Single-epitope T cell-based vaccine protects against SARS-CoV-2 infection in a preclinical animal model.

Currently authorized COVID-19 vaccines induce humoral and cellular responses to epitopes in the SARS-CoV-2 spike protein, though the relative roles of antibodies and T cells in protection are not well understood. To understand the role of vaccine-elicited T cell responses in protection, we established a T cell-only vaccine using a DC-targeted lentiviral vector expressing single CD8+ T cell epitopes of the viral nucleocapsid, spike, and ORF1. Immunization of angiotensin-converting enzyme 2-transgenic mice with ex vivo lentiviral vector-transduced DCs or by direct injection of the vector induced the proliferation of functional antigen-specific CD8+ T cells, resulting in a 3-log decrease in virus load upon live virus challenge that was effective against the ancestral virus and Omicron variants. The Pfizer/BNT162b2 vaccine was also protective in mice, but the antibodies elicited did not cross-react on the Omicron variants, suggesting that the protection was mediated by T cells. The studies suggest that the T cell response plays an important role in vaccine protection. The findings suggest that the incorporation of additional T cell epitopes into current vaccines would increase their effectiveness and broaden protection.

Pre-clinical models to define correlates of protection for SARS-CoV-2.

A defined immune profile that predicts protection against a pathogen-of-interest, is referred to as a correlate of protection (CoP). A validated SARS-CoV-2 CoP has yet to be defined, however considerable insights have been provided by pre-clinical vaccine and animal rechallenge studies which have fewer associated limitations than equivalent studies in human vaccinees or convalescents, respectively. This literature review focuses on the advantages of the use of animal models for the definition of CoPs, with particular attention on their application in the search for SARS-CoV-2 CoPs. We address the conditions and interventions required for the identification and validation of a CoP, which are often only made possible with the use of appropriate in vivo models.

Effect of SARS-CoV-2 mRNA-Vaccine on the Induction of Myocarditis in Different Murine Animal Models.

In the course of the SARS-CoV-2 pandemic, vaccination safety and risk factors of SARS-CoV-2 mRNA-vaccines were under consideration after case reports of vaccine-related side effects, such as myocarditis, which were mostly described in young men. However, there is almost no data on the risk and safety of vaccination, especially in patients who are already diagnosed with acute/chronic (autoimmune) myocarditis from other causes, such as viral infections, or as a side effect of medication and treatment. Thus, the risk and safety of these vaccines, in combination with other therapies that could induce myocarditis (e.g., immune checkpoint inhibitor (ICI) therapy), are still poorly assessable. Therefore, vaccine safety, with respect to worsening myocardial inflammation and myocardial function, was studied in an animal model of experimentally induced autoimmune myocarditis. Furthermore, it is known that ICI treatment (e.g., antibodies (abs) against PD-1, PD-L1, and CTLA-4, or a combination of those) plays an important role in the treatment of oncological patients. However, it is also known that treatment with ICIs can induce severe, life-threatening myocarditis in some patients. Genetically different A/J (most susceptible strain) and C57BL/6 (resistant strain) mice, with diverse susceptibilities for induction of experimental autoimmune myocarditis (EAM) at various age and gender, were vaccinated twice with SARS-CoV-2 mRNA-vaccine. In an additional A/J group, an autoimmune myocarditis was induced. In regard to ICIs, we tested the safety of SARS-CoV-2 vaccination in PD-1-/- mice alone, and in combination with CTLA-4 abs. Our results showed no adverse effects related to inflammation and heart function after mRNA-vaccination, independent of age, gender, and in different mouse strains susceptible for induction of experimental myocarditis. Moreover, there was no worsening effect on inflammation and cardiac function when EAM in susceptible mice was induced. However, in the experiments with vaccination and ICI treatment, we observed, in some mice, low elevation of cardiac troponins in sera, and low scores of myocardial inflammation. In sum, mRNA-vaccines are safe in a model of experimentally induced autoimmune myocarditis, but patients undergoing ICI therapy should be closely monitored when vaccinated.

Development of animal models for emerging infectious diseases by breaking the barrier of species susceptibility to human pathogens.

