Preclinical efficacy and safety analysis of gamma-irradiated inactivated SARS-CoV-2 vaccine candidates.

COVID-19 outbreak caused by SARS-CoV-2 created an unprecedented health crisis since there is no vaccine for this novel virus. Therefore, SARS-CoV-2 vaccines have become crucial for reducing morbidity and mortality. In this study, in vitro and in vivo safety and efficacy analyzes of lyophilized vaccine candidates inactivated by gamma-irradiation were performed. The candidate vaccines in this study were OZG-3861 version 1 (V1), an inactivated SARS-CoV-2 virus vaccine, and SK-01 version 1 (V1), a GM-CSF adjuvant added vaccine. The candidate vaccines were applied intradermally to BALB/c mice to assess toxicity and immunogenicity. Preliminary results in vaccinated mice are reported in this study. Especially, the vaccine models containing GM-CSF caused significant antibody production with neutralization capacity in absence of the antibody-dependent enhancement feature, when considered in terms of T and B cell responses. Another important finding was that the presence of adjuvant was more important in T cell in comparison with B cell response. Vaccinated mice showed T cell response upon restimulation with whole inactivated SARS-CoV-2 or peptide pool. This study shows that the vaccines are effective and leads us to start the challenge test to investigate the gamma-irradiated inactivated vaccine candidates for infective SARS-CoV-2 virus in humanized ACE2 + mice.

SARS-CoV-2 inactivated vaccine (Vero cells) shows good safety in repeated administration toxicity test of Sprague Dawley rats.

The outbreak of COVID-19 has posed a serious threat to global public health. Vaccination may be the most effective way to prevent and control the spread of the virus. The safety of vaccines is the focus of preclinical research, and the repeated dose toxicity test is the key safety test to evaluate the vaccine before clinical trials. The purpose of this study was (i) to observe the toxicity and severity of an inactivated SARS-CoV-2 vaccine (Vero cells) in rodent Sprague Dawley rats after multiple intramuscular injections under the premise of Good Laboratory Practice principles and (ii) to provide a basis for the formulation of a clinical trial scheme. The results showed that all animals in the experimental group were in good condition, no regular changes related to the vaccine were found in the detection of various toxicological indexes, and no noticeable stimulating reaction related to the vaccine was found in the injected local tissues. The neutralizing antibodies in the low- and high-dose vaccine groups began to appear 14 days after the last administration. In the negative control group, no neutralizing antibodies were observed from the administration period to the recovery period. Therefore, the repeated administration toxicity test of the inactivated SARS-CoV-2 vaccine (Vero cells) in Sprague Dawley rats showed no obvious toxic reaction. It was preliminarily confirmed that the vaccine can stimulate production of neutralizing antibodies and is safe in Sprague Dawley rats.

Basic information for the development of a toxicity assay in inactivated bacterial vaccines.

This study dealt with the toxicity of inactivated bacteria intended for veterinary autogenous vaccines toward a suitable control assay. Two in vitro methods were used. The [3-(4, 5 -dimethylthiazol-2-yl) -2,5 -diphenyltetrazolium bromide] (MTT) test, based on the metabolic reaction of a tetrazolium salt in vital cells, was adopted on the basis of previous positive results. The Interleukin (IL)-1 beta release assay on monocyte-derived pig macrophages was carried out for comparative purposes, to evaluate the possible role of the inflammatory response. MTT and IL-1 beta responses showed a significant correlation (P < 0.05) at defined test dilutions of bacterial antigens, whereas no correlation was demonstrated using MTT responses normalized on bacterial cell concentration. Furthermore, the toxic effects shown in the MTT test were positively correlated to the extracellular protein content. On the whole, the above results could be a useful basis for the development of a toxicity assay on inactivated bacterial vaccines. Also, our data point at bacterial autolysis as a major component underlying toxicity.

Repeated dose toxicity evaluation of a cold chain-free, live, attenuated oral cholera vaccine in Sprague Dawley rats.

