Human body constitutes unique biological system containing specific fluid mechanics and biomechanics. Traditional cell culture techniques of 2D and 3D do not recapitulate these specific natures of the human system. In addition, they lack the spatiotemporal conditions of representing the cells. Moreover, they do not enable the study of cell-cell interactions in multiple cell culture platforms. Therefore, establishing biological system of dynamic cell culture was of great interest. Organs on chips systems were fabricated proving their concept to mimic specific organs functions. Therefore, it paves the way for validating new drugs and establishes mechanisms of emerging diseases. It has played a key role in validating suitable vaccines for Coronavirus disease (COVID-19). Herein, the concept of organs on chips, fabrication methodology and their applications are discussed.

After the COVID-19 pandemic, nonhuman primate (NHP) models, which are necessary for the rapid development of vaccines and new medical therapies, have become important in studies on infectious diseases because of their genetic, metabolic, and immunological similarities to humans. Our group has long been using NHP models in studies on infectious diseases including H1N1 influenza pandemic and COVID-19. Despite limitations such as the limited number of animals and the husbandry requirements, NHP models have contributed to the prediction of the pathogenicity of emerging viruses and the evaluation of the efficacy of vaccines and therapeutics due to the similarity of NHP models to humans before starting clinical trials to select good candidates of vaccines and drugs. In this review, the findings obtained in NHP infectious disease models of influenza and COVID-19 are summarized to clarify the benefits of NHP models for studies on infectious diseases. We believe that this review will support future research in exploring new perspectives for the development of vaccines and therapies targeting influenza, COVID-19, and infectious diseases in future pandemics.

Acute respiratory viral infections (ARVI) are one of the most significant infections affecting the breeding of monkeys, especially among imported and captive primates. Respiratory diseases are also an important cause of morbidity and mortality in wild populations, and most of these infections can affect humans. Many anthropoid species, including apes, are susceptible to ARVI. Outbreaks of spontaneous respiratory infections have been described in many zoos and primatological centers around the world. Moreover, the study of spontaneous and experimental infection in laboratory primates provides an invaluable source of information on the biology and pathogenesis of ARVI and remains an indispensable tool for testing vaccines and drugs. The aim of this literature review was to summarize and analyze published data on the circulation of ARVI causative agents (parainfluenza viruses, adenoviruses, respiratory syncytial virus, influenza viruses, rhinoviruses, coronaviruses, metapneumoviruses, bocaviruses) among wild and captive primates, as well as the results of experimental modeling these infections in monkeys.

Antibody-dependent enhancement (ADE) is an immunological paradox whereby sensitization following a primary viral infection results in the subsequent enhancement of a similar secondary infection. This idiosyncratic immune response has been established in dengue virus infections, driven by four antigenically related serotypes co-circulating in endemic regions. Several coronaviruses exhibit antibody-mediated mechanisms of viral entry, which has led to speculation of an ADE capacity for SARS-CoV-2, though in vivo and epidemiological evidence do not currently support this phenomenon. Three distinct antibody-dependent mechanisms for SARS-CoV-2 entry have recently been demonstrated: 1. FcR-dependent, 2. ACE2-FcR-interdependent, and 3. FcR-independent. These mechanisms of viral entry may be dependent on SARS-CoV-2 antibody specificity; antibodies targeting the receptor binding domain (RBD) typically result in Fc-dependent and ACE2-FcR-interdependent entry, whereas antibodies targeting the N-terminal domain can induce a conformational change to the RBD that optimizes ACE2-receptor binding domain interactions independent of Fc receptors. Whether these antibody-dependent entry mechanisms of SARS-CoV-2 result in the generation of infectious progenies and enhancement of infection has not been robustly demonstrated. Furthermore, ADE of SARS-CoV-2 mediated by antigenic seniority remains a theoretical concern, as no evidence suggests that SARS-CoV-2 imprinting blunts a subsequent immune response, contributing to severe COVID-19 disease.

Post-acute sequelae of COVID-19 (PASC) are a diverse set of symptoms and syndromes driven by dysfunction of multiple organ systems that can persist for years and negatively impact the quality of life for millions of individuals. We currently lack specific therapeutics for patients with PASC, due in part to an incomplete understanding of its pathogenesis, especially for non-pulmonary sequelae. Here, we discuss three animal models that have been utilized to investigate PASC: non-human primates (NHPs), hamsters, and mice. We focus on neurological, gastrointestinal, and cardiovascular PASC and highlight advances in mechanistic insight that have been made using these animal models, as well as discussing the sequelae that warrant continued and intensive research.

Given the low levels of coronavirus disease 2019 (COVID-19) vaccine coverage in sub-Saharan Africa (sSA), despite high levels of natural severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) exposures, strategies for extending the breadth and longevity of naturally acquired immunity are warranted. Designing such strategies will require a good understanding of naturally acquired immunity.

We measured whole-spike immunoglobulin G (IgG) and spike-receptor binding domain (RBD) total immunoglobulins (Igs) on 585 plasma samples collected longitudinally over five successive time points within six months of COVID-19 diagnosis in 309 COVID-19 patients. We measured antibody-neutralising potency against the wild-type (Wuhan) SARS-CoV-2 pseudovirus in a subset of 51 patients over three successive time points. Binding and neutralising antibody levels and potencies were then tested for correlations with COVID-19 severities.

Rates of seroconversion increased from day 0 (day of PCR testing) to day 180 (six months) (63.6% to 100 %) and (69.3 % to 97%) for anti-spike-IgG and anti-spike-RBD binding Igs, respectively. Levels of these binding antibodies peaked at day 28 (p<0.01) and were subsequently maintained for six months without significant decay (p>0.99). Similarly, antibody-neutralising potencies peaked at day 28 (p<0.01) but declined by three-fold, six months after COVID-19 diagnosis (p<0.01). Binding antibody levels were highly correlated with neutralising antibody potencies at all the time points analysed (r>0.60, p<0.01). Levels and potencies of binding and neutralising antibodies increased with disease severity.

