Development and preclinical evaluation of virus-like particle vaccine against COVID-19 infection.

BACKGROUND: Vaccines that incorporate multiple SARS-CoV-2 antigens can further broaden the breadth of virus-specific cellular and humoral immunity. This study describes the development and immunogenicity of SARS-CoV-2 VLP vaccine that incorporates the four structural proteins of SARS-CoV-2. METHODS: VLPs were generated in transiently transfected HEK293 cells, purified by multimodal chromatography, and characterized by tunable-resistive pulse sensing, AFM, SEM, and TEM. Immunoblotting studies verified the protein identities of VLPs. Cellular and humoral immune responses of immunized animals demonstrated the immune potency of the formulated VLP vaccine. RESULTS: Transiently transfected HEK293 cells reproducibly generated vesicular VLPs that were similar in size to and expressing all four structural proteins of SARS-CoV-2. Alum adsorbed, K3-CpG ODN-adjuvanted VLPs elicited high titer anti-S, anti-RBD, anti-N IgG, triggered multifunctional Th1-biased T-cell responses, reduced virus load, and prevented lung pathology upon live virus challenge in vaccinated animals. CONCLUSION: These data suggest that VLPs expressing all four structural protein antigens of SARS-CoV-2 are immunogenic and can protect animals from developing COVID-19 infection following vaccination.

[Shedding of SARS-CoV-2 Virus in COVID-19 Patients and Neutralizing Antibody Level]. / COVID-19 Hastalarinda SARS-CoV-2 Virüs Saçilimi ve Nötralizan Antikor Düzeyleri.

The coronavirus disease 2019 (COVID-19) turned into a pandemic shortly after emerging in December 2019, in the city of Wuhan, China. In this study, it was aimed to investigate the presence of severe acute respiratory system coronavirus-2 (SARS-CoV-2) RNA in various clinical samples and the scattering profile of the virus and the variation of anti-SARS-CoV-2 IgG and neutralizing antibody levels over time in infected patients during and after the period of COVID-19 disease. The study included COVID-19 patients from the community (CCP) (n= 47) (May-June 2020) and healthcare workers (HCWP) (n= 30) (November-December 2020). To investigate the presence of SARS-CoV-2 in clinical samples, oropharynx (OF), nasopharynx (NF), sputum, stool, blood and urine samples were taken from the CCP group on days 0, 3, 7, 14 and 28. For the detection of anti SARS-CoV-2 IgG and neutralizing antibodies serum samples were taken from the CCP group on days 0, 3, 7, 14, 28, 60, 90 and 120 and on days 14, 28, 60, 90, 120 and 150 from HCWP group. Virus RNA was detected by reverse transcription polymerase chain reaction (RT-PCR), anti SARS-CoV-2 IgG antibody levels by enzyme-linked immunosorbent assay (ELISA), neutralizing antibody levels (NAb) by cell culture neutralization and representative neutralization test (sVNT) methods. With the onset of the vaccination program in our country, 11 of the HCWP group patients had SARS-CoV-2 vaccine after the second month serum samples were taken, the remaining HCWP group patients did not get vaccinated during the study period. SARS-CoV-2 RNA was detected with the highest rates in NF (100%), stool (65.8%), sputum (45.7%), OF (41.3%), blood (5.3%), and urine (2.2%) samples, respectively. It was found that viral shedding continued for 14 days in respiratory tract samples and up to 60 days in stool samples, and no virus was detected in blood samples after the third day. It was observed that the viral load was highest at the time of diagnosis in both upper and lower respiratory tract samples, peaking on the seventh day in stool samples and following an irregular course throughout the disease. Anti-SARS-CoV-2 IgG antibody positivity was found in 41.4% of CCP group patients on the first day of diagnosis, and seroconversion was observed in all patients at the fourth month. During the study period, seropositivity was detected in only 82.1% of the patients in the HCWP group. It was observed that the IgG antibody levels peaked at the 7th day in the CCP group patients and at the third month in the HCWP group patients (S/Co: 9.6 and 2.8, respectively). Anti-SARS-CoV-2 IgG antibody levels detected in the CCP group were found to be significantly higher than the HCWP group (p<0.05). At the end of the first month, NAb was detected in all (100%) patients in the CCP group. It was found that NAb titers peaked (1/256) on the 28th day and showed a decreasing trend from the second month. NAb median titers were observed to peak earlier in the severe HCWP group (14 days in the severe group, 28 days in the mild group, p> 0.05). It was observed that 6 (26.1%) of HCWP group patients had low, 11 (47.8%) moderate, 6 (26.1%) high titers of representative NAb. The distribution of representative NAb levels by vaccine status was examined and no statistically significant difference was found (p= 0.400, p= 0.077 and p= 0.830, respectively). As a result; SARS-CoV-2 RNA was detected in many samples such as sputum, stool, blood and urine, and it was observed that viral shedding in stool samples could continue for months. Anti-SARS-CoV-2 IgG antibody positivity was observed in most of the patients in the fourth month, and it was found that the antibody titers decreased after the third month. It was determined that protective antibody levels continued in the fourth month. These findings are important in vaccination strategies and in the fight against the pandemic. However, considering the emergence of new mutant forms of the virus in today's conditions where the pandemic continues, more detailed and comprehensive studies are needed for viral shedding and antibody titer studies.

