Pre-existing T cell-mediated cross-reactivity to SARS-CoV-2 cannot solely be explained by prior exposure to endemic human coronaviruses.

T-cell-mediated immunity to SARS-CoV-2-derived peptides in individuals unexposed to SARS-CoV-2 has been previously reported. This pre-existing immunity was suggested to largely derive from prior exposure to 'common cold' endemic human coronaviruses (HCoVs). To test this, we characterised the sequence homology of SARS-CoV-2-derived T-cell epitopes reported in the literature across the full proteome of the Coronaviridae family. 54.8% of these epitopes had no homology to any of the HCoVs. Further, the proportion of SARS-CoV-2-derived epitopes with any level of sequence homology to the proteins encoded by any of the coronaviruses tested is well-predicted by their alignment-free phylogenetic distance to SARS-CoV-2 (Pearson's r = -0.958). No coronavirus in our dataset showed a significant excess of T-cell epitope homology relative to the proportion of expected random matches, given their genetic similarity to SARS-CoV-2. Our findings suggest that prior exposure to human or animal-associated coronaviruses cannot completely explain the T-cell repertoire in unexposed individuals that recognise SARS-CoV-2 cross-reactive epitopes.

Innate immune sensing of coronavirus and viral evasion strategies.

The innate immune system is the first line of the host defense program against pathogens and harmful substances. Antiviral innate immune responses can be triggered by multiple cellular receptors sensing viral components. The activated innate immune system produces interferons (IFNs) and cytokines that perform antiviral functions to eliminate invading viruses. Coronaviruses are single-stranded, positive-sense RNA viruses that have a broad range of animal hosts. Coronaviruses have evolved multiple means to evade host antiviral immune responses. Successful immune evasion by coronaviruses may enable the viruses to adapt to multiple species of host organisms. Coronavirus transmission from zoonotic hosts to humans has caused serious illnesses, such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and coronavirus disease-2019 (COVID-19), resulting in global health and economic crises. In this review, we summarize the current knowledge of the mechanisms underlying host sensing of and innate immune responses against coronavirus invasion, as well as host immune evasion strategies of coronaviruses.

Cross-reactive memory T cells and herd immunity to SARS-CoV-2.

Immunity is a multifaceted phenomenon. For T cell-mediated memory responses to SARS-CoV-2, it is relevant to consider their impact both on COVID-19 disease severity and on viral spread in a population. Here, we reflect on the immunological and epidemiological aspects and implications of pre-existing cross-reactive immune memory to SARS-CoV-2, which largely originates from previous exposure to circulating common cold coronaviruses. We propose four immunological scenarios for the impact of cross-reactive CD4+ memory T cells on COVID-19 severity and viral transmission. For each scenario, we discuss its implications for the dynamics of herd immunity and on projections of the global impact of SARS-CoV-2 on the human population, and assess its plausibility. In sum, we argue that key potential impacts of cross-reactive T cell memory are already incorporated into epidemiological models based on data of transmission dynamics, particularly with regard to their implications for herd immunity. The implications of immunological processes on other aspects of SARS-CoV-2 epidemiology are worthy of future study.

SARS-CoV-2 infections in children and young people.

Though recent reports link SARS-CoV-2 infections with hyper-inflammatory states in children, most children experience no/mild symptoms, and hospitalization and mortality rates are low in the age group. As symptoms are usually mild and seroconversion occurs at low frequencies, it remains unclear whether children significantly contribute to community transmission. Several hypotheses try to explain age-related differences in disease presentation and severity. Possible reasons for milder presentations in children as compared to adults include frequent contact to seasonal coronaviruses, presence of cross-reactive antibodies, and/or co-clearance with other viruses. Increased expression of ACE2 in young people may facilitate virus infection, while limiting inflammation and reducing the risk of severe disease. Further potential factors include recent vaccinations and a more diverse memory T cell repertoire. This manuscript reviews age-related host factors that may protect children from COVID-19 and complications associated, and addresses the confusion around seropositivity and immunity.

HCoV- and SARS-CoV-2 Cross-Reactive T Cells in CVID Patients.