Most COVID-19 patients generated SARS-CoV-2 specific binding antibodies that remained stable in the first six months of infection. However, the respective neutralising antibodies decayed three-fold by month-six of COVID-19 diagnosis suggesting that they are short-lived, consistent with what has been observed elsewhere in the world. Thus, regular vaccination boosters are required to sustain the high levels of anti-SARS-CoV-2 naturally acquired neutralising antibody potencies in our population.

An unprecedented breadth of longitudinal viral and multi-scale immunological data has been gathered during SARS-CoV-2 infection. However, due to the high complexity, non-linearity, multi-dimensionality, mixed anatomic sampling, and possible autocorrelation of available immune data, it is challenging to identify the components of the innate and adaptive immune response that drive viral elimination. Novel mathematical models and analytical approaches are required to synthesize contemporaneously gathered cytokine, transcriptomic, flow cytometry, antibody response, and viral load data into a coherent story of viral control, and ultimately to discriminate drivers of mild versus severe infection.

We investigated a dataset describing innate, SARS-CoV-2 specific T cell, and antibody responses in the lung during early and late stages of infection in immunologically naïve rhesus macaques. We used multi-model inference and ensemble modeling approaches from ecology and weather forecasting to compare and combine various competing models.

Model outputs suggest that the innate immune response plays a crucial role in controlling early infection, while SARS-CoV-2 specific CD4+ T cells correspond to later viral elimination, and anti-spike IgG antibodies do not impact viral dynamics. Among the numerous genes potentially contributing to the innate response, we identified IFI27 as most closely linked to viral load decline. A 90% knockdown of the innate response from our validated model resulted in a ~10-fold increase in peak viral load during infection. Our approach provides a novel methodological framework for future analyses of similar complex, non-linear multi-component immunologic data sets.

Respiratory viruses ravage the world and seriously threaten people's health. Despite intense research efforts, the immune mechanism underlying respiratory virus-induced acute lung injury (ALI) and pulmonary fibrosis (PF) has not been fully elucidated. Here, the cascade of monocyte-derived macrophages to influenza A virus infection is monitored on an optimized human alveolus chip to reveal the role of macrophages in the development of ALI and PF. We find that viral infection causes damage to the alveolar air-liquid barrier and the release of inflammatory cytokines, which induce the M0 macrophages to gather and polarize to the M1 phenotype at the damaged site through recruitment, adhesion, migration, and activation, leading to ALI. Afterward, M1 macrophages polarize into the M2 phenotype, and then transform into myofibroblasts, followed by enhanced secretion of various anti-inflammatory cytokines and profibrotic cytokines, to promote PF. Our study provides an insight into the pathogenesis of virus-induced ALI and PF, which will assist in the development of therapeutic strategies and drugs for treating influenza and other respiratory virus infections.

Influenza and SARS-CoV-2 viruses are two highly variable pathogens. We have developed a candidate bivalent live vaccine based on the strain of licensed A/Leningrad/17-based cold-adapted live attenuated influenza vaccine (LAIV) of H3N2 subtype, which expressed SARS-CoV-2 immunogenic T-cell epitopes. A cassette encoding fragments of S and N proteins of SARS-CoV-2 was inserted into the influenza NA gene using the P2A autocleavage site. In this study, we present the results of preclinical evaluation of the developed bivalent vaccine in a non-human primate model.

Rhesus macaques (Macaca mulatta) (n = 3 per group) were immunized intranasally with 7.5 lg EID50 of the LAIV/CoV-2 bivalent vaccine, a control non-modified H3N2 LAIV or a placebo (chorioallantoic fluid) using a sprayer device, twice, with a 28-day interval. The blood samples were collected at days 0, 3, 28 and 35 for hematological and biochemical assessment. Safety was also assessed by monitoring body weight, body temperature and clinical signs of the disease. Immune responses to influenza virus were assessed both by determining serum antibody titers in hemagglutination inhibition assay, microneutralization assay and IgG ELISA. T-cell responses were measured both to influenza and SARS-CoV-2 antigens using ELISPOT and flow cytometry. Three weeks after the second immunization, animals were challenged with 105 PFU of Delta SARS-CoV-2. The body temperature, weight and challenge virus shedding were monitored for 5 days post-challenge. In addition, virus titers in various organs and histopathology were evaluated on day 6 after SARS-CoV-2 infection.

There was no toxic effect of the immunizations on the hematological and coagulation hemostasis of animals. No difference in the dynamics of the average weight and thermometry results were found between the groups of animals. Both LAIV and LAIV/CoV-2 variants poorly replicated in the upper respiratory tract of rhesus macaques. Nevertheless, despite this low level of virus shedding, influenza-specific serum IgG responses were detected in the group of monkeys immunized with the LAIV/CoV-2 bivalent but not in the LAIV group. Furthermore, T-cell responses to both influenza and SARS-CoV-2 viruses were detected in the LAIV/CoV-2 vaccine group only. The animals were generally resistant to SARS-CoV-2 challenge, with minimal virus shedding in the placebo and LAIV groups. Histopathological changes in vaccinated animals were decreased compared to the PBS group, suggesting a protective effect of the chimeric vaccine candidate.

The candidate bivalent vaccine was safe and immunogenic for non-human primates and warrants its further evaluation in clinical trials.