Outbreaks of emerging infectious diseases pose a serious threat to public health security, human health and economic development. After an outbreak, an animal model for an emerging infectious disease is urgently needed for studying the etiology, host immune mechanisms and pathology of the disease, evaluating the efficiency of vaccines or drugs against infection, and minimizing the time available for animal model development, which is usually hindered by the nonsusceptibility of common laboratory animals to human pathogens. Thus, we summarize the technologies and methods that induce animal susceptibility to human pathogens, which include viral receptor humanization, pathogen-targeted tissue humanization, immunodeficiency induction and screening for naturally susceptible animal species. Furthermore, the advantages and deficiencies of animal models developed using each method were analyzed, and these will guide the selection of susceptible animals and potentially reduce the time needed to develop animal models during epidemics.

Animal Models for the Study of Neurologic Manifestations Of COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the worldwide coronavirus (COVID-19) pandemic, has infected an estimated 525 million people with over 6 million deaths. Although COVID-19 is primarily a respiratory disease, an escalating number of neurologic symptoms have been reported in humans. Some neurologic symptoms, such as loss of smell or taste, are mild. However, other symptoms, such as meningoencephalitis or stroke, are potentially fatal. Along with surveys and postmortem evaluations on humans, scientists worked with several animal species to try to elucidate the causes of neurologic symptoms. Neurologic sequelae remain challenging to study due to the complexity of the nervous system and difficulties in identification and quantification of neurologic signs. We reviewed animal models used in the study of neurologic COVID-19, specifically research in mice, hamsters, ferrets, and nonhuman primates. We summarized findings on the presence and pathologic effects of SARS-CoV-2 on the nervous system. Given the need to increase understanding of COVID-19 and its effects on the nervous system, scientists must strive to obtain new information from animals to reduce mortality and morbidity with neurologic complications in humans.

Efficacy of an unmodified bivalent mRNA vaccine against SARS-CoV-2 variants in female small animal models.

Combining optimized spike (S) protein-encoding mRNA vaccines to target multiple SARS-CoV-2 variants could improve control of the COVID-19 pandemic. We compare monovalent and bivalent mRNA vaccines encoding B.1.351 (Beta) and/or B.1.617.2 (Delta) SARS-CoV-2 S-protein in a transgenic mouse and a Wistar rat model. The blended low-dose bivalent mRNA vaccine contains half the mRNA of each respective monovalent vaccine, but induces comparable neutralizing antibody titres, enrichment of lung-resident memory CD8+ T cells, antigen-specific CD4+ and CD8+ responses, and protects transgenic female mice from SARS-CoV-2 lethality. The bivalent mRNA vaccine significantly reduces viral replication in both Beta- and Delta-challenged mice. Sera from bivalent mRNA vaccine immunized female Wistar rats also contain neutralizing antibodies against the B.1.1.529 (Omicron BA.1 and BA.5) variants. These data suggest that low-dose and fit-for-purpose multivalent mRNA vaccines encoding distinct S-proteins are feasible approaches for extending the coverage of vaccines for emerging and co-circulating SARS-CoV-2 variants.

Animal Models, Zoonotic Reservoirs, and Cross-Species Transmission of Emerging Human-Infecting Coronaviruses.

Over the past three decades, coronavirus (CoV) diseases have impacted humans more than any other emerging infectious disease. The recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 (coronavirus disease 2019), has resulted in huge economic disruptions and loss of human lives. The SARS-CoV-2 genome was found to mutate more rapidly due to sustained transmission in humans and potentially animals, resulting in variants of concern (VOCs) that threaten global human health. However, the primary difficulties are filling in the current knowledge gaps in terms of the origin and modalities of emergence for these viruses. Because many CoVs threatening human health are suspected to have a zoonotic origin, identifying the animal hosts implicated in the spillover or spillback events would be beneficial for current pandemic management and to prevent future outbreaks. In this review, wesummarize the animal models, zoonotic reservoirs, and cross-species transmission of the emerging human CoVs. Finally, we comment on potential sources of SARS-CoV-2 Omicron VOCs and the new SARS-CoV-2 recombinants currently under investigation.

A generalized classification and coding system of Human Disease Animal Model Resource data with a case study to show improving database retrieval efficiency.