The inability of patients with CVID to mount specific antibody responses to pathogens has raised concerns on the risk and severity of SARS-CoV-2 infection, but there might be a role for protective T cells in these patients. SARS-CoV-2 reactive T cells have been reported for SARS-CoV-2 unexposed healthy individuals. Until now, there is no data on T cell immunity to SARS-CoV-2 infection in CVID. This study aimed to evaluate reactive T cells to human endemic corona viruses (HCoV) and to study pre-existing SARS-CoV-2 reactive T cells in unexposed CVID patients. We evaluated SARS-CoV-2- and HCoV-229E and -OC43 reactive T cells in response to seven peptide pools, including spike and nucleocapsid (NCAP) proteins, in 11 unexposed CVID, 12 unexposed and 11 post COVID-19 healthy controls (HC). We further characterized reactive T cells by IFNγ, TNFα and IL-2 profiles. SARS-CoV-2 spike-reactive CD4+ T cells were detected in 7 of 11 unexposed CVID patients, albeit with fewer multifunctional (IFNγ/TNFα/IL-2) cells than unexposed HC. CVID patients had no SARS-CoV-2 NCAP reactive CD4+ T cells and less reactive CD8+ cells compared to unexposed HC. We observed a correlation between T cell reactivity against spike of SARS-CoV-2 and HCoVs in unexposed, but not post COVID-19 HC, suggesting cross-reactivity. T cell responses in post COVID-19 HC could be distinguished from unexposed HC by higher frequencies of triple-positive NCAP reactive CD4+ T cells. Taken together, SARS-CoV-2 reactive T cells are detectable in unexposed CVID patients albeit with lower recognition frequencies and polyfunctional potential. Frequencies of triple-functional reactive CD4+ cells might provide a marker to distinguish HCoV cross-reactive from SARS-CoV-2 specific T cell responses. Our data provides evidence, that anti-viral T cell immunity is not relevantly impaired in most CVID patients.

Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a lineage C betacoronavirus that since its emergence in 2012 has caused outbreaks in human populations with case-fatality rates of ∼36%. As in other coronaviruses, the spike (S) glycoprotein of MERS-CoV mediates receptor recognition and membrane fusion and is the primary target of the humoral immune response during infection. Here we use structure-based design to develop a generalizable strategy for retaining coronavirus S proteins in the antigenically optimal prefusion conformation and demonstrate that our engineered immunogen is able to elicit high neutralizing antibody titers against MERS-CoV. We also determined high-resolution structures of the trimeric MERS-CoV S ectodomain in complex with G4, a stem-directed neutralizing antibody. The structures reveal that G4 recognizes a glycosylated loop that is variable among coronaviruses and they define four conformational states of the trimer wherein each receptor-binding domain is either tightly packed at the membrane-distal apex or rotated into a receptor-accessible conformation. Our studies suggest a potential mechanism for fusion initiation through sequential receptor-binding events and provide a foundation for the structure-based design of coronavirus vaccines.

RNA-virus proteases counteracting host innate immunity.

Virus invasion triggers host immune responses, in particular, innate immune responses. Pathogen-associated molecular patterns of viruses (such as dsRNA, ssRNA, or viral proteins) released during virus replication are detected by the corresponding pattern-recognition receptors of the host, and innate immune responses are induced. Through production of type-I and type-III interferons as well as various other cytokines, the host innate immune system forms the frontline to protect host cells and inhibit virus infection. Not surprisingly, viruses have evolved diverse strategies to counter this antiviral system. In this review, we discuss the multiple strategies used by proteases of positive-sense single-stranded RNA viruses of the families Picornaviridae, Coronaviridae, and Flaviviridae, when counteracting host innate immune responses.

Early endonuclease-mediated evasion of RNA sensing ensures efficient coronavirus replication.

