Immunogenicity and safety of RAZI recombinant spike protein vaccine (RCP) as a booster dose after priming with BBIBP-CorV: a parallel two groups, randomized, double blind trial.

BACKGROUND: The immunity induced by primary vaccination is effective against COVID-19; however, booster vaccines are needed to maintain vaccine-induced immunity and improve protection against emerging variants. Heterologous boosting is believed to result in more robust immune responses. This study investigated the safety and immunogenicity of the Razi Cov Pars vaccine (RCP) as a heterologous booster dose in people primed with Beijing Bio-Institute of Biological Products Coronavirus Vaccine (BBIBP-CorV). METHODS: We conducted a randomized, double-blind, active-controlled trial in adults aged 18 and over primarily vaccinated with BBIBP-CorV, an inactivated SARS-CoV-2 vaccine. Eligible participants were randomly assigned (1:1) to receive a booster dose of RCP or BBIBP-CorV vaccines. The primary outcome was neutralizing antibody activity measured by a conventional virus neutralization test (cVNT). The secondary efficacy outcomes included specific IgG antibodies against SARS-CoV-2 spike (S1 and receptor-binding domain, RBD) antigens and cell-mediated immunity. We measured humoral antibody responses at 2 weeks (in all participants) and 3 and 6 months (a subgroup of 101 participants) after the booster dose injection. The secondary safety outcomes were solicited and unsolicited immediate, local, and systemic adverse reactions. RESULTS: We recruited 483 eligible participants between December 7, 2021, and January 13, 2022. The mean age was 51.9 years, and 68.1% were men. Neutralizing antibody titers increased about 3 (geometric mean fold increase, GMFI = 2.77, 95% CI 2.26-3.39) and 21 (GMFI = 21.51, 95% CI 16.35-28.32) times compared to the baseline in the BBIBP-CorV and the RCP vaccine groups. Geometric mean ratios (GMR) and 95% CI for serum neutralizing antibody titers for RCP compared with BBIBP-CorV on days 14, 90, and 180 were 6.81 (5.32-8.72), 1.77 (1.15-2.72), and 2.37 (1.62-3.47) respectively. We observed a similar pattern for specific antibody responses against S1 and RBD. We detected a rise in gamma interferon (IFN-γ), tumor necrosis factor (TNF-α), and interleukin 2 (IL-2) following stimulation with S antigen, particularly in the RCP group, and the flow cytometry examination showed an increase in the percentage of CD3 + /CD8 + lymphocytes. RCP and BBIBP-CorV had similar safety profiles; we identified no vaccine-related or unrelated deaths. CONCLUSIONS: BBIBP-CorV and RCP vaccines as booster doses are safe and provide a strong immune response that is more robust when the RCP vaccine is used. Heterologous vaccines are preferred as booster doses. TRIAL REGISTRATION: This study was registered with the Iranian Registry of Clinical Trial at www.irct.ir , IRCT20201214049709N4. Registered 29 November 2021.

Phase II, Safety and Immunogenicity of RAZI Cov Pars (RCP) SARS Cov-2 Vaccine in Adults Aged 18-70 Years; A Randomized, Double-Blind Clinical Trial.

BACKGROUND: This study explores the safety and immunogenicity of the Razi-Cov-Pars (RCP) SARS Cov-2 recombinant spike protein vaccine. METHOD: In a randomized, double-blind, placebo-controlled trial, adults aged 18-70 were randomly allocated to receive selected 10 µg/200 µl vaccine strengths or placebo (adjuvant). It included two intramuscular injections at days 0 and 21, followed by an intranasal dose at day 51. Immediate and delayed solicited local and systemic adverse reactions after each dose up to a week, and specific IgG antibodies against SARS Cov-2 spike antigens two weeks after the 2nd dose were assessed as primary outcomes. Secondary safety outcomes were abnormal laboratory findings and medically attended adverse events (MAAE) over six months follow up. Secondary immunogenicity outcomes were neutralizing antibody activity and cell-mediated immune response. RESULT: Between May 27th and July 15th, 2021, 500 participants were enrolled. Participants' mean (SD) age was 37.8 (9.0), and 67.0 % were male. No immediate adverse reaction was observed following the intervention. All solicited local and systemic adverse events were moderate (Grade I-II). Specific IgG antibody response against S antigen in the vaccine group was 5.28 times (95 %CI: 4.02-6.94) the placebo group with a 75 % seroconversion rate. During six months of follow-up, 8 SAEs were reported, unrelated to the study intervention. The participants sustained their acquired humoral responses at the end of the sixth month. The vaccine predominantly resulted in T-helper 1 cell-mediated immunity, CD8+ cytotoxic T-cell increase, and no increase in inflammatory IL-6 cytokine. CONCLUSION: RCP vaccine is safe and creates strong and durable humoral and cellular immunity. TRIAL REGISTRATION: (IRCT20201214049709N2).