To adequately prepare for potential hazards caused by emerging and reemerging infectious diseases, the WHO has issued a list of high-priority pathogens that are likely to cause future outbreaks and for which research and development (R&D) efforts are dedicated, known as paramount R&D blueprints. Within R&D efforts, the goal is to obtain effective prophylactic and therapeutic approaches, which depends on a comprehensive knowledge of the etiology, epidemiology, and pathogenesis of these diseases. In this process, the accessibility of animal models is a priority bottleneck because it plays a key role in bridging the gap between in-depth understanding and control efforts for infectious diseases. Here, we reviewed preclinical animal models for high priority disease in terms of their ability to simulate human infections, including both natural susceptibility models, artificially engineered models, and surrogate models. In addition, we have thoroughly reviewed the current landscape of vaccines, antibodies, and small molecule drugs, particularly hopeful candidates in the advanced stages of these infectious diseases. More importantly, focusing on global trends and novel technologies, several aspects of the prevention and control of infectious disease were discussed in detail, including but not limited to gaps in currently available animal models and medical responses, better immune correlates of protection established in animal models and humans, further understanding of disease mechanisms, and the role of artificial intelligence in guiding or supplementing the development of animal models, vaccines, and drugs. Overall, this review described pioneering approaches and sophisticated techniques involved in the study of the epidemiology, pathogenesis, prevention, and clinical theatment of WHO high-priority pathogens and proposed potential directions. Technological advances in these aspects would consolidate the line of defense, thus ensuring a timely response to WHO high priority pathogens.

Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), as a typical tumor marker, has been found to exert immunomodulatory effects in many diseases. We previously reported the clinical and molecular evidences supporting that SARS-Cov-2 infected the gastrointestinal (GI) tract and found a reduction of CEACAM5 in COVID-19 patients' feces which associated with gut dysbiosis. Yet the role of CEACAM5 in GI infection is ill-defined.

Mice models were established through intraperitoneally injecting with recombinant viral spike-Fc to mimic the intestinal inflammation. We collected duodenum, jejunum, ileum and colon samples after 6h, 2 days, 4 days and 7 days of spike-Fc or control-Fc injection to perform proteomic analysis. Blood was collected from healthy donors and peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation, then CD4+ T cells were isolated with magnetic beads and co-cultured with Caco-2 cells.

In addition to intestinal CEACAM5, the expression of tight junction and the percent of CD4+ T lymphocytes were significantly decreased in spike-Fc group compared to control (p < 0.05), accompanied with increased level of inflammatory factors. The KEGG analysis revealed differentially expressed proteins were mainly enriched in the coronavirus disease (COVID-19), tight junction, focal adhesion, adherens junction and PI3K-Akt signaling pathway. Protein-protein interaction (PPI) network analysis identified the interaction between CEACAM5 and Galectin-9 that was also verified by molecular docking and co-IP assay. We further confirmed a reduction of CEACAM5 in SARS-CoV-2 spike stimulated enterocytes could promote the expression of Galectin-9 protein in CD4+T cells. Then it gave rise to the increasing release of inflammatory factors and increased apoptosis of CD4+T cells by inhibition of PI3K/AKT/mTOR pathway. Ultimately intestinal barrier dysfunction happened.

Our results indicated that CEACAM5 overexpression and Galectin-9 knockdown played a protective role in intestinal barrier injury upon spike-Fc stimulation. Collectively, our findings identified firstly that SARS-CoV-2 spike induced intestinal barrier dysfunction through the interaction between CEACAM5 and Galectin-9. The result provides potential therapeutic targets in intestinal barrier dysfunction for treating severe COVID patients.

Evidence suggests associations between COVID-19 patients or vaccines and glycometabolic dysfunction and an even higher risk of the occurrence of diabetes. Herein, we retrospectively analyzed pancreatic lesions in autopsy tissues from 67 SARS-CoV-2 infected non-human primates (NHPs) models and 121 vaccinated and infected NHPs from 2020 to 2023 and COVID-19 patients. Multi-label immunofluorescence revealed direct infection of both exocrine and endocrine pancreatic cells by the virus in NHPs and humans. Minor and limited phenotypic and histopathological changes were observed in adult models. Systemic proteomics and metabolomics results indicated metabolic disorders, mainly enriched in insulin resistance pathways, in infected adult NHPs, along with elevated fasting C-peptide and C-peptide/glucose ratio levels. Furthermore, in elder COVID-19 NHPs, SARS-CoV-2 infection causes loss of beta (β) cells and lower expressed-insulin in situ characterized by islet amyloidosis and necrosis, activation of α-SMA and aggravated fibrosis consisting of lower collagen in serum, an increase of pancreatic inflammation and stress markers, ICAM-1 and G3BP1, along with more severe glycometabolic dysfunction. In contrast, vaccination maintained glucose homeostasis by activating insulin receptor α and insulin receptor β. Overall, the cumulative risk of diabetes post-COVID-19 is closely tied to age, suggesting more attention should be paid to blood sugar management in elderly COVID-19 patients.

The COVID-19 pandemic, which was caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a worldwide health crisis due to its transmissibility. SARS-CoV-2 infection results in severe respiratory illness and can lead to significant complications in affected individuals. These complications encompass symptoms such as coughing, respiratory distress, fever, infectious shock, acute respiratory distress syndrome (ARDS), and even multiple-organ failure. Animal models serve as crucial tools for investigating pathogenic mechanisms, immune responses, immune escape mechanisms, antiviral drug development, and vaccines against SARS-CoV-2. Currently, various animal models for SARS-CoV-2 infection, such as nonhuman primates (NHPs), ferrets, hamsters, and many different mouse models, have been developed. Each model possesses distinctive features and applications. In this review, we elucidate the immune response elicited by SARS-CoV-2 infection in patients and provide an overview of the characteristics of various animal models mainly used for SARS-CoV-2 infection, as well as the corresponding immune responses and applications of these models. A comparative analysis of transcriptomic alterations in the lungs from different animal models revealed that the K18-hACE2 and mouse-adapted virus mouse models exhibited the highest similarity with the deceased COVID-19 patients. Finally, we highlighted the current gaps in related research between animal model studies and clinical investigations, underscoring lingering scientific questions that demand further clarification.