Coronaviruses are of veterinary and medical importance and include highly pathogenic zoonotic viruses, such as SARS-CoV and MERS-CoV. They are known to efficiently evade early innate immune responses, manifesting in almost negligible expression of type-I interferons (IFN-I). This evasion strategy suggests an evolutionary conserved viral function that has evolved to prevent RNA-based sensing of infection in vertebrate hosts. Here we show that the coronavirus endonuclease (EndoU) activity is key to prevent early induction of double-stranded RNA (dsRNA) host cell responses. Replication of EndoU-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of the infection. In macrophages we found immediate induction of IFN-I expression and RNase L-mediated breakdown of ribosomal RNA. Accordingly, EndoU-deficient viruses can retain replication only in cells that are deficient in IFN-I expression or sensing, and in cells lacking both RNase L and PKR. Collectively our results demonstrate that the coronavirus EndoU efficiently prevents simultaneous activation of host cell dsRNA sensors, such as Mda5, OAS and PKR. The localization of the EndoU activity at the site of viral RNA synthesis-within the replicase complex-suggests that coronaviruses have evolved a viral RNA decay pathway to evade early innate and intrinsic antiviral host cell responses.

Development of monoclonal antibodies and serological assays including indirect ELISA and fluorescent microsphere immunoassays for diagnosis of porcine deltacoronavirus.

BACKGROUND: A novel porcine deltacoronavirus (PDCoV), also known as porcine coronavirus HKU15, was reported in China in 2012 and identified in the U.S. in early 2014. Since then, PDCoV has been identified in a number of U.S. states and linked with clinical disease including acute diarrhea and vomiting in the absence of other identifiable pathogens. Since PDCoV was just recently linked with clinical disease, few specific antibody-based reagents were available to assist in diagnosis of PDCoV and limited serological capabilities were available to detect an antibody response to this virus. Therefore, the overall objective of this project was to develop and validate selected diagnostic reagents and assays for PDCoV antigen and antibody detection. RESULTS: The nucleoprotein of PDCoV was expressed as a recombinant protein and purified for use as an antigen to immunize mice for polyclonal, hyperimmune sera and monoclonal antibody (mAb) production. The resulting mAbs were evaluated for use in fluorescent antibody staining methods to detect PDCoV infected cells following virus isolation attempts and for immunohistochemistry staining of intestinal tissues of infected pigs. The same antigen was used to develop serological tests to detect the antibody response to PDCoV in pigs following infection. Serum samples from swine herds with recent documentation of PDCoV infection and samples from expected naïve herds were used for initial assay optimization. The tests were optimized in a checkerboard fashion to reduce signal to noise ratios using samples of known status. Statistical analysis was performed to establish assay cutoff values and assess diagnostic sensitivities and specificities. At least 629 known negative serum samples and 311 known positive samples were evaluated for each assay. The enzyme linked immunosorbent assay (ELISA) showed diagnostic sensitivity (DSe) of 96.1% and diagnostic specificity (DSp) of 96.2%. The fluorescent microsphere immunoassay (FMIA) showed a DSe of 95.8% and DSp of 98.1%. Both ELISA and FMIA detected seroconversion of challenged pigs between 8-14 days post-infection (DPI). An indirect fluorescent antibody (IFA) test was also developed using cell culture adapted PDCoV for comparative purposes. CONCLUSION: These new, specific reagents and serological assays will allow for improved diagnosis of PDCoV. Since many aspects of PDCoV infection and transmission are still not fully understood, the reagents and assays developed in this project should provide valuable tools to help understand this disease and to aid in the control and surveillance of porcine deltacoronavirus outbreaks.

SARS-like WIV1-CoV poised for human emergence.

Outbreaks from zoonotic sources represent a threat to both human disease as well as the global economy. Despite a wealth of metagenomics studies, methods to leverage these datasets to identify future threats are underdeveloped. In this study, we describe an approach that combines existing metagenomics data with reverse genetics to engineer reagents to evaluate emergence and pathogenic potential of circulating zoonotic viruses. Focusing on the severe acute respiratory syndrome (SARS)-like viruses, the results indicate that the WIV1-coronavirus (CoV) cluster has the ability to directly infect and may undergo limited transmission in human populations. However, in vivo attenuation suggests additional adaptation is required for epidemic disease. Importantly, available SARS monoclonal antibodies offered success in limiting viral infection absent from available vaccine approaches. Together, the data highlight the utility of a platform to identify and prioritize prepandemic strains harbored in animal reservoirs and document the threat posed by WIV1-CoV for emergence in human populations.

A recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay to detect antibodies against porcine deltacoronavirus.

Recently, porcine deltacoronavirus (PDCoV) has been proven to be associated with enteric disease in piglets. Diagnostic tools for serological surveys of PDCoV remain in the developmental stage when compared with those for other porcine coronaviruses. In our study, an indirect enzyme-linked immunosorbent assay (ELISA) (rPDCoV-N-ELISA) was developed to detect antibodies against PDCoV using a histidine-tagged recombinant nucleocapsid (N) protein as an antigen. The rPDCoV-N-ELISA did not cross-react with antisera against porcine epidemic diarrhea virus, swine transmissible gastroenteritis virus, porcine group A rotavirus, classical swine fever virus, porcine circovirus-2, porcine pseudorabies virus, and porcine reproductive and respiratory syndrome virus; the receiver operating characteristic (ROC) curve analysis revealed 100% sensitivity and 90.4% specificity of the rPDCoV-N-ELISA based on samples of known status (n=62). Analyses of field samples (n=319) using the rPDCoV-N-ELISA indicated that 11.59% of samples were positive for antibodies against PDCoV. These data demonstrated that the rPDCoV-N-ELISA can be used for epidemiological investigations of PDCoV and that PDCoV had a low serum prevalence in pig population in Heilongjiang province, northeast China.

RNA virus reverse genetics and vaccine design.

RNA viruses are capable of rapid spread and severe or potentially lethal disease in both animals and humans. The development of reverse genetics systems for manipulation and study of RNA virus genomes has provided platforms for designing and optimizing viral mutants for vaccine development. Here, we review the impact of RNA virus reverse genetics systems on past and current efforts to design effective and safe viral therapeutics and vaccines.

Evaluation of serologic and antigenic relationships between middle eastern respiratory syndrome coronavirus and other coronaviruses to develop vaccine platforms for the rapid response to emerging coronaviruses.

BACKGROUND: Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012, causing severe acute respiratory disease and pneumonia, with 44% mortality among 136 cases to date. Design of vaccines to limit the virus spread or diagnostic tests to track newly emerging strains requires knowledge of antigenic and serologic relationships between MERS-CoV and other CoVs. METHODS: Using synthetic genomics and Venezuelan equine encephalitis virus replicons (VRPs) expressing spike and nucleocapsid proteins from MERS-CoV and other human and bat CoVs, we characterize the antigenic responses (using Western blot and enzyme-linked immunosorbent assay) and serologic responses (using neutralization assays) against 2 MERS-CoV isolates in comparison with those of other human and bat CoVs. RESULTS: Serologic and neutralization responses against the spike glycoprotein were primarily strain specific, with a very low level of cross-reactivity within or across subgroups. CoV N proteins within but not across subgroups share cross-reactive epitopes with MERS-CoV isolates. Our findings were validated using a convalescent-phase serum specimen from a patient infected with MERS-CoV (NA 01) and human antiserum against SARS-CoV, human CoV NL63, and human CoV OC43. CONCLUSIONS: Vaccine design for emerging CoVs should involve chimeric spike protein containing neutralizing epitopes from multiple virus strains across subgroups to reduce immune pathology, and a diagnostic platform should include a panel of nucleocapsid and spike proteins from phylogenetically distinct CoVs.

Engineering a replication-competent, propagation-defective Middle East respiratory syndrome coronavirus as a vaccine candidate.