Nanoadjuvants Produced by Advanced Nanochelating Technology in the Inactivated-Severe Acute Respiratory Syndrome Coronavirus-2 Vaccine Formulation: Preliminary Results on Cytokines and IgG Responses.

Despite the great success of vaccines in various infectious diseases, most current vaccines are not effective enough, and on the contrary, clinically approved alum adjuvants cannot induce sufficient immune responses, including a potent cellular immune response to confer protection. In this study, we used Nanochelating Technology to develop novel nanoadjuvants to boost the potency of the alum-adjuvanted inactivated severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccine. BALB/c mice were immunized twice over 2 weeks with different doses of adjuvanted-vaccine formulations and immune responses were assessed. The analysis results of IFN-γ and IL-17 cytokines demonstrated the effectiveness of the nanoadjuvants produced by the Nanochelating Technology in shifting the alum-based vaccine toward a stronger Th1 pattern. In addition, these nanoadjuvants improved IL-2 cytokine response, which shows the efficacy of these novel formulations in inducing specific T lymphocyte proliferation. Using these nanoadjuvants increased IL-10 cytokine secretion that may be representative of a better immunoregulatory impact and may also potentially prevent immunopathology responses. Moreover, specific IgG titer analysis revealed the potency of these nanoadjuvants in improving humoral immune responses. The enzyme-linked immunosorbent assay of receptor-binding domain (RBD)-specific IgG response showed that the developed novel formulations induced strong IgG responses against this protein. This study shows that the nanostructures produced by the Advanced Nanochelating Technology have potent adjuvant effects on alum-based SARS-CoV-2 vaccines to not only compensate for alum weakness in inducing the cellular immune responses by smart regulation of the immune system but also significantly improve the humoral and cellular immune responses simultaneously.

Immunogenicity and Safety of a Combined Intramuscular/Intranasal Recombinant Spike Protein COVID-19 Vaccine (RCP) in Healthy Adults Aged 18 to 55 Years Old: A Randomized, Double-Blind, Placebo-Controlled, Phase I Trial.

Objectives: This study aimed to determine the safety and immunogenicity of a combined intramuscular/intranasal recombinant spike protein COVID-19 vaccine (RCP). Methods: We conducted a randomized, double-blind, placebo-controlled, phase I trial. Three vaccine strengths were compared with an adjuvant-only preparation. It included two intramuscular and a third intranasal dose. Eligible participants were followed for adverse reactions. Specific IgG, secretory IgA, neutralizing antibodies, and cell-mediated immunity were assessed. Results: A total of 153 participants were enrolled (13 sentinels, 120 randomized, 20 non-randomized open-labeled for IgA assessment). No related serious adverse event was observed. The geometric mean ratios (GMRs) and 95% CI for serum neutralizing antibodies compared with placebo two weeks after the second injection were 5.82 (1.46-23.13), 11.12 (2.74-45.09), and 20.70 (5.05-84.76) in 5, 10, and 20 µg vaccine groups, respectively. The GMR for anti-RBD IgA in mucosal fluid two weeks after the intranasal dose was 23.27 (21.27-25.45) in the 10 µg vaccine group. The humoral responses were sustained for up to five months. All vaccine strengths indicated a strong T-helper 1 response. Conclusion: RCP is safe and creates strong and durable humoral and cellular immunity and good mucosal immune response in its 10 µg /200 µL vaccine strengths. Trial registration: IRCT20201214049709N1.