Considerable research has been done in investigating SARS-CoV-2 infection, its characteristics, and host immune response. However, debate is still ongoing over the emergence of post-acute sequelae of SARS-CoV-2 infection (PASC). A multitude of long-lasting symptoms have been reported several weeks after the primary acute SARS-CoV-2 infection that resemble several other viral infections. Thousands of research articles have described various post-COVID-19 conditions. Yet, the evidence around these ongoing health problems, the reasons behind them, and their molecular underpinnings are scarce. These persistent symptoms are also known as long COVID-19. The persistence of SARS-CoV-2 and/or its components in host tissues can lead to long COVID. For example, the presence of viral nucleocapsid protein and RNA was detected in the skin, appendix, and breast tissues of some long COVID patients. The persistence of viral RNA was reported in multiple anatomic sites, including non-respiratory tissues such as the adrenal gland, ocular tissue, small intestine, lymph nodes, myocardium, and sciatic nerve. Distinctive viral spike sequence variants were also found in non-respiratory tissues. Interestingly, prolonged detection of viral subgenomic RNA was observed across all tissues, sometimes in multiple tissues of the same patient, which likely reflects recent but defective viral replication. Moreover, the persistence of SARS-CoV-2 RNA was noticed throughout the brain at autopsy, as late as 230 days following symptom onset among unvaccinated patients who died of severe infection. Here, we review the persistence of SARS-CoV-2 and its components as an intrinsic factor behind long COVID. We also highlight the immunological consequences of this viral persistence.

Coronavirus infections have been known to cause disease in animals since as early as the 1920s. However, only seven coronaviruses capable of causing human disease have been identified thus far. These Human Coronaviruses (HCoVs) include the causes of the common cold, but more recent coronaviruses that have emerged (i.e. SARS-CoV, MERS-CoV and SARS-CoV-2) are associated with much greater morbidity and mortality. HCoVs have been relatively under-studied compared to other common respiratory infections, as historically they have presented with mild symptoms. This has led to a relatively limited understanding of their animal reservoirs, transmission and determinants of immune protection. To address this, human infection challenge studies with HCoVs have been performed that enable a detailed clinical and immunological analysis of the host response at specific time points under controlled conditions with standardised viral inocula. Until recently, all such human challenge studies were conducted with common cold HCoVs, with the study of SARS-CoV and MERS-CoV unacceptable due to their greater pathogenicity. However, with the emergence of SARS-CoV-2 and the COVID-19 pandemic during which severe outcomes in young healthy adults have been rare, human challenge studies with SARS-CoV-2 are now being developed. Two SARS-CoV-2 human challenge studies in the UK studying individuals with and without pre-existing immunity are underway. As well as providing a platform for testing of antivirals and vaccines, such studies will be critical for understanding the factors associated with susceptibility to SARS-CoV-2 infection and thus developing improved strategies to tackle the current as well as future HCoV pandemics. Here, we summarise the major questions about protection and pathogenesis in HCoV infection that human infection challenge studies have attempted to answer historically, as well as the knowledge gaps that aim to be addressed with contemporary models.

Hybrid immunity (vaccination + natural infection) to SARS-CoV-2 provides superior protection to re-infection. We performed immune profiling studies during breakthrough infections in mRNA-vaccinated hamsters to evaluate hybrid immunity induction. The mRNA vaccine, BNT162b2, was dosed to induce binding antibody titers against ancestral spike, but inefficient serum virus neutralization of ancestral SARS-CoV-2 or variants of concern (VoCs). Vaccination reduced morbidity and controlled lung virus titers for ancestral virus and Alpha but allowed breakthrough infections in Beta, Delta and Mu-challenged hamsters. Vaccination primed for T cell responses that were boosted by infection. Infection back-boosted neutralizing antibody responses against ancestral virus and VoCs. Hybrid immunity resulted in more cross-reactive sera, reflected by smaller antigenic cartography distances. Transcriptomics post-infection reflects both vaccination status and disease course and suggests a role for interstitial macrophages in vaccine-mediated protection. Therefore, protection by vaccination, even in the absence of high titers of neutralizing antibodies in the serum, correlates with recall of broadly reactive B- and T-cell responses.

SARS-CoV-2 ribonucleic acid (RNA) has been detected in feces, but RNA is not infectious. This systematic review aims to answer if fecal SARS-CoV-2 is experimentally infectious and if evidence of human fecal-oral SARS-CoV-2 transmission exists.

On September 19, 2022, we searched PubMed, Embase, Web of Science, medRxiv, and bioRxiv. Biomedical studies inoculating SARS-CoV-2 from feces, rectal, or anal swabs in cells, tissue, organoids, or animals were included. Epidemiological studies of groups differing in exposure to fecal SARS-CoV-2 were included. Risk of bias was assessed using standardized tools. Results were summarized by vote counting, tabulation, and a harvest plot. PROSPERO registration no. CRD42020221719.

A total of 4,874 studies were screened; 26 studies were included; and 13 out of 23 biomedical studies (56.5%) succeeded in infection. Two (66.7%) epidemiological studies found limited evidence suggesting fecal-oral transmission. All studies had concerns about the risk of bias.

It is possible to experimentally infect cell cultures, organoids, and animals with fecal SARS-CoV-2. No strong epidemiologic evidence was found to support human fecal-oral transmission. We advise future research to study fecal infectivity at different time points during infection, apply appropriate controls, use in vivo models, and study fecal exposure as a risk factor of transmission in human populations.

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.