BACKGROUND: Currently there is no unified data classification and coding standard for the existing human disease animal model resource data worldwide. Different data classification and coding systems produce different retrieval methods. Some of these methods are inefficient and difficult to use. This research investigated the rules for the classification and coding of such data based on the Replication Methodology of Animal Models for Human Disease, the Classification and Coding Rules for Health Information Data Set (WS/T 306-2009), the Science and Technology Resource Identification (GB/T 32843-2016), the Scientific Data Management Measures (000014349/2018-00052), and The Generic Description Specification for Natural Science and Technology Resources. This research aimed to develop a classification and coding system for data obtained from human disease animal model resource based on the Internet environment to provide a standardized and unified foundation for the collection, saving, retrieval, and sharing of data from this resource. RESULTS: A complete data classification and coding table compiled in the form of letters and numbers was produced, with a classification infrastructure that expanded layer by layer according to the three dimensions (namely, system diseases, animal species, and modeling methods) and essential attributes. When necessary, it adopted the hierarchy of major, intermediate, and minor categories for certain layer and also one-to-one matched the code and classification result. CONCLUSION: Through this study, a sharing and joint construction mechanism for data from this resource can be developed by all research institutes in this field. As a case study, this research also offered technical support for constructing the database for the National Human Disease Animal Model Resource Center. The technological innovation of this paper is to derive a research oriented retrieval method, which provides technical support for the research on the current COVID-19 epidemic and on possible future epidemics.

Short-term ex vivo tissue culture models help study human lung infectionsA review.

Most studies on human lung infection have been performed using animal models, formalin or other fixed tissues, and in vitro cultures of established cell lines. However, the experimental data and results obtained from these studies may not completely represent the complicated molecular events that take place in intact human lung tissue in vivo. The newly developed ex vivo short-term tissue culture model can mimic the in vivo microenvironment of humans and allow investigations of different cell types that closely interact with each other in intact human lung tissues. Therefore, this kind of model may be a promising tool for future studies of different human lung infections, owing to its special advantages in providing more realistic events that occur in vivo. In this review, we have summarized the preliminary applications of this novel short-term ex vivo tissue culture model, with a particular emphasis on its applications in some common human lung infections.

Automatic air volume control system for ventilation of two patients using a single ventilator: a large animal model study.

The COVID-19 pandemic outbreak led to a global ventilator shortage. Hence, various strategies for using a single ventilator to support multiple patients have been considered. A device called Ventil previously validated for independent lung ventilation was used in this study to evaluate its usability for shared ventilation. We performed experiments with a total number of 16 animals. Eight pairs of pigs were ventilated by a ventilator or anesthetic machine and by Ventil for up to 27 h. In one experiment, 200 ml of saline was introduced to one subject's lungs to reduce their compliance. The experiments were analyzed in terms of arterial blood gases and respiratory parameters. In addition to the animal study, we performed a series of laboratory experiments with artificial lungs (ALs). The resistance and compliance of one AL (affected) were altered, while the tidal volume (TV) and peak pressure (Ppeak) in the second (unaffected) AL were analyzed. In addition, to assess the risk of transmission of pathogens between AL respiratory tracts, laboratory tests were performed using phantoms of virus particles. The physiological level of analyzed parameters in ventilated animals was maintained, except for CO2 tension, for which a permissive hypercapnia was indicated. Experiments did not lead to injuries in the animal's lungs except for one subject, as indicated by CT scan analysis. In laboratory experiments, changes in TV and Ppeak in the unaffected AL were less than 11%, except for 2 cases where the TV change was 20%. No cross-contamination was found in simulations of pathogen transmission. We conclude that ventilation using Ventil can be considered safe in patients undergoing deep sedation without spontaneous breathing efforts.

Characteristics of animal models for COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), the most consequential pandemic of this century, threatening human health and public safety. SARS-CoV-2 has been continuously evolving through mutation of its genome and variants of concern have emerged. The World Health Organization R&D Blueprint plan convened a range of expert groups to develop animal models for COVID-19, a core requirement for the prevention and control of SARS-CoV-2 pandemic. The animal model construction techniques developed during the SARS-CoV and MERS-CoV pandemics were rapidly deployed and applied in the establishment of COVID-19 animal models. To date, a large number of animal models for COVID-19, including mice, hamsters, minks and nonhuman primates, have been established. Infectious diseases produce unique manifestations according to the characteristics of the pathogen and modes of infection. Here we classified animal model resources around the infection route of SARS-CoV-2, and summarized the characteristics of the animal models constructed via transnasal, localized, and simulated transmission routes of infection.