PPD in HBsAg vaccine formulation suppressed IFN-γ and IL-4 cytokine responses and induced long-lived humoral immune responses: Results from 220-day monitoring of specific IgG responses.

Objectives: Here, immune responses and long-lived IgG responses of HBsAg-Alum, HBsAg-MF59, as well as HBsAg-MF59 were compared when formulated with PPD. Materials and Methods: BALB/c mice were vaccinated subcutaneously three times with a two-week -interval. Then, specific IgG, long-lived IgG responses up to 220 days, and IgG1/IgG2a isotypes, and IFN-γ and IL-4 on spleen cell culture supernatant were assessed using ELISA. Results: IFN-γ cytokine response between MF59- and Alum-adjuvanted vaccines did not show a significant difference. HBsAg-Alum revealed an increase in IL-4 cytokine versus HBsAg-MF59 at borderline (P=0.0553). In addition, HBsAg-MF59+PPD 10 µg showed a significant decrease in IL-4 and IFN-γ cytokines versus HBsAg-MF59. Furthermore, HBsAg-MF59+PPD10 µg showed a significant increase in the IL-2/IL-4 ratio versus HBsAg-MF59 (P=0.0339). Specific IgG antibody showed a significant increase in HBsAg-MF59, as compared with HBsAg-Alum. Furthermore, HBsAg-MF59 plus PPD showed a significant increase in IgG responses versus HBsAg-MF59 and HBsAg-Alum groups. Long-lived IgG responses showed a significant increase in HBsAgMF59 versus HBsAg-Alum group and PPD in the HBsAg-MF59 vaccine formulation, resulting in a significant increase in IgG responses versus HBsAg-MF59 group. In addition, HBsAg-MF59 plus PPD suppressed IgG1 response versus HBsAg-Alum. However, HBsAg-MF59 showed a significant increase in IgG2α versus the HBsAg-Alum group (P=0.0190). Immunization with HBsAg-MF59+PPD (10 µg) showed a significant increase versus the HBsAg-MF59 group (P=0.0040). IgG2a/IgG1 ratio in HBsAg-MF59+PPD1µg and HBsAg-MF59+PPD10 µg groups showed a significant increase versus HBsAg-MF59 groups (P<0.0345). Conclusion: PPD leads to a more potent long-lived IgG responses in the HBsAg vaccine, highlighting its potential as a component of a complex adjuvant.

Different Formulations of Inactivated SARS-CoV-2 Vaccine Candidates in Human Compatible Adjuvants: Potency Studies in Mice Showed Different Platforms of Immune Responses.

Several inactivated SARS-CoV-2 vaccines have been approved for human use, but are not highly potent. In this study, different formulations of the inactivated SARS-CoV-2 virus were developed in Alum, Montanide 51VG, and Montanide ISA720VG adjuvants, followed by assessment of immune responses. The SARS-CoV-2 virus was inactivated with formalin and formulated in the adjuvants. BALB/c mice were immunized subcutaneously with 4 µg of vaccines on days 0 and 14; (IL-4) and (IFN-g), cytotoxic T lymphocyte (CTL) activity, and specific immunoglobulin G (IgG) titer and IgG1, IgG2a, and IgG2a/IgG1 ratio, and anti-receptor-binding domain (RBD) IgG response were assessed 2 weeks after the final immunization. Immunization with SARS-CoV-2-Montanide ISA51VG showed a significant increase in the IFN-γ cytokine versus SARS-CoV-2-Alum, SARS-CoV-2-Montanide ISA720VG, and control groups (p < 0.0033). Cytokine IL-4 response in SARS-CoV-2-Alum group showed a significant increase compared with SARS-CoV-2-Montanide ISA51VG, SARS-CoV-2-Montanide ISA720VG, and control groups (p < 0.0206). In addition, SARS-CoV-2-Montanide ISA51VG vaccine induced the highest IFN-γ/IL-4 cytokine ratio versus other groups (p < 0.0004). CTL activity in SARS-CoV-2-Montanide ISA51VG and SARS-CoV-2-Montanide ISA720VG groups showed a significant increase compared with SARS-CoV-2-Alum and control groups (p < 0.0075). Specific IgG titer in SARS-CoV-2-Montanide ISA51 VG and SARS-CoV-2-Montanide ISA720VG showed a significant increase compared with SARS-CoV-2-Alum and control groups (p < 0.0143). Results from specific IgG1and IgG2a in SARS-CoV-2-Alum, SARS-CoV-2-Montanide ISA51VG, and SARS-CoV-2-Montanide ISA720VG vaccine showed a significant increase compared with phosphate buffer saline (PBS) group (p < 0.0001), but SARS-CoV-2-Montanide ISA51VG and SARS-CoV-2-Montanide ISA 720VG groups showed the highest IgG2a/IgG1 ratio and a significant increase compared with SARS-CoV-2-Alum group (p < 0.0379). Moreover, inactivated SARS-CoV-2+Alum and SARS-CoV-2-Montanide ISA 720VG groups demonstrated a significant increase in anti-RBD IgG response versus the SARS-CoV-2-Montanide ISA51VG group. It seems that the type of vaccine formulation is a critical parameter, influencing the immunologic pattern and vaccine potency and human-compatible oil-based adjuvants were more potent than Alum adjuvant in the vaccine formulation.

