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.

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.

Diphtheria toxoid nanoparticles improve learning and memory impairment in animal model of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder involving memory. The present study aimed at evaluating the effects of encapsulated diphtheria toxoid (DT) on behavioral learning impairment, and XBP1 mRNA splicing in AD. METHODS: A DT-loaded nanoparticle (NP) carrier was prepared using the ionic gelation method. Sixty-three rats were divided into nine groups: (1) healthy, (2-4) sham, and (5-9) AD models: (5) AD was induced by intracerebroventricular injection of amyloid beta (Aß) 1-42. (6) The rats received a subcutaneous diphtheria vaccine only 28 days before Aß injection. (7) The rats received an intranasal diphtheria vaccine, in group 8, induced by administering empty chitosan NPs. 9) it was induced by administering chitosan NPs carrying DT. Morris water maze (MWM) test was used to examine the animals' learning and memory. Also, X-box binding protein 1 (XBP-1) mRNA gene splicing was studied in the hippocampus by reverse-transcription polymerase chain reaction (RT-PCR). RESULTS: For the first time, chitosan NPs were prepared with an average diameter size of 40 nm and the effectiveness of approximately 70% during DT encapsulation. In comparison with the healthy group, the AD models exhibited significant impairment of learning and memory (P < 0.05), while DT-administrated animals showed significant improvements in learning and memory impairment (P < 0.05). XBP-1 mRNA gene splicing was only detected in an untreated AD group, while encapsulated DT completely inhibited splicing. CONCLUSION: The therapeutic effects of DT chitosan NPs against learning and memory impairment were observed in this study, and XBP1 mRNA splicing was reported in the animal models.

Bordetella pertussis antigens encapsulated into N-trimethyl chitosan nanoparticulate systems as a novel intranasal pertussis vaccine.

The mucosal immune system serves as the first line of defense against Bordetella pertussis. Intranasal vaccination, due to its potential to induce systemic and mucosal immune responses, appears to prevent the initial adherence and colonization of the bacteria at the first point of contact. In the present study, two B. pertussis antigens, pertussis Toxoid (PTd) and Filamentous hemagglutinin (FHA), which play a very significant role in virulence and protection against pertussis, were encapsulate into N-trimethyl chitosan (TMC) nanoparticulate systems. After preparation of TMC nanoparticles (NPs), the NPs were characterized and their ability to induce efficient immune responses against B. pertussis was studied in a mouse model. Our findings showed that PTd + FHA-loaded TMC NPs have strong ability to induce IL-4, IL-17, IFN-γ, IgG, and IgA in the mouse model. Results from this study suggest that nasal administration of the PTd + FHA-loaded TMC NPs induced not only a systemic immune response but also a local mucosal response, which may improve the efficacy of pertussis prevention through respiratory tract transmission.

Preparation and nanoencapsulation of l-asparaginase II in chitosan-tripolyphosphate nanoparticles and in vitro release study.

This paper describes the production, purification, and immobilization of l-asparaginase II (ASNase II) in chitosan nanoparticles (CSNPs). ASNase II is an effective antineoplastic agent, used in the acute lymphoblastic leukemia chemotherapy. Cloned ASNase II gene (ansB) in pAED4 plasmid was transformed into Escherichia coli BL21pLysS (DE3) competent cells and expressed under optimal conditions. The lyophilized enzyme was loaded into CSNPs by ionotropic gelation method. In order to get optimal entrapment efficiency, CSNP preparation, chitosan/tripolyphosphate (CS/TPP) ratio, and protein loading were investigated. ASNase II loading into CSNPs was confirmed by Fourier transform infrared (FTIR) spectroscopy, and morphological observation was carried out by transmission electron microscopy. Three absolute CS/TPP ratios were studied. Entrapment efficiency and loading capacity increased with increasing CS and TPP concentration. The best ratio was applied for obtaining optimal ASNase II-loaded CSNPs with the highest entrapment efficiency. Size, zeta potential, entrapment efficiency, and loading capacity of the optimal ASNase II-CSNPs were 340 ± 12 nm, 21.2 ± 3 mV, 76.2% and 47.6%, respectively. The immobilized enzyme showed an increased in vitro half-life in comparison with the free enzyme. The pH and thermostability of the immobilized enzyme was comparable with the free enzyme. This study leads to a better understanding of how to prepare CSNPs, how to achieve high encapsulation efficiency for a high molecular weight protein, and how to prolong the release of protein from CSNPs. A conceptual understanding of biological responses to ASNase II-loaded CSNPs is needed for the development of novel methods of drug delivery.

Foot and Mouth Disease virus-loaded fungal chitosan nanoparticles for intranasal administration: impact of formulation on physicochemical and immunological characteristics.

Nasal vaccination is a promising, needle-free alternative route for parenteral vaccination. This study introduces a simple, scalable nasal vaccine delivery formulation for Foot and Mouth Disease virus (FMDv) using chitosan (CS) nanoparticles and assesses the potential of fungal CS for use as nanocarriers for mucosal vaccines. Fungal CS was extracted from fungal biomass and physiochemically characterized. FMDv-loaded CS nanoparticles, prepared using an ionic gelation technique, were characterized for particle size, zeta potential, morphology, loading efficiency and virus particle release. The immunogenicities of nasally applied FMDv-loaded fungal or commercial shrimp CS were compared with intraperitoneally administered fluid vaccine in guinea pigs. The nanoparticles had varied sizes (221.9-281.2 nm), positive electrical charge (+7 to +13 mV) and excellent antigen-loading capacity (93-97%). In vitro release studies revealed a biphasic virus particle release for all CS nanoparticles. Higher serum titers were developed with CS formulations than with free virus and were comparable with the titers for intraperitoneally administered fluid vaccine. Significantly higher IgA levels were found after the administration of nasal vaccine than after fluid vaccine or free virus. Overall, CS-FMDv nanoparticles stimulated humoral and mucosal immunity following intranasal administration. Fungal CS polymers were potent mucosal immunoadjuvants and showed promise as alternative sources of CS for mucosal vaccine formulations.

Cloning, expression, and immunogenicity of novel fusion protein of Mycobacterium tuberculosis based on ESAT-6 and truncated C-terminal fragment of HSP70.

Tuberculosis (TB) remains as a major public health problem worldwide. Identification and selection of immunodominant antigens of Mycobacterium tuberculosis (MTB), capable of efficiently inducing a protective immune response is the ultimate goal of TB vaccine development studies. Accordingly, this study was designed to produce a novel M. tuberculosis fusion protein consisted of MTB ESAT-6 (early secreted antigenic target-6 kDa), as a potent immunogenic protein, fused to C-terminus of MTB HSP70 (HSP70(359-610)), as an appropriate carrier and adjuvant. The constructed gene was inserted into a prokaryotic expression vector (pQE30); consequently, the recombinant fusion protein with a 6xHis-tag was successfully over expressed in Escherichia coli M15. Inclusion bodies from bacterial cell lysates were solubilized and the recombinant fusion protein was easily purified by Ni-NTA affinity chromatography under denaturing conditions followed by urea gradient dialysis. The purified and refolded protein was then applied for immunization of mice that resulted in the detection of high titers of specific antibodies, high level of IFN-γ and cell proliferation. The results of our study could confirm the capability of E6H70C fusion protein, as a potential tuberculosis vaccine candidate, for the efficient induction of specific immune responses in a mouse model. However, further investigation need to evaluate the protectivity of this recombinant protein in host model.