Prospects of animal models and their application in studies on adaptive immunity to SARS-CoV-2.

The adaptive immune response induced by SARS-CoV-2 plays a key role in the antiviral process and can protect the body from the threat of infection for a certain period of time. However, owing to the limitations of clinical studies, the antiviral mechanisms, protective thresholds, and persistence of the immune memory of adaptive immune responses remain unclear. This review summarizes existing research models for SARS-CoV-2 and elaborates on the advantages of animal models in simulating the clinical symptoms of COVID-19 in humans. In addition, we systematically summarize the research progress on the SARS-CoV-2 adaptive immune response and the remaining key issues, as well as the application and prospects of animal models in this field. This paper provides direction for in-depth analysis of the anti-SARS-CoV-2 mechanism of the adaptive immune response and lays the foundation for the development and application of vaccines and drugs.

Squalene in oil-based adjuvant improves the immunogenicity of SARS-CoV-2 RBD and confirms safety in animal models.

COVID-19 pandemic has accelerated the development of vaccines against its etiologic agent, SARS-CoV-2. However, the emergence of new variants of the virus lead to the generation of new alternatives to improve the current sub-unit vaccines in development. In the present report, the immunogenicity of the Spike RBD of SARS-CoV-2 formulated with an oil-in-water emulsion and a water-in-oil emulsion with squalene was evaluated in mice and hamsters. The RBD protein was expressed in insect cells and purified by chromatography until >95% purity. The protein was shown to have the appropriate folding as determined by ELISA and flow cytometry binding assays to its receptor, as well as by its detection by hamster immune anti-S1 sera under non-reducing conditions. In immunization assays, although the cellular immune response elicited by both adjuvants were similar, the formulation based in water-in-oil emulsion and squalene generated an earlier humoral response as determined by ELISA. Similarly, this formulation was able to stimulate neutralizing antibodies in hamsters. The vaccine candidate was shown to be safe, as demonstrated by the histopathological analysis in lungs, liver and kidney. These results have shown the potential of this formulation vaccine to be evaluated in a challenge against SARS-CoV-2 and determine its ability to confer protection.

Little in vitro effect of remdesivir on mitochondrial respiration and monoamine oxidase activity in isolated mitochondria.

Remdesivir (RDV) is a novel antiviral drug whose mitochondrial effects are not well known. In vitro effects of RDV on the mitochondrial respiration, individual respiratory complexes, and the activity of monoamine oxidase (MAO-A and MAO-B) were measured in isolated mitochondria. At micromolar RDV concentrations, minimal or no inhibitory effects on the studied mitochondrial enzymes were found. At very high concentrations of RDV, there was partial inhibition of complex I- (IC50 675 µmol/L, residual activity 39.4 %) and complex II-linked (IC50 81.8 µmol/L, residual activity 40.7 %) respiration, without inhibition of complex IV-linked respiration, and partial inhibition both of MAO-A (IC50 26.6 µmol/L, residual activity 35.2 %) and MAO-B (IC50 89.8 µmol/L, residual activity 34.0 %) activity. Individual respiratory complexes (I, II + III, and IV) were partially inhibited at a high drug concentration. The active metabolite of RDV (GS-443902) had very little effect on mitochondrial oxygen consumption rate with residual activity of 87.0 % for complex I-linked respiration, 90.3 % for complex II-linked respiration, and with no inhibition of complex IV-linked respiration. In conclusion, measurement of the effect of RDV and its active metabolite on isolated mitochondria shows that there is very little direct effect on mitochondrial respiration occurs at therapeutic drug concentration.

Cell and Animal Models for SARS-CoV-2 Research.

During the last two years following the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, development of potent antiviral drugs and vaccines has been a global health priority. In this context, the understanding of virus pathophysiology, the identification of associated therapeutic targets, and the screening of potential effective compounds have been indispensable advancements. It was therefore of primary importance to develop experimental models that recapitulate the aspects of the human disease in the best way possible. This article reviews the information concerning available SARS-CoV-2 preclinical models during that time, including cell-based approaches and animal models. We discuss their evolution, their advantages, and drawbacks, as well as their relevance to drug effectiveness evaluation.

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.