Coronaviruses and SARS-COV-2

Coronaviruses (CoVs) cause a broad spectrum of diseases in domestic and wild animals, poultry, and rodents, ranging from mild to severe enteric, respiratory, and systemic disease, and also cause the common cold or pneumonia in humans. Seven coronavirus species are known to cause human infection, 4 of which, HCoV 229E, HCoV NL63, HCoV HKU1 and HCoV OC43, typically cause cold symptoms in immunocompetent individuals. The others namely SARS-CoV (severe acute respiratory syndrome coronavirus), MERS-CoV (Middle East respiratory syndrome coronavirus) were zoonotic in origin and cause severe respiratory illness and fatalities. On 31 December 2019, the existence of patients with pneumonia of an unknown aetiology was reported to WHO by the national authorities in China. This virus was officially identified by the coronavirus study group as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the present outbreak of a coronavirus-associated acute respiratory disease was labelled coronavirus disease 19 (COVID-19). COVID-19's first cases were seen in Turkey on March 10, 2020 and was number 47,029 cases and 1006 deaths after 1 month. Infections with SARS-CoV-2 are now widespread, and as of 10 April 2020, 1,727,602 cases have been confirmed in more than 210 countries, with 105,728 deaths.

Humoral immune response profile of a cattle herd vaccinated with 5- and 10-times Bakirköy strain sheep pox vaccine under field conditions.

Vaccination is the most effective control measure for Lumpy Skin Disease (LSD). The Bakirköy strain-derived sheep pox vaccine (SPPV) has been used against LSD in Türkiye since 2013. In this study, a cattle herd was vaccinated with SPPV and 35 cattle, of which 9 and 26 received 10 and 5 sheep doses, respectively, were followed for 200 days for humoral immune responses. Additionally, maternal antibodies in colostrum-fed calves were investigated. The humoral immune responses of naive and previously vaccinated cattle were compared to determine the effects of annual re-vaccination. Furthermore, the compatibility of the VNT and ELISA tests was analyzed. According to the results, on day 30 post-vaccination, 19 and 13 out of 35 cattle were positive for VNT and ELISA, respectively. The number of seropositive cattle was higher in the group that had been vaccinated in previous years than in naive cattle. No significant differences were observed in the number of positive cattle between the groups vaccinated with the 5- and 10- doses. In colostrum-fed calves grouped according to age, the seropositivity rate was 87 % (41/47) in the one-week-old group, while this rate was only 18 % (3/16) in the 3-month-old group. It was determined that vaccination at different stages in the last four months of pregnancy did not cause any difference in the number of seropositive calves in one-week-old calves fed with colostrum. The concordance between VNT and ELISA tests was lower in 5-dose vaccinated group than 10-dose vaccinated and colostrum-fed calves groups. This study provides insights into the effect of the vaccination strategy followed by Türkiye during its combat of LSD and revealed that annual repeated vaccination using heterologous vaccine has significant positive effects on humoral immun response at the herd level.

