N1 expressing Brucella outer membrane protein (Omp) 16, L7/L12, Omp19 or Cu-Zn SOD proteins has been developed. This paper presents the results of the study of protection of the vaccine using on guinea pigs, including various options of administering, dose and frequency. Provided data of the novel vaccine candidate will contribute to its further movement into the preclinical stage study. METHODS: General states of guinea pigs was assessed based on behavior and dynamics of a guinea pig weight-gain test. The effectiveness of the new anti-brucellosis vector vaccine was determined by studying its protective effect after conjunctival, intranasal and sublingual administration in doses 105 EID50, 106 EID50 and 107 EID50 during prime and boost vaccinations of animals, followed by challenge with a virulent strain of B. melitensis 16 M infection. For sake of comparison, the commercial B. melitensis Rev.1 vaccine was used as a control. The protective properties of vaccines were assessed by quantitation of Brucella colonization in organs and tissues of infected animals and compared to the control groups. RESULTS: It was observed a gradual increase in body weight of guinea pigs after prime and booster immunization with the vaccine using conjunctival, intranasal and sublingual routes of administration, as well as after using various doses of vaccine. The most optimal way of using the vaccine has been established: double intranasal immunization of guinea pigs at a dose of 106 EID50, which provides 80% protection of guinea pigs from B. melitensis 16 M infection (P < 0.05), which is comparable to the results of the effectiveness of the commercial B. melitensis Rev.1 vaccine. CONCLUSIONS: We developed effective human vaccine candidate against brucellosis and developed its immunization protocol in guinea pig model. We believe that because of these studies, the proposed vaccine has achieved the best level of protection, which in turn provides a basis for its further promotion.

Development of Human Vectored Brucellosis Vaccine Formulation: Assessment of Safety and Protectiveness of Influenza Viral Vectors Expressing Brucella Immunodominant Proteins in Mice and Guinea Pigs.

In this paper, we first used recombinant influenza viral vector (rIVV) subtype H5N1 expressing from the open reading frame of NS1 80 and NS1 124 amino acids of Brucella outer membrane proteins (Omp) 16 and 19, ribosomal L7/L12, and Cu-Zn superoxide dismutase (SOD) proteins to develop a human brucellosis vaccine. We made 18 combinations of IVVs in mono-, bi-, and tetravalent vaccine formulations and tested them on mice to select the safest and most effective vaccine samples. Then, the most effective vaccine candidates were further tested on guinea pigs. Safety of the rIVV-based vaccine candidate was evaluated by a mouse weight-gain test. Mice and guinea pigs were challenged with the virulent strain B. melitensis 16M. The protective effect of the rIVV-based vaccine candidate was assessed by quantitation of Brucella colonization in tissues and organs of challenged animals. All vaccine formulations were safe in mice. Tested vaccine formulations, as well as the commercial B. melitensis Rev.1 vaccine, have been found to protect mice from B. melitensis 16M infection within the range of 1.6 to 2.97 log10 units (P < 0.05). Tetravalent vaccine formulations from the position of NS1 80 amino acids (0.2 ± 0.4), as well as the commercial B. melitensis Rev.1 vaccine (1.2 ± 2.6), have been found to protect guinea pigs from B. melitensis 16M infection at a significant level (P < 0.05). Thus, tetravalent vaccine formulation Flu-NS1-80-Omp16+Flu-NS1-80-L7/L12+Flu-NS1-80-Omp19+Flu-NS1-80-SOD was chosen as a potential vaccine candidate for further development of an effective human vaccine against brucellosis. These results show a promising future for the development of a safe human vaccine against brucellosis based on rIVVs.

First evaluation of an influenza viral vector based Brucella abortus vaccine in sheep and goats: Assessment of safety, immunogenicity and protective efficacy against Brucella melitensis infection.

Previously we developed and evaluated a candidate influenza viral vector based Brucella abortus vaccine (Flu-BA) administered with a potent adjuvant Montanide Gel01 in cattle, which was found safe and highly effective. This study was aimed to establish a proof-of-concept of the efficacy of Flu-BA vaccine formulation in sheep and goats. We vaccinated sheep and goats with Flu-BA vaccine and as a positive control vaccinated a group of animals with a commercial B. melitensis Rev.1 vaccine. Clinically, both Flu-BA and Rev.1 vaccines were found safe. Serological analysis showed the animals received Flu-BA vaccine did not induce antibody response against Brucella Omp16 and L7/L12 proteins during the period of our study (56days post-initial vaccination, PIV). But observed significant antigen-specific T cell response indicated by increased lymphocyte stimulation index and enhanced secretion of IFN-γ at day 56 PIV in Flu-BA group. The Flu-BA vaccinated animals completely protected 57.1% of sheep and 42.9% of goats against B. melitensis 16M challenge. The severity of brucellosis in terms of infection index and colonization of Brucella in tissues was significantly lower in the Flu-BA group compared to negative control animals group. Nevertheless, positive control commercial Rev.1 vaccine provided strong antigen-specific T cell immunity and protection against B. melitensis 16M infection. We conclude that the Flu-BA vaccine induces a significant antigen-specific T-cell response and provides complete protection in approximately 50% of sheep and goats against B. melitensis 16M infection. Further investigations are needed to improve the efficacy of Flu-BA and explore its practical application in small ruminants.

Influenza viral vectors expressing the Brucella OMP16 or L7/L12 proteins as vaccines against B. abortus infection.

