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 Pathogens among Ticks Collected from Livestock in Kazakhstan.

Ticks carry and transmit a wide variety of pathogens (bacteria, viruses and protozoa) that pose a threat to humans and animals worldwide. The purpose of this work was to study ticks collected in different regions of Kazakhstan for the carriage of various pathogens. The collected ticks were examined by PCR for the carriage of various pathogens. A total of 3341 tick samples parasitizing three animal species (cattle, sheep and horses) were collected at eight regions of Kazakhstan. Eight tick species were found infesting animals: Dermacentor marginatus (28.08%), Hyalomma asiaticum (21.28%), Hyalomma anatolicum (17.18%), Dermacentor reticulatus (2.01%), Ixodes ricinus (3.35%), Ixodes persulcatus (0.33%), Hyalomma scupense (12.87%) and Hyalomma marginatum (14.90%). Ticks collected from livestock animals were examined for the pathogen spectrum of transmissible infections to determine the degree of their infection. Four pathogen DNAs (lumpy skin disease virus (LSDV), Coxiella burnetti, Teileria annulata, and Babesia caballi) were detected by PCR in Dermacentor marginatus, Hyalomma asiaticum, Hyalomma scupense, Hyalomma anatolicum. The infection of ticks Dermacentor marginatus and Hyalomma asiaticum collected on cattle in the West Kazakhstan region with LSDV was 14.28% and 5.71%, respectively. Coxiella burnetti was found in the ticks Dermacentor marginatus (31.91%) in the Turkestan region and Hyalomma anatolicum (52.63%) in the Zhambyl region. Theileria annulata was found in ticks Hyalomma scupense (7.32%) and Dermacentor marginatus (6.10%) from cattle in the Turkestan region. Babesia caballi was isolated only from the species Hyalomma scupense (17.14%) in the Turkestan region. There were no PCR-positive tick samples collected from sheep. RNA/DNAs of tick-borne encephalitis virus (TBEV), African swine fever virus (ASFV), Hantavirus hemorrhagic fever with renal syndrome (HFRS), and chlamydia pathogens were not found in ticks. The new data give a better understanding of the epidemiology of tick-borne pathogens and the possibility of the emergence of tick-borne animal diseases in Kazakhstan.

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.

The Prevalence and Genetic Variants of the CCHF Virus Circulating among Ticks in the Southern Regions of Kazakhstan.

Crimean-Congo hemorrhagic fever (CCHF) disease cases are registered annually in endemic regions of Kazakhstan. To study the prevalence of various Crimean-Congo hemorrhagic fever virus (CCHFV) genotypes, a total of 694 ticks were collected from southern regions of Kazakhstan in 2021. Hyalomma marginatum (n = 323) (46.5%), Hyalomma anatolicum (n = 138) (19.9%), Hyalomma asiaticum (n = 126) (18.2%), Hyalomma scupense (n = 80) (11.5%) and Ixodes ricinus (n = 27) (3.9%) were collected using the standardized flagging technique from the environment. All the tick samples were analyzed for the presence of CCHFV RNA by RT-PCR. The CCHF-positive samples were found within three Hyalomma asiaticum and one Ixodes ricinus tick sample. For the first time in Kazakhstan, infection of the Ixodes ricinus tick with CCHFV was detected. The results of sequencing and analysis of the S-gene fragment showed that the Asia 1 and Asia 2 CCHF genotypes circulate in the southern regions of Kazakhstan. Viruses isolated in the Zhambyl and Turkestan regions are assigned to the Asia-2 genotype, whereas the virus isolated in the Kyzylorda region to the Asia-1 genotype.

Evidence for flock transmission of individual subtypes and strains of avian influenza viruses: A monitoring study of wild birds in Kazakhstan.

An active surveillance study of avian influenza viruses (AIVs) in wild birds was carried out in Kazakhstan in 2018-2019. In total, 866 samples were collected from wild birds and analyzed for influenza viruses using molecular and virological tests. Genome segments of Asian, European, and Australian lineages were detected in 25 (4.6%) out of 541 waterfowl samples positive for subtype H3N8, and in two (0.6%) out of 325 H3N8 positive samples from terrestrial birds. No highly pathogenic avian influenza virus (AIV) was detected. The results indicated transmission of closely related strains or identical subtypes of AIVs by a flock-unit of migratory birds or annual cyclical pattern of subtype dominance. The simultaneous circulation of genome segments of the Asian, European and Australian genetic lineages of H3N8 AIVs in wild birds in Kazakhstan indicated the important role of Central Asia as a transmission hub of AI viruses linking the East Asian migratory flyways with European flyways and vice versa.

Genome Sequence of Atyrau-5BJN(IL18), a Recombinant Lumpy Skin Disease Virus with Knockout of Virulence Genes.

Here, we present the coding sequence of the genome of the recombinant lumpy skin disease virus (LSDV) Atyrau-5BJN(IL18), obtained by knocking out four genes in the genome of a virulent field LSDV isolate. Genome sequencing confirmed the deletion of genes and the insertion of a foreign sequence in the viral genome.

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.

Engineering of Recombinant Sheep Pox Viruses Expressing Foreign Antigens.