Immunogenicity and protection efficacy of a COVID-19 DNA vaccine encoding spike protein with D614G mutation and optimization of large-scale DNA vaccine production.

Severe acute respiratory syndrome coronavirus 2 had devastating consequences for human health. Despite the introduction of several vaccines, COVID-19 continues to pose a serious health risk due to emerging variants of concern. DNA vaccines gained importance during the pandemic due to their advantages such as induction of both arms of immune response, rapid development, stability, and safety profiles. Here, we report the immunogenicity and protective efficacy of a DNA vaccine encoding spike protein with D614G mutation (named pcoSpikeD614G) and define a large-scale production process. According to the in vitro studies, pcoSpikeD614G expressed abundant spike protein in HEK293T cells. After the administration of pcoSpikeD614G to BALB/c mice through intramuscular (IM) route and intradermal route using an electroporation device (ID + EP), it induced high level of anti-S1 IgG and neutralizing antibodies (P < 0.0001), strong Th1-biased immune response as shown by IgG2a polarization (P < 0.01), increase in IFN-γ levels (P < 0.01), and increment in the ratio of IFN-γ secreting CD4+ (3.78-10.19%) and CD8+ (5.24-12.51%) T cells. Challenging K18-hACE2 transgenic mice showed that pcoSpikeD614G administered through IM and ID + EP routes conferred 90-100% protection and there was no sign of pneumonia. Subsequently, pcoSpikeD614G was evaluated as a promising DNA vaccine candidate and scale-up studies were performed. Accordingly, a large-scale production process was described, including a 36 h fermentation process of E. coli DH5α cells containing pcoSpikeD614G resulting in a wet cell weight of 242 g/L and a three-step chromatography for purification of the pcoSpikeD614G DNA vaccine.

Immune response against lumpy skin disease after simultaneous vaccination of cattle with sheep pox and goat pox and foot and mouth disease vaccines.

Foot and mouth disease (FMD) and Lumpy skin disease (LSD) are contagious viral diseases that cause significant economic damage in the livestock industry of countries. Cattle are vaccinated two times a year with FMD and sheep pox and goat pox vaccines (SGP) within 30-day intervals to combat both diseases in Türkiye. However, vaccinations in different periods increase vaccination costs, labor, and distress on animals. Therefore, it was aimed to determine the effects of simultaneous vaccination of FMD and SGP vaccines on the immunity against LSD and FMD in cattle. For this purpose, animals were divided into 4 groups; SGP vaccinated group (Group 1, n = 10), FMD vaccinated group (Group 2, n = 10), FMD and SGP simultaneously vaccinated group (Group 3, n = 10), and the unvaccinated control group (Group 4, n = 6). Blood samples were collected and analyzed to detect the antibody response against the LSD via Capripoxvirus (CaPV) ELISA and FMD by Virus Neutralisation test (VNT) and Liquid Phase Blocking ELISA (LPBE). A live virus challenge study was performed to determine the immune response against LSD. The mean antibody titers were determined protective levels on 28 days post vaccination (DPV) against FMDV serotypes O and A, respectively. The logarithmic difference of skin lesions was calculated log10 titer > 2.5. LSD genome could not be detected in the blood, eyes, and nose swap samples of the challenged animals on the 15th day via PCR. In conclusion, adequate protective immune response was provided against LSD when the SGP and FMD vaccines were used simultaneously in cattle.

Process development for an effective COVID-19 vaccine candidate harboring recombinant SARS-CoV-2 delta plus receptor binding domain produced by Pichia pastoris.

Recombinant protein-based SARS-CoV-2 vaccines are needed to fill the vaccine equity gap. Because protein-subunit based vaccines are easier and cheaper to produce and do not require special storage/transportation conditions, they are suitable for low-/middle-income countries. Here, we report our vaccine development studies with the receptor binding domain of the SARS-CoV-2 Delta Plus strain (RBD-DP) which caused increased hospitalizations compared to other variants. First, we expressed RBD-DP in the Pichia pastoris yeast system and upscaled it to a 5-L fermenter for production. After three-step purification, we obtained RBD-DP with > 95% purity from a protein yield of > 1 g/L of supernatant. Several biophysical and biochemical characterizations were performed to confirm its identity, stability, and functionality. Then, it was formulated in different contents with Alum and CpG for mice immunization. After three doses of immunization, IgG titers from sera reached to > 106 and most importantly it showed high T-cell responses which are required for an effective vaccine to prevent severe COVID-19 disease. A live neutralization test was performed with both the Wuhan strain (B.1.1.7) and Delta strain (B.1.617.2) and it showed high neutralization antibody content for both strains. A challenge study with SARS-CoV-2 infected K18-hACE2 transgenic mice showed good immunoprotective activity with no viruses in the lungs and no lung inflammation for all immunized mice.