The repeated dose toxicity of a prototype cold chain-free, live, attenuated oral cholera vaccine containing 5 × 106 CFU/mL of the VCUSM14P strain was evaluated in Sprague Dawley (SD) rats (single dose administered daily for 30 days) to ascertain its safety for clinical use. Repeated dose toxicity studies for cholera vaccines in the literature have administered 2 or 3 fixed doses at 7, 14, 21 or 69 day intervals. The present study reports an evaluation of 30 repeated doses of cholera vaccine administered at three different concentrations (Group II (1.25 × 106 CFU), Group III (2.5 × 106 CFU) and Group IV (5 × 106 CFU)) in SD rats. The liquid vaccine was administered orally to the rats with the respective dose every day, and normal saline was administered to the control group (Group I). No significant difference (P > 0.05) was observed in the body weights and biochemical parameters of the rats after 15 and 30 repeated doses compared to those of the control group. However, compared to those of Group I, a significant increase (P < 0.05) in the organ to body weight ratios of the lungs, ureter, liver, kidney, heart and spleen was found in G-II, G-III and G-IV. In the haematological analysis, a significant increase in the WBC was observed in G-II and G-IV compared to that in G-I. The histopathological findings indicated mild to moderate degeneration in the liver, kidney, heart and spleen in the treated rats. Mild to moderate lymphocytic infiltration in the lungs was observed in the G-II and G-III rats, and severe infiltration was observed in the G-IV rats. These histopathological findings may be attributed to the 30 doses of vaccine given in daily succession without an interval. In the acute toxicity study, a single dose of vaccine up to 10 × 106 CFU did not cause any adverse effects and lethality in SD rats.

Evaluation of G2 Citric Acid-Based Dendrimer as an Adjuvant in Veterinary Rabies Vaccine.

For induction of an appropriate immune response, especially in the case of an inactivated vaccine, the use of an adjuvant is crucial. In this study, adjuvanticity effect of G2 dendrimer in veterinary rabies vaccine has been investigated. A nonlinear globular G2 dendrimer comprising citric acid and polyethylene glycol 600 (PEG-600) was synthesized and the toxicity was studied in vitro on the J774A.1 cell line. The adjuvanticity effect of the dendrimer was then investigated on rabies virus in NMRI mice as a model. Different concentrations of dendrimer were used to determine the best formulation for the survival of the mice after virus challenge. The rise of neutralizing antibody was also checked by rapid fluorescent focus inhibition test (RFFIT). The relative potency of the prepared formulation was finally calculated using standard NIH test and the results were compared (and discussed) with the commercially available rabies vaccine. The accuracy of dendrimer synthesis was confirmed using Fourier transform infrared (FT-IR), size, and zeta potential analysis. The in vitro toxicity assay revealed that no significant toxic effect is observed in cells when data are compared with the control group. The in vivo assay showed that a higher survival rate in the mice received a special formulation due to adjuvanticity effect of dendrimer, which is also confirmed by RFFIT. However, the relative potency of that formulation does not give expected results when compared with the alum-containing rabies vaccine. In the current investigation, the adjuvanticity effect of G2 dendrimer was demonstrated for the first time in rising of neutralizing antibodies against rabies virus. Our data confirm that nanoparticles can enhance immune responses in an appropriate manner. Moreover, engineered nanoparticles will enable us to develop novel potent multivalent adjuvants in vaccine technology.

Cross-protection studies with Pasteurella multocida bacterins prepared from bacteria propagated in iron-depleted medium.

Strains X-73 (serotype 1) and P-1059 (serotype 3) of Pasteurella multocida, avian origin, expressed additional membrane proteins (MPs) when grown in brain-heart infusion (BHI) broth containing the iron chelator dipyridyl and when grown in BHI broth treated with the iron chelator Chelex 100. These additional MPs were not detected when both strains were grown in BHI broth. Chickens and turkeys were vaccinated twice with inactivated oil-emulsion vaccines containing bacterial cells expressing these MPs or with vaccines containing bacterial cells grown in BHI broth. Two weeks after the final vaccination, all birds were challenged to determine whether bacterins made from P. multocida that had been propagated in conditions of iron deprivation would induce heterologous serotype immunity. The bacterins produced in medium low in iron did not consistently induce significant protection against heterologous challenge.

