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.

Phylogenetic analysis of prospective M. bovis antigens with the aim of developing candidate vaccines for bovine tuberculosis.

BACKGROUND: Bovine Tuberculosis is a respiratory disease caused by the pathogen Mycobacterium bovis (M. bovis) that infects cattle. Though rare, this disease can also affect humans, as well as domestic and wild animals, making it a serious concern. Therefore, searching for alternative and new vaccines with high efficiency and safety is the main goal in bovine tuberculosis prophylaxis. New vaccines, known as vector vaccines, have the potential to become safe and effective alternatives to the traditional BCG vaccine. In this study, two major immunodominant proteins of M. bovis Esat-6 and TB10.4 were utilized to create a vector vaccine for bovine tuberculosis. METHODS: The Esat-6 and TB10.4 genes were amplified by PCR. The amplified and purified PCR products were sequenced by the Sanger method. Assembly and multiple alignments of amplicon nucleotides were carried out in the MEGA 11 software. RESULT: Two genes of the local strain 0078-M. bovis-8/RIBSP were sequenced. The nucleotide sequences were deposited in the GenBank database. Comparative analysis of the nucleotide sequences of the ESAT-6 and TB10.4 genes established 100% identity of the compared strains of Mycobacterium. CONCLUSION: Through the use of phylogenetic analysis, it has been confirmed that the amplified genes are related to the mycobacteria genus. This discovery allows the development of a vector vaccine against bovine tuberculosis utilising these genes.

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.

Discovery and Characterization of Novel Bat Coronavirus Lineages from Kazakhstan.

Coronaviruses are positive-stranded RNA viruses that infect a variety of hosts, resulting in a range of symptoms from gastrointestinal illness to respiratory distress. Bats are reservoirs for a high diversity of coronaviruses, and focused surveillance detected several strains genetically similar to MERS-coronavirus, SARS-coronavirus, and the human coronaviruses 229E and NL63. The bat fauna of central Asia, which link China to eastern Europe, are relatively less studied than other regions of the world. Kazakhstan is the world's ninth largest country; however, little is understood about the prevalence and diversity of bat-borne viruses. In this study, bat guano was collected from bat caves in three different sites of southern Kazakhstan that tested positive for coronaviruses. Our phylogenetic reconstruction indicates these are novel bat coronaviruses that belong to the genus Alphacoronavirus. In addition, two distinct lineages of Kazakhstan bat coronaviruses were detected. Both lineages are closely related to bat coronaviruses from China, France, Spain, and South Africa, suggesting that co-circulation of coronaviruses is common in multiple bat species with overlapping geographical distributions. Our study highlights the need for collaborative efforts in understudied countries to increase integrated surveillance capabilities toward better monitoring and detection of infectious diseases.

Biological characterization of Pasteurella multocida present in the Saiga population.

BACKGROUND: This study provides biochemical and molecular genetic characteristics of P. multocida isolated from dead saigas in 1988, 2010-2015 on the territory of the Republic of Kazakhstan. RESULTS: Bacteriological samples taken from carcasses of saiga antelope during mortality events recorded in West Kazakhstan in both 2010 and 2011 and in Kostanay in 2012 and 2015 confirmed the presence of P. multocida, according to morphological and biochemical characterisation. Only in the event of 2015 was the agent proven to be the causative agent of the disease observed, haemorrhagic septicaemia. In the other mortality events it is not certain if the organism was a primary aetiology or an incidental finding as confirmatory pathological investigation was not undertaken. The implemented phylogenetic analysis of ribosomal RNA 16S gene allowed us to identify Pasteurella strains isolated in 2010-2015 as P. multocida subspecies multocida. Capsular typing by PCR showed that the studied strains isolated from dead saiga in 2010, 2011, 2012 and 2015 belonged to serotype B. MLST analysis showed that these strains of P. multocida are of the capsule type B and form one clonal grouping with isolates ST64, ST44, ST45, ST46, ST44, ST47 which isolated from cases of hemorrhagic septicemia of animals in Hungary, Burma, Sri Lanka, Pakistan and Spain. Sixteen virulence genes of the five strains of P. multocida, isolated from saigas were studied using multiplex PCR. ptfA, ompA, ompH, oma87, plpB, fimA, hsf-2, pfhA, exbB, tonB, hgbA, fur, nanB, nanH and pmHAS genes were detected in all strains. The toxA gene was not identified in the studied strains. The phylogenies of these isolates is compared across saiga populations and years and the 2015 isolate was compared to that of an isolate from a disease outbreak in 1988 and the findings suggest that these isolated bacteria are stable commensals, opportunistically pathogenic, being phylogenetically uniform with very little genetic variation notable over the last 4 decades. CONCLUSION: Isolation, phenotypic and genetic characterization of the P. multocida isolates inform understanding of the epidemiology of infection in saigas and predict virulent potential of these opportunistic bacteria.