Improvement of the inactivated SARS-CoV-2 vaccine potency through formulation in alum/naloxone adjuvant; Robust T cell and anti-RBD IgG responses.

Objectives: SARS-CoV-2, emerging as a major threat to public health, has to be controlled through vaccination. Naloxone (NLX), an opioid receptor antagonist, demonstrated its adjuvant activity for microbial vaccines. In this study, inactivated SARS-CoV-2 was developed in the Alum/NLX adjuvant to increase the potency of the inactivated SARS-CoV-2 vaccine. Materials and Methods: BALB/c mice were immunized on days 0 and 14 with inactivated SARS-CoV-2-Alum, -Alum + NLX 3 mg/kg, -Alum + NLX 10 mg/kg, and -Freund adjuvant, as well as PBS. IFN-γ and IL-4 cytokines and Granzyme-B release were assessed with ELISA. In addition, specific total IgG, IgG1/IgG2a isotypes, and ratio as well as anti-RBD IgG responses were assessed with an optimized ELISA. Results: SARS-CoV-2-Alum-NLX10 group showed a significant increase in the IFN-γ cytokine response versus SARS-CoV-2-Alum, SARS-CoV-2-Alum-NLX3, and PBS groups. The SARS-CoV-2-Alum-NLX3 group exhibited a significant decrease in IL-4 cytokine versus SARS-CoV-2-Alum. The mice immunized with SARS-CoV-2-Alum-NLX10 showed a significant increase in CTL activity versus SARS-CoV-2-Alum and PBS. In addition, mice immunized with SARS-CoV-2-Alum-NLX3, SARS-CoV-2-Alum-NLX10 and SARS-CoV-2-Freund demonstrated an increase in IgG response, as compared with SARS-CoV-2-Alum and PBS group. Furthermore, all formulations of SARS-CoV-2 vaccines could induce both IgG1 and IgG2a isotypes. But, the IgG2a/IgG1 ratio in SARS-CoV-2-Freund and SARS-CoV-2-Alum-NLX10 revealed an increase as compared with that of the SARS-CoV-2-Alum group. Anti-RBD IgG response in the SARS-CoV-2-Alum-NLX10 group showed a significant increase as compared with the Alum-based vaccine. Conclusion: Formulation of inactivated SARS-CoV-2 virus in NLX/alum adjuvant improved the potency of humoral and, especially, cellular responses.

Safety and Efficacy of Combined Intramuscular/Intranasal RAZI-COV PARS Vaccine Candidate Against SARS-CoV-2: A Preclinical Study in Several Animal Models.