Development of local morphological changes after intradermal inoculation of Q-fever chemovaccine.

Development of morphological changes was studied in the site of intradermal inoculation of Q-fever chemovaccine. The chemovaccine was obtained by trichloroacetic acid extraction from Coxiella burnetii, strain Nine mile, phase I cells. The effect of two vaccine doses (0.2 mg and 1.0 mg) was compared in two groups of guinea pigs (each consisting of 21 animals). After inoculation of 1.0 mg vaccine abscesses formed in the dermis and in subcutaneous tissue along with dystrophic changes of skeletal muscles. Reparation of these lesions progressed sufficiently slowly and was not completed before 60 days after vaccine inoculation. The regeneration of skeletal muscles was of budding type. After inoculation of 0.2 mg of chemovaccine minimal lesions occurred in the subcutaneous tissue only. However, already within 48 hr this vaccine dose evoked a strong reparation response. Interesting was the finding of Kurloff cells in the haemorrhagic foci. The results suggest that the low-reactogenic chemovaccine dose of 0.2 mg might be sufficiently immunogenic after subcutaneous administration.

Quality control tests for vaccines in leprosy vaccine trial, Avadi.

All the vaccines supplied for the large scale comparative leprosy vaccine trial of ICRC bacilli, M.w, BCG plus killed M. leprae (candidate vaccines), BCG and normal saline (control arms) at CJIL Field Unit, Chennai were tested for quality control by the suppliers following the procedures laid down in the WHO protocol for killed M. leprae. Quality control for BCG was carried out at BCG vaccine laboratory as per protocol. Toxicity and sterility tests were done on all the vaccine batches/lots received. As part of the quality control, bacterial count, and protein estimation were also done. Studies showed that the bacterial content and protein concentration were comparable with the original preparations. Vaccines were free from micro-organisms, toxic materials and safe for human use. Thus the quality of all vaccine preparations was satisfactory.

[A comparative study of the immunogenicity of a bacterial mass and of the cell membranes of Vibrio cholerae grown in vivo and in vitro]. / Sravnitel'noe izuchenie immunogennosti bakterial'noi massy i kletochnykh obolochek Vibrio cholerae, vyrashchennykh in vivo i in vitro.

The comparative study of the immunogenic and protective properties of V. cholerae, grown in vivo and in vitro, and cell walls and lysates obtained from these organisms. In the mouse protection test the efficacy of preparations obtained from vibrios grown in vivo did not exceed that of the preparations obtained from V. cholerae agar cultures.

[The immunobiological properties of a corpuscular pertussis vaccine inactivated by gamma irradiation]. / Immunobiologicheskie svoistva korpuskuliarnoi kokliushnoi vaktsiny, inaktivirovannoi gamma-oblucheniem.

As a result of the high-dose gamma-irradiation (50 kGy) of corpuscular pertussis vaccine (CPV), pertussis radiovaccine (PRV) was obtained. Compared with CPV, PRV was shown to possess reduced toxicity and higher protective potency. Its adjuvant properties remained at the initial level, while its leukocytosis-promoting and histamine-sensitizing activities drastically decreased. Moreover, PRV produced less pronounced stimulating effect on the proliferation of hematopoietic stem cells, but, in contrast to CPV, was shown to capable of protecting mice from lethal radiation.

[The evaluation of the mutagenic activity of pertussis vaccinal preparations]. / Otsenka mutagennoi aktivnosti kokliushnykh vaktsinal'nykh preparatov.