Immunomodulation of Mesenchymal Stem Cells in Acute Lung Injury: From Preclinical Animal Models to Treatment of Severe COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been a major public health challenge worldwide. Owing to the emergence of novel viral variants, the risks of reinfections and vaccine breakthrough infections has increased considerably despite a mass of vaccination. The formation of cytokine storm, which subsequently leads to acute respiratory distress syndrome, is the major cause of mortality in patients with COVID-19. Based on results of preclinical animal models and clinical trials of acute lung injury and acute respiratory distress syndrome, the immunomodulatory, tissue repair, and antiviral properties of MSCs highlight their potential to treat COVID-19. This review article summarizes the potential mechanisms and outcomes of MSC therapy in COVID-19, along with the pathogenesis of the SARS-CoV-2 infection. The properties of MSCs and lessons from preclinical animal models of acute lung injury are mentioned ahead. Important issues related to the use of MSCs in COVID-19 are discussed finally.

Advances in Animal Models and Cutting-Edge Research in Alternatives: Proceedings of the Second International Conference on 3Rs Research and Progress, Hyderabad, 2021.

The fact that animal models fail to replicate human disease faithfully is now being widely accepted by researchers across the globe. As a result, they are exploring the use of alternatives to animal models. The time has come to refine our experimental practices, reduce the numbers and eventually replace the animals used in research with human-derived and human-relevant 3-D disease models. Oncoseek Bio-Acasta Health, which is an innovative biotechnology start-up company based in Hyderabad and Vishakhapatnam, India, organises an annual International Conference on 3Rs Research and Progress. In 2021, this conference was on 'Advances in Research Animal Models and Cutting-Edge Research in Alternatives'. This annual conference is a platform that brings together eminent scientists and researchers from various parts of the world, to share recent advances from their research in the field of alternatives to animals including new approach methodologies, and to promote practices to help refine animal experiments where alternatives are not available. This report presents the proceedings of the conference, which was held in hybrid mode (i.e. virtual and in-person) in November 2021.

De Novo-Whole Genome Assembly of the Roborovski Dwarf Hamster (Phodopus roborovskii) Genome: An Animal Model for Severe/Critical COVID-19.

The Roborovski dwarf hamster Phodopus roborovskii belongs to the Phodopus genus, one of the seven within Cricetinae subfamily. Like other rodents such as mice, rats, or ferrets, hamsters can be important animal models for a range of diseases. Whereas the Syrian hamster from the genus Mesocricetus is now widely used as a model for mild-to-moderate coronavirus disease 2019, Roborovski dwarf hamster shows a severe-to-lethal course of disease upon infection with the novel human coronavirus severe acute respiratory syndrome coronavirus 2.

Targeting intra-viral conserved nucleocapsid (N) proteins as novel vaccines against SARS-CoVs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global pandemic of the Coronavirus disease in late 2019 (COVID-19). Vaccine development efforts have predominantly been aimed at 'Extra-viral' Spike (S) protein as vaccine vehicles, but there are concerns regarding 'viral immune escape' since multiple mutations may enable the mutated virus strains to escape from immunity against S protein. The 'Intra-viral' Nucleocapsid (N-protein) is relatively conserved among mutant strains of coronaviruses during spread and evolution. Herein, we demonstrate novel vaccine candidates against SARS-CoV-2 by using the whole conserved N-protein or its fragment/peptides. Using ELISA assay, we showed that high titers of specific anti-N antibodies (IgG, IgG1, IgG2a, IgM) were maintained for a reasonably long duration (> 5 months), suggesting that N-protein is an excellent immunogen to stimulate host immune system and robust B-cell activation. We synthesized three peptides located at the conserved regions of N-protein among CoVs. One peptide showed as a good immunogen for vaccination as well. Cytokine arrays on post-vaccination mouse sera showed progressive up-regulation of various cytokines such as IFN-γ and CCL5, suggesting that TH1 associated responses are also stimulated. Furthermore, vaccinated mice exhibited an elevated memory T cells population. Here, we propose an unconventional vaccine strategy targeting the conserved N-protein as an alternative vaccine target for coronaviruses. Moreover, we generated a mouse monoclonal antibody specifically against an epitope shared between SARS-CoV and SARS-CoV-2, and we are currently developing the First-in-Class humanized anti-N-protein antibody to potentially treat patients infected by various CoVs in the future.