Each year, SARS-CoV-2 is infecting an increasingly unprecedented number of species. In the present article, we combine mammalian phylogeny with the genetic characteristics of isolates found in mammals to elaborate on the host-range potential of SARS-CoV-2. Infections in nonhuman mammals mirror those of contemporary viral strains circulating in humans, although, in certain species, extensive viral circulation has led to unique genetic signatures. As in other recent studies, we found that the conservation of the ACE2 receptor cannot be considered the sole major determinant of susceptibility. However, we are able to identify major clades and families as candidates for increased surveillance. On the basis of our findings, we argue that the use of the term panzootic could be a more appropriate term than pandemic to describe the ongoing scenario. This term better captures the magnitude of the SARS-CoV-2 host range and would hopefully inspire inclusive policy actions, including systematic screenings, that could better support the management of this worldwide event.

COVID-19 (Coronavirus Disease 2019) is an infectious disease caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and has globally infected 768 million people and caused over 6 million deaths. COVID-19 primarily affects the respiratory system but increasing reports of neurologic symptoms associated with COVID-19 have been reported in the literature. The exact mechanism behind COVID-19 neurologic pathophysiology remains poorly understood due to difficulty quantifying clinical neurologic symptoms in humans and correlating them to findings in human post-mortem samples and animal models. Thus, robust preclinical experimental models for COVID-19 neurologic manifestations are urgently needed. Here, we review recent advances in in vitro, in vivo, and other models and technologies for studying COVID-19 including primary cell cultures, pluripotent stem cell-derived neurons and organoids, rodents, nonhuman primates, 3D bioprinting, artificial intelligence, and multiomics. We specifically focus our discussion on the contribution, recent advancements, and limitations these preclinical models have on furthering our understanding of COVID-19's neuropathic physiology. We also discuss these models' roles in the screening and development of therapeutics, vaccines, antiviral drugs, and herbal medicine, and on future opportunities for COVID-19 neurologic research and clinical management.

SARS-CoV-2 variants and seasonal coronaviruses continue to cause disease and coronaviruses in the animal reservoir pose a constant spillover threat. Importantly, understanding of how previous infection may influence future exposures, especially in the context of seasonal coronaviruses and SARS-CoV-2 variants, is still limited. Here we adopted a step-wise experimental approach to examine the primary immune response and subsequent immune recall toward antigenically distinct coronaviruses using male Syrian hamsters. Hamsters were initially inoculated with seasonal coronaviruses (HCoV-NL63, HCoV-229E, or HCoV-OC43), or SARS-CoV-2 pango B lineage virus, then challenged with SARS-CoV-2 pango B lineage virus, or SARS-CoV-2 variants Beta or Omicron. Although infection with seasonal coronaviruses offered little protection against SARS-CoV-2 challenge, HCoV-NL63-infected animals had an increase of the previously elicited HCoV-NL63-specific neutralizing antibodies during challenge with SARS-CoV-2. On the other hand, primary infection with HCoV-OC43 induced distinct T cell gene signatures. Gene expression profiling indicated interferon responses and germinal center reactions to be induced during more similar primary infection-challenge combinations while signatures of increased inflammation as well as suppression of the antiviral response were observed following antigenically distant viral challenges. This work characterizes and analyzes seasonal coronaviruses effect on SARS-CoV-2 secondary infection and the findings are important for pan-coronavirus vaccine design.

There is an imperative choice to develop a secure feasible strategy to address evasion dynamics of refractory tumors and SARS-CoV-2-variants, while stem cell-based protocol may be more reliable as its unique ability for resetting multifunctional immunity to address progressive tumor and the constantly-evolving virus. In this study, spheroid-embryonoid stem cells from mature somatic cells were engineered as multifunctional biologics (3D-E/BSC) and inoculated in senile rhesus to identify secure potential against immune-evasion from viral-variants. Meanwhile, a cohort of eligible patients with stage IV NSCLC were approved for phase I clinical trials. Subsequently, long-lasting security and efficacy were validated by primate and clinical trials (p < 0.01) in that it could not only stimulate serological immunity, but also reset core immunity for hosts to address variant evasion after 3D-E/BSC withdrawal. Particularly, illustrated by single-cell evolving trajectory, 3D-E/BSC had securely reset senile thymus of aging hosts to remodel core immunity by rearranging naive rhythm to evolve TRGC2+/JCHAIN+NKT clusters to abolish tumoral and viral evasion dynamics with path-feedbacks of NSCLC and COVID-19 simultaneously activated, leading to continuous blockade of breakthrough infection of viral-mutants and long-term survival in one-third of terminal patients without adjuvant required. Our study may pioneer a practical multifunctional strategy to eliminate evasion of SARS-CoV-2 variants and refractory NSCLC so as for victims to restart a new life-equation.

Since its emergence in late 2019, coronavirus disease 2019 (COVID-19) has caused millions of deaths and socioeconomic losses. Although vaccination significantly reduced disease mortality, it has been shown that protection wanes over time, and that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) may escape vaccine-derived immunity. Therefore, serological studies are necessary to assess protection in the population and guide vaccine regimens. A common measure of protective immunity is the presence of neutralizing antibodies (nAbs). However, the gold standard for measuring nAbs (plaque reduction neutralization test, or PRNT) is laborious and time-consuming, limiting its large-scale applicability. We developed a high-throughput fluorescence reduction neutralization assay (FRNA) to detect SARS-CoV-2 nAbs. Because the assay relies on immunostaining, we developed and characterized monoclonal antibodies (mAbs) to lower costs and reduce the assay's vulnerability to reagent shortages. Using samples of individuals vaccinated with COVID-19 and unvaccinated/pre-pandemic samples, we showed that FRNA results using commercial and in-house mAbs strongly correlated with those of the PRNT method while providing results in 70% less time. In addition to providing a fast, reliable, and high-throughput alternative for measuring nAbs, the FRNA can be easily customized to assess SARS-CoV-2 VOCs. Additionally, the mAb we produced was able to detect SARS-CoV-2 in pulmonary tissues by immunohistochemistry assays.