BACKGROUND: We generated novel, effective candidate vaccine against Brucella abortus based on recombinant influenza viruses expressing the Brucella ribosomal protein L7/L12 or outer membrane protein (Omp)-16 from the NS1 open reading frame. The main purpose of this work was to evaluate the safety, immunogenicity and protectiveness of vaccine candidate in laboratory animals. METHODS AND RESULTS: Four recombinant influenza A viral constructs of the subtypes Н5N1 or H1N1 expressing the Brucella proteins L7/L12 or Omp16 were obtained by a reverse genetics method: Flu-NS1-124-L7/L12-H5N1, Flu-NS1-124-Omp16-H5N1, Flu-NS1-124-L7/L12-H1N1 and Flu-NS1-124-Omp16-H1N1. Despite of substantial modification of NS1 gene, all constructs replicated well and were retain their Brucella inserts over five passages in embryonated chicken eggs (CE). Administration of the mono- or bivalent vaccine formulation via prime-boost intranasal (i.n.), conjunctival (c.) or subcutaneous (s.c.) immunization was safe in mice; no deaths, body weight loss or pathomorphological changes were observed over 56 days. Moreover, guinea pigs vaccinated i.n. with vaccine vectors did not shed the vaccine viruses through their upper respiratory tract after the prime and booster vaccination. These findings confirmed the replication-deficient phenotype of viral vectors. The highest antibody response to Brucella antigen was obtained with constructs expressing L7/L12 (ELISA, GMT 242.5-735.0); whereas the highest T-cell immune response- with construct expressing Omp16 (ELISPOT, 337 ± 52-651 ± 45 spots/4×105cells), which was comparable (P > 0.05) to the response induced by the commercial vaccine B. abortus 19. Interestingly, c. immunization appeared to be optimal for eliciting T-cell immune response. In guinea pigs, the highest protective efficacy after challenge with B. abortus 544 was achieved with Omp16 expressing constructs in both monovalent or bivalent vaccine formulations; protective efficacy was comparable to those induced by a commercial live B. abortus 19 vaccine. CONCLUSION: Thus, influenza vectors expressing Brucella protective antigens can be developed as novel influenza vectored vaccine against B. abortus infection.

Novel influenza virus vectors expressing Brucella L7/L12 or Omp16 proteins in cattle induced a strong T-cell immune response, as well as high protectiveness against B. abortus infection.

This paper presents the results of a study of the immunogenicity and protectiveness of new candidate vector vaccine against Brucella abortus - a bivalent vaccine formulation consisting of a mixture of recombinant influenza A subtype H5N1 or H1N1 (viral constructs vaccine formulation) viruses expressing Brucella ribosomal protein L7/L12 and Omp16, in cattle. To increase the effectiveness of the candidate vaccine, adjuvants such as Montanide Gel01 or chitosan were included in its composition. Immunization of cattle (heifers aged 1-1.5 years, 5 animals per group) with the viral constructs vaccine formulation only, or its combination with adjuvants Montanide Gel01 or chitosan, was conducted via the conjunctival method using cross prime (influenza virus subtype H5N1) and booster (influenza virus subtype H1N1) vaccination schedules at an interval of 28 days. Vaccine candidates were evaluated in comparison with the positive (B. abortus S19) and negative (PBS) controls. The viral constructs vaccine formulations, particularly in combination with Montanide Gel01 adjuvant promoted formation of IgG antibodies (with a predominance of antibodies of isotype IgG2a) against Brucella L7/L12 and Omp16 proteins in ELISA. Moreover, these vaccines in cattle induced a strong antigen-specific T-cell immune response, as indicated by a high number of CD4(+) and CD8(+) cells, as well as the concentration of IFN-γ, and most importantly provided a high level of protectiveness comparable to the commercial B. abortus S19 vaccine and superior to the B. abortus S19 vaccine in combination with Montanide Gel01 adjuvant. Based on these findings, we recommended the bivalent vaccine formulation containing the adjuvant Montanide Gel01 for practical use in cattle.

Immunogenic and protective properties of the first Kazakhstan vaccine against pandemic influenza A (H1N1) pdm09 in ferrets.

This paper presents the results of a pre-clinical study of the immunogenicity and efficacy of an egg-derived, inactivated, whole-virion adjuvanted vaccine (Refluvac®) on ferret models. For this purpose, groups of eight ferrets (6 to 7 months old) were injected with 0.5 mL of vaccine specimens containing 3.75, 7.5 or 15.0 µg of virus hemagglutinin. Administration was intramuscular and given either as a single dose or as two doses 14 days apart. All vaccine specimens manifested immunogenicity in ferrets for single (HI titer, from 51 ± 7 to 160 ± 23) and double (HI titer, from 697 ± 120 to 829 ± 117) administrations. To assess the protective effects of the vaccine, ferrets from the vaccinated and control groups were infected intranasally with pandemic virus A/California/7/09 (H1N1) pdm09 at a dose of 10(6) EID(50)/0.5 mL. Fourteen days post-infection, the ferrets inoculated with single or double vaccines containing 3.75, 7.5 or 15.0 µg of hemagglutinin per dose showed no signs of influenza infection, weight loss, or body temperature rise, and no premature deaths occurred. The number of vaccinated ferrets shedding the virus via the upper airway, as well as the amount of virus shed after infection, was significantly reduced in comparison with animals from the control group. Based on our results, we suggest that a single vaccination at a dose of 3.75 or 7.5 µg hemagglutinin be used for Phase I clinical trials.