Capripoxviruses with a host range limited to ruminants have the great potential to be used as vaccine vectors. The aim of this work was to evaluate attenuated sheep pox virus (SPPV) vaccine strain NISKHI as a vector expressing several genes. Open reading frames SPPV020 (ribonucleotide kinase) and SPPV066 (thymidine kinase) were selected as sites for the insertion of foreign genes. Two integration plasmids with expression cassette were designed and constructed. Recombinant SPPVs expressing an enhanced green fluorescent protein (EGFP) (rSPPV(RRΔ)EGFP and rSPPV(TKΔ)EGFP), Foot-and-mouth disease virus capsid protein (VP1), and Brucella spp. outer membrane protein 25 (OMP25) (rSPPV(RRΔ)VP1A-(TKΔ)OMP25) were generated under the transient dominant selection method. The insertion of foreign genes into the SPPV020 and SPPV066 open reading frames did not influence the replication of the recombinant viruses in the cells. Successful foreign gene expression in vitro was assessed by luminescent microscopy (EGFP) and Western blot (VP1 and OMP25). Our results have shown that foreign genes were expressed by rSPPV both in permissive (lamb testicles) and non-permissive (bovine kidney, saiga kidney, porcine kidney) cells. Mice immunized with rSPPV(RRΔ)VP1A-(TKΔ)OMP25 elicited specific antibodies to both SPPV and foreign genes VP1 and OMP25. Thus, SPPV NISKHI may be used as a potential safe immunogenic viral vector for the development of polyvalent vaccines.

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.

Complete Genome Sequence of the Highly Pathogenic Strain A/Domestic Goose/Pavlodar/1/05 (H5N1) of the Avian Influenza Virus, Isolated in Kazakhstan in 2005.

Here, we present the complete genome sequence of a highly pathogenic strain of avian influenza A virus/domestic goose/Pavlodar/1/05 (H5N1) (GS/1/05), which belongs to clade 2.2. This strain of the influenza virus was isolated in northern Kazakhstan in 2005.

New oligonucleotide microarray for rapid diagnosis of avian viral diseases.

BACKGROUND: We developed a new oligonucleotide microarray comprising 16 identical subarrays for simultaneous rapid detection of avian viruses: avian influenza virus (AIV), Newcastle disease virus (NDV), infection bronchitis virus (IBV), and infectious bursal disease virus (IBDV) in single- and mixed-virus infections. The objective of the study was to develop an oligonucleotide microarray for rapid diagnosis of avian diseases that would be used in the course of mass analysis for routine epidemiological surveillance owing to its ability to test one specimen for several infections. METHODS AND RESULTS: The paper describes the technique for rapid and simultaneous diagnosis of avian diseases such as avian influenza, Newcastle disease, infectious bronchitis and infectious bursal disease with use of oligonucleotide microarray, conditions for hybridization of fluorescent-labelled viral cDNA on the microarray and its specificity tested with use of AIV, NDV, IBV, IBDV strains as well as biomaterials from poultry. Sensitivity and specificity of the developed microarray was evaluated with use of 122 specimens of biological material: 44 cloacal swabs from sick birds and 78 tissue specimens from dead wild and domestic birds, as well as with use of 15 AIV, NDV, IBV and IBDV strains, different in their origin, epidemiological and biological characteristics (RIBSP Microbial Collection). This microarray demonstrates high diagnostic sensitivity (99.16% within 95% CI limits 97.36-100%) and specificity (100%). Specificity of the developed technique was confirmed by direct sequencing of NP and M (AIV), VP2 (IBDV), S1 (IBV), NP (NDV) gene fragments. CONCLUSION: Diagnostic effectiveness of the developed DNA microarray is 99.18% and therefore it can be used in mass survey for specific detection of AIV, NDV, IBV and IBDV circulating in the region in the course of epidemiological surveillance. Rather simple method for rapid diagnosis of avian viral diseases that several times shortens duration of assay versus classical diagnostic methods is proposed.

Recombinant Sheep Pox Virus Proteins Elicit Neutralizing Antibodies.

The aim of this work was to evaluate the immunogenicity and neutralizing activity of sheep pox virus (SPPV; genus Capripoxvirus, family Poxviridae) structural proteins as candidate subunit vaccines to control sheep pox disease. SPPV structural proteins were identified by sequence homology with proteins of vaccinia virus (VACV) strain Copenhagen. Four SPPV proteins (SPPV-ORF 060, SPPV-ORF 095, SPPV-ORF 117, and SPPV-ORF 122), orthologs of immunodominant L1, A4, A27, and A33 VACV proteins, respectively, were produced in Escherichia coli. Western blot analysis revealed the antigenic and immunogenic properties of SPPV-060, SPPV-095, SPPV-117 and SPPV-122 proteins when injected with adjuvant into experimental rabbits. Virus-neutralizing activity against SPPV in lamb kidney cell culture was detected for polyclonal antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. To our knowledge, this is the first report demonstrating the virus-neutralizing activities of antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins.

Molecular and genetic analysis of NS gene from high pathogenic strains of the avian influenza (H5N1) virus isolated in Kazakhstan.

The high pathogenic strains of the avian influenza H5N1 virus isolated in Kazakhstan have NS of different genotypes. The influenza virus strains isolated in 2005 is of NS1E Qinghai genotype. A/swan/Mangystau/3/2006 strain is of NS2A genotype that is typical for Gs/Gd-like strains. The results of the analysis allow assuming that A/swan/Mangystau/3/2006 strain has been brought onto the territory of Kazakhstan from the European part of the continent along the Black Sea-Mediterranean flyway.