The novel coronavirus disease (COVID-19) continues to endanger human health, and its therapeutic drugs are under intensive research and development. Identifying the efficacy and toxicity of drugs in animal models is helpful for further screening of effective medications, which is also a prerequisite for drugs to enter clinical trials. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) invades host cells mainly by the S protein on its surface. After the SARS-CoV-2 RNA genome is injected into the cells, M protein will help assemble and release new viruses. RdRp is crucial for virus replication, assembly, and release of new virus particles. This review analyzes and discusses 26 anti-SARS-CoV-2 drugs based on their mechanism of action, effectiveness and safety in different animal models. We propose five drugs to be the most promising to enter the next stage of clinical trial research, thus providing a reference for future drug development.

Respiratory infections cause tremendous morbidity and mortality worldwide. Amongst these diseases, tuberculosis (TB), a bacterial illness caused by Mycobacterium tuberculosis which often affects the lung, and coronavirus disease 2019 (COVID-19) caused by the Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2), stand out as major drivers of epidemics of global concern. Despite their unrelated etiology and distinct pathology, these infections affect the same vital organ and share immunopathogenesis traits and an imperative demand to model the diseases at their various progression stages and localizations. Due to the clinical spectrum and heterogeneity of both diseases experimental infections were pursued in a variety of animal models. We summarize mammalian models employed in TB and COVID-19 experimental investigations, highlighting the diversity of rodent models and species peculiarities for each infection. We discuss the utility of non-human primates for translational research and emphasize on the benefits of non-conventional experimental models such as livestock. We epitomize advances facilitated by animal models with regard to understanding disease pathophysiology and immune responses. Finally, we highlight research areas necessitating optimized models and advocate that research of pulmonary infectious diseases could benefit from cross-fertilization between studies of apparently unrelated diseases, such as TB and COVID-19.

A key question in the coronavirus disease 2019 (COVID-19) pandemic is the duration of specific T cell responses against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) post primary infection, which is difficult to address due to the large-scale COVID-19 vaccination and re-exposure to the virus. Here, we conducted an analysis of the long-term SARS-CoV-2-specific T cell responses in a unique cohort of convalescent individuals (CIs) that were among the first to be infected worldwide and without any possible antigen re-exposure since then. The magnitude and breadth of SARS-CoV-2-specific T cell responses correlated inversely with the time that had elapsed from disease onset and the age of those CIs. The mean magnitude of SARS-CoV-2-specific CD4 and CD8 T cell responses decreased about 82% and 76%, respectively, over the time period of ten months after infection. Accordingly, the longitudinal analysis also demonstrated that SARS-CoV-2-specific T cell responses waned significantly in 75% of CIs during the follow-up. Collectively, we provide a comprehensive characterization of the long-term memory T cell response in CIs, suggesting that robust SARS-CoV-2-specific T cell immunity post primary infection may be less durable than previously expected.

This study reports on the successful conjugation of SARS-CoV-2 S1 spike protein fragments with gold nanoparticles (AuNPs) that were synthesised with Ultrasonic Spray Pyrolysis (USP). This method enables the continuous synthesis of AuNPs with a high degree of purity, round shapes, and the formation of a surface that allows various modifications. The conjugation mechanism of USP synthesized AuNPs with SARS-CoV-2 S1 spike protein fragments was investigated. A gel electrophoresis experiment confirmed the successful conjugation of AuNPs with SARS-CoV-2 S1 fragments indirectly. X-ray Photoelectron Spectroscopy (XPS) analysis confirmed the presence of characteristic O1s and N1s peaks, which indicated that specific binding between AuNPs and SARS-CoV-2 S1 spike protein fragments takes place via a peptide bond formed with the citrate stabiliser. This bond is coordinated to the AuNP's surface and the N-terminals of the protein, with the conjugate displaying the expected response within a prototype LFIA test. This study will help in better understanding the behaviour of AuNPs synthesised with USP and their potential use as sensors in colorimetric or electrochemical sensors and LFIA tests.

COVID-19 outbreak was first reported in 2019, Wuhan, China. The spillover of the disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), to a wide range of pet, zoo, wild, and farm animals has emphasized potential zoonotic and reverse zoonotic viral transmission. Furthermore, it has evoked inquiries about susceptibility of different animal species to SARS-CoV-2 infection and role of these animals as viral reservoirs. Therefore, studying susceptible and non-susceptible hosts for SARS-CoV-2 infection could give a better understanding for the virus and will help in preventing further outbreaks. Here, we review structural aspects of SARS-CoV-2 spike protein, the effect of the different mutations observed in the spike protein, and the impact of ACE2 receptor variations in different animal hosts on inter-species transmission. Moreover, the SARS-CoV-2 spillover chain was reviewed. Combination of SARS-CoV-2 high mutation rate and homology of cellular ACE2 receptors enable the virus to transcend species barriers and facilitate its transmission between humans and animals.