Two vaccine preparations obtained from Bordetella pertussis, whole-cell vaccine constituting one of the components of adsorbed DPT vaccine and acellular vaccine, were tested for mutagenicity. The doses of the preparations covered the range 1-100 ED50. Ames' test and the metaphase analysis of marrow cells of C57BL/6J mice were used. The acellular preparation was also tested on thymectomized mice, taking into consideration chromosomal aberrations in marrow metaphases. Whole-cell and acellular pertussis vaccines did not induce mutations in Salmonella typhimurium and chromosomal aberrations in mouse marrow cells.

[Synaptonemal complexes in vaccinated mice]. / Issledovanie sinaptonemnykh kompleksov vaktsinirovannykh myshei.

Synaptonemal complexes of spermatocytes I obtained from C57BL/6j male mice treated with inactivated bacterial vaccines were spread over the hypotonic phase and then were investigated using light microscope. The slides of synaptonemal complexes of mice treated with cyclophosphamide were used as positive control. It is shown possible in principle to reveal synaptonemal complex abnormalities by means of light microscopy. These abnormalities were not more frequent in vaccinated animals than in intact ones. Cyclophosphamide at doses of 100-200 mg/kg induced synaptonemal complex damage practically in 100% of cells 96 hours after the injection.

Pharmacology and toxicology of an oral tablet whole cells inactivated cholera vaccine in Sprague Dawley rats.

Here we further investigate the pharmacological and toxicological properties of a cholera vaccine based on inactivated whole cells presented in either enteric coated (COA) or uncoated (U/C) tablet formulation from Vibrio cholerae C7258 strain. Tablets were dispersed in 2mL drinking water and administered orally to Sprague Dawley rats distributed in five groups (I COA7, II U/C7 immunized at 0, 7, 69days and III COA14, IV U/C14 immunized at 0, 14, 69days and V control group). Serum vibriocidal antibody response was measured after the administration of two doses with an interval of 7-14days. To further investigate the toxicological aspects a third dose was applied 10 weeks after the initial one. Animals were observed daily and water and food consumption was measured every other day. Periodic blood extractions were performed for hematology, biochemistry, and the titer of serum vibriocidal antibodies was determined. Anatomopathological analysis was performed at days 3 or 14 after the third dose. Results from clinical observations, as well as from water and food consumption and body weigh indicated no toxicity of the vaccine product. Meanwhile, no biological differences were found among different groups in hematological, hemo-chemistry, and anatomopathological studies. Moreover, enteric coated and uncoated tablets against human cholera were found to induce an immune response in rats.

Anti-IFN-gamma-treated mice--a model for testing safety of live Salmonella vaccines.

Salmonella enterica serovars Typhimurium and Enteritidis are facultative intracellular pathogens which cause in mice a disease similar to human typhoid fever caused by serovar Typhi. An essential phase in the infection process is bacterial replication inside cells of the liver and spleen; the rate of replication is restricted by interferon-gamma (IFN-gamma) produced early in the infection. In the present study the effect of IFN-gamma was neutralized in vivo with monoclonal antibody and the fate of bacteria in the liver and spleen in these mice compared with that in control mice after intravenous challenge. The depletion of IFN-gamma remarkably sensitized the mice to Typhimurium infection. Five different Typhimurium and Enteritidis candidate vaccine strains were tested. Only one of them, the aroA mutant SL3261, was avirulent also in anti-IFN-gamma treated mice. This finding may have important implications for the safety of live attenuated Salmonella vaccines since immunosuppression is likely to cause a state of reduced production of IFN-gamma.

Preclinical investigations of the safety, immunogenicity and efficacy of a purified, inactivated tick-borne encephalitis vaccine.

A new tick-borne encephalitis (TBE) vaccine for human use has been developed. TBE virus (TBEV) was propagated in primary chick embryo cells, inactivated by formalin and purified by continuous-flow density gradient centrifugation. The TBE vaccine was tested for innocuity, immunogenicity and protective capacity in a series of laboratory tests. The results indicated that the vaccine is outstandingly well tolerated, highly immunogenic in various laboratory animals, and induces protective immunity in mice. These data suggest that this new vaccine should be studied in clinical trials.

