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.

The Prevalence of Viral Pathogens among Bats in Kazakhstan.

Bats carry thousands of viruses from 28 different families. To determine the presence of various pathogens in bat populations in Kazakhstan, 1149 samples (393 oropharyngeal swabs, 349 brain samples, 407 guano) were collected. The samples were collected from four species of bats (Vespertilio murinus, Nyctalus noctula, Myotis blythii, Eptesicus serotinus) in nine regions. The Coronavirus RNA was found in 38 (4.75%) samples, and the rabies virus in 27 (7.74%) samples from bats. Coronaviruses and the rabies virus were found in bats in six out of nine studied areas. The RNAs of SARS-CoV-2, MERS, TBE, CCHF, WNF, influenza A viruses were not detected in the bat samples. The phylogeny of the RdRp gene of 12 samples made it possible to classify them as alphacoronaviruses and divide them into two groups. The main group (n = 11) was closely related to bat coronaviruses from Ghana, Zimbabwe and Kenya. The second group (n = 1) was closely related to viruses previously isolated in the south of Kazakhstan. The phylogeny of the N gene sequence from a bat from west Kazakhstan revealed its close relationship with isolates from the Cosmopolitan group of rabies viruses (Central Asia). These results highlight the need for a continuous monitoring of volatile populations to improve the surveillance and detection of infectious diseases.

Lumpy Skin Disease Virus with Four Knocked Out Genes Was Attenuated In Vivo and Protects Cattle from Infection.

Vaccination with live attenuated vaccines is a key element in the prevention of lumpy skin disease. The mechanism of virus attenuation by long-term passaging in sensitive systems remains unclear. Targeted inactivation of virulence genes is the most promising way to obtain attenuated viruses. Four virulence genes in the genome of the lumpy skin disease virus (LSDV) Dermatitis nodulares/2016/Atyrau/KZ were sequentially knocked out by homologous recombination under conditions of temporary dominant selection. The recombinant LSDV Atyrau-5BJN(IL18) with a knockout of the LSDV005, LSDV008, LSDV066 and LSDV142 genes remained genetically stable for ten passages and efficiently replicated in cells of lamb testicles, saiga kidney and bovine kidney. In vivo experiments with cattle have shown that injection of the LSDV Atyrau-5BJN(IL18) at a high dose does not cause disease in animals or other deviations from the physiological norm. Immunization of cattle with the LSDV Atyrau-5BJN(IL18) induced the production of virus-neutralizing antibodies in titers of 4-5 log2. The challenge did not cause disease in immunized animals. The knockout of four virulence genes resulted in attenuation of the virulent LSDV without loss of immunogenicity. The recombinant LSDV Atyrau-5BJN(IL18) is safe for clinical use, immunogenic and protects animals from infection with the virulent LSDV.

Lumpy skin disease in Kazakhstan.

This study describes the registration of the first cases of lumpy skin disease in July 2016 in the Republic of Kazakhstan. In the rural district of Makash, Kurmangazinsky district of Atyrau region, 459 cattle fell ill and 34 died (morbidity 12.9% and mortality 0.96%). To determine the cause of the disease, samples were taken from sick and dead animals, as well as from insects and ticks. LSDV DNA was detected by PCR in all samples from dead animals and ticks (Dermacentor marginatus and Hyalomma asiaticum), in 14.29% of samples from horseflies (Tabanus bromius), and in one of the samples from two Stomoxys calcitrans flies. The reproductive LSD virus was isolated from organs of dead cattle and insects in the culture of LT and MDBK cells. The virus accumulated in cell cultures of LT and MDBK at the level of the third passage with titers in the range of 5.5-5.75 log 10 TCID50/cm3. Sequencing of the GPCR gene allowed us to identify this virus as a lumpy skin disease virus.