The surge of fast-spreading SARS-CoV-2 mutated variants highlights the need for fast, broad-spectrum strategies to counteract viral infections. In this work, we report a physical barrier against SARS-CoV-2 infection based on an inhalable bioadhesive hydrogel, named spherical hydrogel inhalation for enhanced lung defence (SHIELD). Conveniently delivered via a dry powder inhaler, SHIELD particles form a dense hydrogel network that coats the airway, enhancing the diffusional barrier properties and restricting virus penetration. SHIELD's protective effect is first demonstrated in mice against two SARS-CoV-2 pseudo-viruses with different mutated spike proteins. Strikingly, in African green monkeys, a single SHIELD inhalation provides protection for up to 8 hours, efficiently reducing infection by the SARS-CoV-2 WA1 and B.1.617.2 (Delta) variants. Notably, SHIELD is made with food-grade materials and does not affect normal respiratory functions. This approach could offer additional protection to the population against SARS-CoV-2 and other respiratory pathogens.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has infected >600 million people in the ongoing global pandemic. Several variants of the SARS-CoV-2 have emerged in the last >2 years, challenging the continued efficacy of current COVID vaccines. Therefore, there is a crucial need to investigate a highly cross-protective vaccine effective against variants of SARS-CoV-2. In this study, we examined seven lipopeptides derived from highly conserved, immunodominant epitopes from the S, N, and M proteins of SARS-CoV-2, that are predicted to contain epitopes for clinically protective B cells, helper T cells (TH) and cytotoxic T cells (CTL). Intranasal immunization of mice with most of the lipopeptides led to significantly higher splenocyte proliferation and cytokine production, mucosal and systemic antibody responses, and induction of effector B and T lymphocytes in both lungs and spleen, compared to immunizations with the corresponding peptides without lipid. Immunizations with Spike-derived lipopeptides led to cross-reactive IgG, IgM and IgA responses against Alpha, Beta, Delta, and Omicron Spike proteins as well as neutralizing antibodies. These studies support their potential for development as components of a cross-protective SARS-CoV-2 vaccine.

Hybrid immunity to SARS-CoV-2 provides superior protection to re-infection. We performed immune profiling studies during breakthrough infections in mRNA-vaccinated hamsters to evaluate hybrid immunity induction. mRNA vaccine, BNT162b2, was dosed to induce binding antibody titers against ancestral spike, but inefficient serum virus neutralization of ancestral SARS-CoV-2 or variants of concern (VoCs). Vaccination reduced morbidity and controlled lung virus titers for ancestral virus and Alpha but allowed breakthrough infections in Beta, Delta and Mu-challenged hamsters. Vaccination primed T cell responses that were boosted by infection. Infection back-boosted neutralizing antibody responses against ancestral virus and VoCs. Hybrid immunity resulted in more cross-reactive sera. Transcriptomics post-infection reflects both vaccination status and disease course and suggests a role for interstitial macrophages in vaccine-mediated protection. Therefore, protection by vaccination, even in the absence of high titers of neutralizing antibodies in the serum, correlates with recall of broadly reactive B- and T-cell responses.

The aim of this study was to investigate the persistence of SARS-CoV-2 neutralizing antibodies (NAbs) one year after contracting COVID-19.

The study included 38 patients - 34 men and 4 women - suffering from COVID-19 between March 15 and May 26, 2020. The median age in the group was 31 years, ranging from 22 to 67 years. The levels of neutralizing antibodies were measured at three time-points - baseline, 6 months, and 12 months. The primary endpoint was a post-infection positive result for NAbs (> 15 AU/ml; Liaison SARS-CoV-2 S1/S2 IgG quantitative test) 12 months after infection.

The median level of NAbs after 12 months was 26.5 AU/ml. At the end of observation (12 months), 21 of the 38 patients had a NAb level of >15 AU/ml (positive). The median antibody half-life was 5.8 months.

A high percentage of the patients maintained positive levels of antibodies 6 and 12 months after COVID-19 infection. The dynamics of the antibody level decline suggests the need for booster vaccination at least once a year.

Recently some of us used a random-walk Monte Carlo simulation approach to study the spread of COVID-19. The calculations were reasonably successful in describing secondary and tertiary waves of infection, in countries such as the USA, India, South Africa and Serbia. However, they failed to predict the observed third wave for India. In this work we present a more complete set of simulations for India, that take into consideration two aspects that were not incorporated previously. These include the stochastic movement of an erstwhile protected fraction of the population, and the reinfection of some recovered individuals because of their exposure to a new variant of the SARS-CoV-2 virus. The extended simulations now show the third COVID-19 wave for India that was missing in the earlier calculations. They also suggest an additional fourth wave, which was indeed observed during approximately the same time period as the model prediction.

Neutralizing antibodies (nAbs) are considered a valuable marker for measuring humoral immunity against SARS-CoV-2. However, live-virus neutralization tests (NTs) require high-biosafety-level laboratories and are time-consuming. Therefore, surrogate virus neutralization tests (sVNTs) have been widely applied, but unlike most anti-spike (S) antibody assays, NTs and sVNTs are not harmonized, requiring further evaluation and comparative analyses. This study compared seven commercial sVNTs and anti-S-antibody assays with a live-virus NT as a reference, using a panel of 720 single and longitudinal serum samples from 666 convalescent patients after SARS-CoV-2 infection. The sensitivity of these assays for detecting antibodies ranged from 48 to 94% after PCR-confirmed infection and from 56% to 100% relative to positivity in the in-house live-virus NT. Furthermore, we performed receiver operating characteristic (ROC) curve analyses to determine which immunoassays were most suitable for assessing nAb titers exceeding a specific cutoff (NT titer, ≥80) and found that the NeutraLISA and the cPass assays reached the highest area under the curve (AUC), exceeding 0.91. In addition, when the assays were compared for their correlation with nAb kinetics over time in a set of longitudinal samples, the extent of the measured decrease of nAbs after infection varied widely among the evaluated immunoassays. Finally, in vaccinated convalescent patients, high titers of nAbs exceeded the upper limit of the evaluated assays' quantification ranges. Based on data from this study, we conclude that commercial immunoassays are acceptable substitutes for live-virus NTs, particularly when additional adapted cutoffs are employed to detect nAbs beyond a specific threshold titer. IMPORTANCE While the measurement of neutralizing antibodies is considered a valuable tool in assessing protection against SARS-CoV-2, neutralization tests employ live-virus isolates and cell culture, requiring advanced laboratory biosafety levels. Including a large sample panel (over 700 samples), this study provides adapted cutoff values calculated for seven commercial immunoassays (including four surrogate neutralization assays and a protein-based microarray) that robustly correlate with specific titers of neutralizing antibodies.