Several vaccine candidates for COVID-19 have been developed, and few vaccines received emergency approval with an acceptable level of efficacy and safety. We herein report the development of the first recombinant protein-based vaccine in Iran based on the recombinant SARS-CoV-2 spike protein in its monomeric (encompassing amino acid 1-674 for S1 and 685-1211 for S2 subunits) and trimer form (S-Trimer) formulated in the oil-in-water adjuvant system RAS-01 (Razi Adjuvant System-01). The safety and immunity of the candidate vaccine, referred to as RAZI-COV PARS, were evaluated in Syrian hamster, BALB/c mice, Pirbright guinea pig, and New Zeeland white (NZW) rabbit. All vaccinated animals received two intramuscular (IM) and one intranasal (IN) candidate vaccine at 3-week intervals (days 0, 21, and 51). The challenge study was performed intranasally with 5×106 pfu of SARS-CoV-2 35 days post-vaccination. None of the vaccinated mice, hamsters, guinea pigs, or rabbits showed any changes in general clinical observations; body weight and food intake, clinical indicators, hematology examination, blood chemistry, and pathological examination of vital organs. Safety of vaccine after the administration of single and repeated dose was also established. Three different doses of candidate vaccine stimulated remarkable titers of neutralizing antibodies, S1, Receptor-Binding Domain (RBD), and N-terminal domain (NTD) specific IgG antibodies as well as IgA antibodies compared to placebo and control groups (P<0.01). Middle and high doses of RAZI-COV PARS vaccine significantly induced a robust and quick immune response from the third-week post-immunization. Histopathological studies on vaccinated hamsters showed that the challenge with SARS-CoV-2 did not induce any modifications in the lungs. The protection of the hamster was documented by the absence of lung pathology, the decreased virus load in the lung, rapid clearance of the virus from the lung, and strong humoral and cellular immune response. These findings confirm the immunogenicity and efficacy of the RAZI-COV PARS vaccine. Of the three tested vaccine regimens, the middle dose of the vaccine showed the best protective immune parameters. This vaccine with heterologous prime-boost vaccination method can be a good candidate to control the viral infection and its spread by stimulating central and mucosal immunity.

Complete genome sequencing and molecular characterization of SARS-COV-2 from COVID-19 cases in Alborz province in Iran.

Iran was among countries which was hard hit at the early stage of the coronavirus disease 2019 (COVID-19) pandemic and dealt with the second wave of the pandemic in May and June 2020; however, there are a very limited number of complete genome sequences of acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from Iran. In this study, complete genome sequences of the virus in the samples obtained from three patients in Alborz province in May and June 2020 were generated and analyzed using bioinformatic methods. The sequenced genomes were positioned in a cluster with B.4 lineage along with the sequences from other countries namely, United Arab Emirates and Oman. There were seven single nucleotide variations (SNVs) in common in all samples and only one of the sequenced genomes showed the D614G amino acid substitution. Three SNVs, 1397 G > A, 28688T > C, 29742 G > T, which had already been reported in February, were found with high frequency in all the sequenced genomes in this study, implying that viral diversity reflected in the early stages of viral transmission in Iran were established in the second wave. Considering the importance of molecular epidemiology in response to ongoing pandemic, there is an urgent need for more complete genome sequencing and comprehensive analyses to gain insight into the transmission, adaptation and evolution of the virus in Iran.

Preparation of FMD type A87/IRN inactivated vaccine by gamma irradiation and the immune response on guinea pig.

FMD is one of the most economically damaging diseases that affect livestock animals. In this study FMD Virus type A87/IRN was multiplied on BHK21 cells. The virus was titrated by TCID50 method, it was 10(7.5)/ml. The FMD virus samples were inactivated by gamma ray from 60Co source at -20°C. Safety test was done by IBRS2 monolayer cell culture method, also antigenicity of irradiated and un-irradiated virus samples were studied by Complement Fixation Test. The Dose/Survival curve for irradiated FMD Virus was drawn, the optimum dose range for inactivation of FMDV type A87/IRN and unaltered antigenicity was obtained 40-44 kGy. The inactivated virus samples by irradiation and ethyleneimine (EI) were formulated respectively as vaccine with Al(OH)3 gel and other substances. The vaccines were inoculated to Guinea pigs and the results of Serum Neutralization Test for the normal vaccine and radio-vaccine showed protective titer after 8 months. The potency test of the inactivated vaccines was done, PD50 Value of the vaccines were calculated 7.06 and 5.6 for inactivated vaccine by EI and gamma irradiation respectively.