UNLABELLED: Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging coronavirus infecting humans that is associated with acute pneumonia, occasional renal failure, and a high mortality rate and is considered a threat to public health. The construction of a full-length infectious cDNA clone of the MERS-CoV genome in a bacterial artificial chromosome is reported here, providing a reverse genetics system to study the molecular biology of the virus and to develop attenuated viruses as vaccine candidates. Following transfection with the cDNA clone, infectious virus was rescued in both Vero A66 and Huh-7 cells. Recombinant MERS-CoVs (rMERS-CoVs) lacking the accessory genes 3, 4a, 4b, and 5 were successfully rescued from cDNA clones with these genes deleted. The mutant viruses presented growth kinetics similar to those of the wild-type virus, indicating that accessory genes were not essential for MERS-CoV replication in cell cultures. In contrast, an engineered mutant virus lacking the structural E protein (rMERS-CoV-ΔE) was not successfully rescued, since viral infectivity was lost at early passages. Interestingly, the rMERS-CoV-ΔE genome replicated after cDNA clone was transfected into cells. The infectious virus was rescued and propagated in cells expressing the E protein in trans, indicating that this virus was replication competent and propagation defective. Therefore, the rMERS-CoV-ΔE mutant virus is potentially a safe and promising vaccine candidate to prevent MERS-CoV infection. IMPORTANCE: Since the emergence of MERS-CoV in the Arabian Peninsula during the summer of 2012, it has already spread to 10 different countries, infecting around 94 persons and showing a mortality rate higher than 50%. This article describes the development of the first reverse genetics system for MERS-CoV, based on the construction of an infectious cDNA clone inserted into a bacterial artificial chromosome. Using this system, a collection of rMERS-CoV deletion mutants has been generated. Interestingly, one of the mutants with the E gene deleted was a replication-competent, propagation-defective virus that could only be grown in the laboratory by providing E protein in trans, whereas it would only survive a single virus infection cycle in vivo. This virus constitutes a vaccine candidate that may represent a balance between safety and efficacy for the induction of mucosal immunity, which is needed to prevent MERS-CoV infection.

Cross-reactive antibodies in convalescent SARS patients' sera against the emerging novel human coronavirus EMC (2012) by both immunofluorescent and neutralizing antibody tests.

OBJECTIVES: A severe acute respiratory syndrome (SARS)-like disease due to a novel betacoronavirus, human coronavirus EMC (HCoV-EMC), has emerged recently. HCoV-EMC is phylogenetically closely related to Tylonycteris-bat-coronavirus-HKU4 and Pipistrellus-bat-coronavirus-HKU5 in Hong Kong. We conducted a seroprevalence study on archived sera from 94 game-food animal handlers at a wild life market, 28 SARS patients, and 152 healthy blood donors in Southern China to assess the zoonotic potential and evidence for intrusion of HCoV-EMC and related viruses into humans. METHODS: Anti-HCoV-EMC and anti-SARS-CoV antibodies were detected using screening indirect immunofluorescence (IF) and confirmatory neutralizing antibody tests. RESULTS: Two (2.1%) animal handlers had IF antibody titer of ≥ 1:20 against both HCoV-EMC and SARS-CoV with neutralizing antibody titer of <1:10. No blood donor had antibody against either virus. Surprisingly, 17/28 (60.7%) of SARS patients had significant IF antibody titers with 7/28 (25%) having anti-HCoV-EMC neutralizing antibodies at low titers which significantly correlated with that of HCoV-OC43. Bioinformatics analysis demonstrated a significant B-cell epitope overlapping the heptad repeat-2 region of Spike protein. Virulence of SARS-CoV over other betacoronaviruses may boost cross-reactive neutralizing antibodies against other betacoronaviruses. CONCLUSIONS: Convalescent SARS sera may contain cross-reactive antibodies against other betacoronaviruses and confound seroprevalence study for HCoV-EMC.

Intestinal mucosal immune response in ducklings following oral immunisation with an attenuated Duck enteritis virus vaccine.

To investigate the intestinal mucosal immune responses in ducklings orally inoculated with attenuated Duck enteritis virus (DEV), the kinetics of the viral load, the specific humoral immune responses, and the distribution of immunoglobulin (Ig)A-secreting cells in the intestine were evaluated. Oral inoculation with attenuated DEV stimulated an IgA-dominant response in intestinal secretions and a IgY-dominant response in the serum. The dramatic increase in virus-specific mucosal IgA 15 days after inoculation was accompanied by reductions in the DEV intestinal load, suggesting that the IgA response has a role in controlling viral replication. The kinetics of virus-specific IgA production closely correlated with the presence of IgA+ plasma cells in the intestinal lamina propria.