The frequent emergence of SARS-CoV-2 variants thwarts the prophylactic and therapeutic countermeasures confronting COVID-19. Among them, the Delta variant attracts widespread attention due to its high pathogenicity and fatality rate compared with other variants. However, with the emergence of new variants, studies on Delta variants have been gradually weakened and ignored. In this study, a replication-competent recombinant virus carrying the S protein of the SARS-CoV-2 Delta variant was established based on the vesicular stomatitis virus (VSV), which presented a safe alternative model for studying the Delta variant. The recombinant virus showed a replication advantage in Vero E6 cells, and the viral titers reach 10^7.3 TCID₅₀/mL at 36 h post-inoculation. In the VSV-vectored recombinant platform, the spike proteins of the Delta variant mediated higher fusion activity and syncytium formation than the wild-type strain. Notably, the recombinant virus was avirulent in BALB/c mice, Syrian hamsters, 3-day ICR suckling mice, and IFNAR/GR^-/- mice. It induced protective neutralizing antibodies in rodents, and protected the Syrian hamsters against the SARS-CoV-2 Delta variant infection. Meanwhile, the eGFP reporter of recombinant virus enabled the visual assay of neutralizing antibodies. Therefore, the recombinant virus could be a safe and convenient surrogate tool for authentic SARS-CoV-2. This efficient and reliable model has significant potential for research on viral-host interactions, epidemiological investigation of serum-neutralizing antibodies, and vaccine development.

Emergence of the betacoronavirus SARS-CoV-2 has resulted in a historic pandemic, with millions of deaths worldwide. An unprecedented effort has been made by the medical, scientific, and public health communities to rapidly develop and implement vaccines and therapeutics to prevent and reduce hospitalizations and deaths. Although SARS-CoV-2 infection can lead to disease in many organ systems, the respiratory system is its main target, with pneumonia and acute respiratory distress syndrome as the hallmark features of severe disease. The large number of patients who have contracted COVID-19 infections since 2019 has permitted a detailed characterization of the clinical and pathologic features of the disease in humans. However, continued progress in the development of effective preventatives and therapies requires a deeper understanding of the pathogenesis of infection. Studies using animal models are necessary to complement in vitro findings and human clinical data. Multiple animal species have been evaluated as potential models for studying the respiratory disease caused by SARSCoV-2 infection. Knowing the similarities and differences between animal and human responses to infection is critical for effective translation of animal data into human medicine. This review provides a detailed summary of the respiratory disease and associated pathology induced by SARS-CoV-2 infection in humans and compares them with the disease that develops in 3 commonly used models: NHP, hamsters, and mice. The effective use of animals to study SARS-CoV-2-induced respiratory disease will enhance our understanding of SARS-CoV-2 pathogenesis, allow the development of novel preventatives and therapeutics, and aid in the preparation for the next emerging virus with pandemic potential.

This review provides an overview covering mRNA from its use in the COVID-19 pandemic to cancer immunotherapy, starting from the selection of appropriate antigens, tumor-associated and tumor-specific antigens, neoantigens, the basics of optimizing the mRNA molecule in terms of stability, efficacy, and tolerability, choosing the best formulation and the optimal route of administration, to summarizing current clinical trials of mRNA vaccines in tumor therapy.

Neutralizing antibodies (NAbs) are central players in the humoral immunity that defends the body from SARS-CoV-2 infection by blocking viral entry into host cells and neutralizing their biological effects. Even though NAbs primarily work by neutralizing viral antigens, on some occasions, they may also combat the SARS-CoV-2 virus escaping neutralization by employing several effector mechanisms in collaboration with immune cells like natural killer (NK) cells and phagocytes. Besides their prophylactic and therapeutic roles, antibodies can be used for COVID-19 diagnosis, severity evaluation, and prognosis assessment in clinical practice. Furthermore, the measurement of NAbs could have key implications in determining individual or herd immunity against SARS-CoV-2, vaccine effectiveness, and duration of the humoral protective response, as well as aiding in the selection of suitable individuals who can donate convalescent plasma to treat infected people. Despite all these clinical applications of NAbs, using them in clinical settings can present some challenges. This review discusses the protective functions, possible protective mechanisms against SARS-CoV-2, and potential clinical applications of NAbs in COVID-19. This article also highlights the possible challenges and solutions associated with COVID-19 antibody-based prophylaxis, therapy, and vaccination.

Although COVID-19 has only recently appeared, research studies have already developed and implemented many animal models for deciphering the secrets of the disease and provided insights into the biology of SARS-CoV-2. However, there are several major factors that complicate the study of this virus in model organisms, such as the poor infectivity of clinical isolates of SARS-CoV-2 in some model species, and the absence of persistent infection, immunopathology, severe acute respiratory distress syndrome, and, in general, all the systemic complications which characterize COVID-19 clinically. Another important limitation is that SARS-CoV-2 mainly causes severe COVID-19 in older people with comorbidities, which represents a serious problem when attempting to use young and immunologically naïve laboratory animals in COVID-19 testing. We review here the main animal models developed so far to study COVID-19 and the unique advantages of the zebrafish model that may help to contribute to understand this disease, in particular to the identification and repurposing of drugs to treat COVID-19, to reveal the mechanism of action and side-effects of Spike-based vaccines, and to decipher the high susceptibility of aged people to COVID-19.