Comparison and efficacy of two different sheep pox vaccines prepared from the Bakirköy strain against lumpy skin disease in cattle.

PURPOSE: Lumpy skin disease (LSD) is a highly contagious and economically important viral infection of cattle, which leads to financial losses in the livestock industry of affected countries. Vaccination is the most effective control measure to prevent the disease. Heterologous sheep pox (SP) vaccine was used against LSD in Turkey. In this research, it was aimed to adapt SP Bakirköy vaccine strain attenuated in lamb kidney cells to Madin-Darby bovine kidney (MDBK) cells to provide better protection than commercial SP vaccine in cattle. MATERIALS AND METHODS: To evaluate safety and efficacy of vaccines, while animals were immunized with 10 doses (104.75 50% tissue culture infectious dose [TCID50]) and 5 doses of SP vaccine (104 TCID50) produced in MDBK cells, others were immunized with commercial Penpox-M vaccine (103.9 TCID50). Two cattle were kept as unvaccinated. At day 31 post-vaccination, all animals were challenged with the virulent LSD virus. Blood and swab samples were taken on certain days post-inoculation. Logarithmic differences challenge virus titers between vaccinated and unvaccinated animals were calculated. RESULTS: The clinical sign was not observed in animals immunized with 10 doses of SP vaccine. The differences between the animals immunized with SP vaccine and control group was less than log 2.5 and the viremia occurred in immunized animals. The difference in titer was higher than log 2.5 in animals immunized with the Penpox-M, and viremia did not occur. CONCLUSION: SP vaccine strain propagated in MDBK cells and can be used for immunization to prevent LSD infections. However, SP vaccine strain propagated in MDBK showed poor protection as compared to Penpox-M.

Segment 10 based molecular epidemiology of bluetongue virus (BTV) isolates from Turkey: 1999-2001.

Bluetongue is a significant arbovirus infection that has a negative impact on ruminant productivity in Turkey. Twenty-one Turkish BTV isolates were analyzed phylogenetically, based on genome segment 10 (Seg-10) nucleotide sequences. These analyses were used to explore the epidemiological background of individual isolates from both a regional and global perspective. In the regional analysis, the different BTV strains fell into two groups (Group 1 and Group 2). The Turkish virus isolates were localized in Group 1 which contains two sub-groups. The neighbor-joining analysis revealed that Seg-10 of majority of the Turkish viruses was closely related to certain other virus strains allocated in the eastern lineage. The Seg-10's of two viruses (TR25 and TR26) were more closely related to strains isolated in the Asia-Australia region. These strains belong to the 'eastern' topotype identified by [Maan, S., Maan, N.S., Ross-Smith, N., Batten, C.A., Shaw, A.E., Anthony, S.J., Samuel, A.R., Darpel, K.E., Veronesi, E., Oura, C.A.L., Singh,K.P., Nomikou, K., Potgieter, A.C., Attoui, H., van Rooij, E., van Rijn, P., De Clercq, K., Vandenbussche, F., Zientara, S., Bréard, E., Sailleau, C., Beer, M., Hoffman, B., Mellor, P.S., Mertens, P.P.C., 2008. Sequence analysis of bluetongue virus serotype 8 from the Netherlands 2006 and comparison to other European strains. Virology 377, 308-318]. Comparisons of amino acid sequences deduced from the Seg-10 genes showed a high level of conservation in the NS3/3A proteins from the Turkish viruses. The more frequent amino acid substitutions were identified by multiple alignment analysis, and one of the isolates (TR23) was remarkably found to be genetically quite distinct from the other isolates.