Opportunities to reduce the use of animals in the potency and toxicity testing of toxoid vaccines.

Alternative methods for titrating antitoxin are now available which should, in principle, permit a very large reduction in the numbers of animals required to test the potency of toxoid vaccines. More importantly they make it possible to eliminate the use of animals for the indication of excess toxicity almost completely. The full realisation of this potential is dependent upon the careful validation of all other methods and the introduction of more appropriate standard and reference preparations for titration of antisera and for the assay of DTP. It would be facilitated by a mechanism to facilitate the wider dissemination of relevant monoclonal antibodies and by a restructuring of veterinary vaccine potency tests to make full use of the additional serological information provided by the new methods.

Monophosphoryl lipid A adjuvant reverses a principal histologic parameter of formalin-inactivated respiratory syncytial virus vaccine-induced disease.

The mechanisms by which administration of a formalin-inactivated respiratory syncytial virus vaccine resulted in enhanced disease among children after they later became naturally infected with the virus remains largely undefined. After immunization and live virus challenge, the cotton rat demonstrated the histopathologic marker of the enhanced disease, polymorphonuclear leukocyte infiltration of lung alveolar spaces. We now report that immunization with formalin-inactivated vaccine formulated with the adjuvant, 3-deacylated monophosphoryl lipid A, dramatically reduces or eliminates the polymorphonuclear leukocyte infiltration within the alveoli of cotton rats post-challenge. We suggest, that this or similar adjuvants may be beneficial components of candidate non-replicating respiratory syncytial virus vaccines, whose development has been hampered by safety concerns.

Preparation and immunogenicity of an inactivated hepatitis A vaccine.

A hepatitis A vaccine was prepared by formaldehyde inactivation of purified hepatitis A virus (HAV) LSH/S strain grown on human diploid MRC-5 cells. The vaccine was devoid of residual infectivity in vitro and failed to induce in marmoset monkeys any pathological features or variations of haematological and clinical chemistry values. Infectious HAV particles were not detected in faeces and sera of the vaccinated primates by ELISA or after passages in MRC-5 cells. The immunogenicity of the vaccine was evaluated by injecting guinea-pigs with 0.8, 0.2 or 0.05 micrograms of HAV antigen adsorbed onto 0.5 and 1 mg of Al (OH)3 or 0.3 mg of AlPO4. The antibody response, measured by a competitive radioimmunoassay, was dose- and adjuvant-dependent. One injection of 0.2 micrograms of AlPO4-adsorbed HAV antigen induced seroconversion in 100% of animals and high levels of specific and neutralizing serum antibodies. A further increase of antibody titres was observed after the second and third inoculations. These results show that this vaccine formulation is safe and immunogenic in animal models, and suggest that it should be evaluated further by human clinical studies.

Enhanced pulmonary immunopathology following neonatal priming with formalin-inactivated respiratory syncytial virus but not with the BBG2NA vaccine candidate.

Prevention of respiratory syncytial virus (RSV) disease will implicate neonatal priming. However, neonatal antigen exposure frequently results into Th2-like responses, some of which are critical for formalin-inactivated RSV (FI-RSV)-associated lung immunopathology. Neonatal immunization of mice may thus represent a more stringent model of RSV-enhanced pathology than adults. Indeed, after RSV challenge, lung cell infiltration, lymphocyte activation, and eosinophilia were higher following neonatal compared with adult FI-RSV priming of BALB/c mice. Unexpectedly, similar findings were obtained with Al(OH)(3)-adsorbed live RSV. In contrast, neonatal priming with BBG2Na, a recombinant RSV subunit vaccine candidate, formulated in either Al(OH)(3) or TiterMax (a Th1-driving adjuvant) resulted in predominant Th2- or Th1-like responses, respectively, but never elicited lung immunopathology post-challenge. Importantly, our data emphasize that the induction of Th2-like responses by RSV subunit vaccines do not necessarily imply lung immunopathology.