Inferring Incidence of Unreported SARS-CoV-2 Infections Using Seroprevalence of Open Reading Frame 8 Antigen, Hong Kong.

We tested seroprevalence of open reading frame 8 antigens to infer the number of unrecognized SARS-CoV-2 Omicron infections in Hong Kong during 2022. We estimate 33.6% of the population was infected, 72.1% asymptomatically. Surveillance and control activities during large-scale outbreaks should account for potentially substantial undercounts.

Baseline gut microbiota and metabolome predict durable immunogenicity to SARS-CoV-2 vaccines.

The role of gut microbiota in modulating the durability of COVID-19 vaccine immunity is yet to be characterised. In this cohort study, we collected blood and stool samples of 121 BNT162b2 and 40 CoronaVac vaccinees at baseline, 1 month, and 6 months post vaccination (p.v.). Neutralisation antibody, plasma cytokine and chemokines were measured and associated with the gut microbiota and metabolome composition. A significantly higher level of neutralising antibody (at 6 months p.v.) was found in BNT162b2 vaccinees who had higher relative abundances of Bifidobacterium adolescentis, Bifidobacterium bifidum, and Roseburia faecis as well as higher concentrations of nicotinic acid (Vitamin B) and γ-Aminobutyric acid (P < 0.05) at baseline. CoronaVac vaccinees with high neutralising antibodies at 6 months p.v. had an increased relative abundance of Phocaeicola dorei, a lower relative abundance of Faecalibacterium prausnitzii, and a higher concentration of L-tryptophan (P < 0.05) at baseline. A higher antibody level at 6 months p.v. was also associated with a higher relative abundance of Dorea formicigenerans at 1 month p.v. among CoronaVac vaccinees (Rho = 0.62, p = 0.001, FDR = 0.123). Of the species altered following vaccination, 79.4% and 42.0% in the CoronaVac and BNT162b2 groups, respectively, recovered at 6 months. Specific to CoronaVac vaccinees, both bacteriome and virome diversity depleted following vaccination and did not recover to baseline at 6 months p.v. (FDR < 0.1). In conclusion, this study identified potential microbiota-based adjuvants that may extend the durability of immune responses to SARS-CoV-2 vaccines.

Lack of neutralizing antibodies against influenza A viruses in adults during the 2022/2023 winter season - a serological study using retrospective samples collected in Hong Kong.

OBJECTIVES: Since the onset of the COVID-19 pandemic in 2020, there has been a significant decline in seasonal influenza infection cases in Hong Kong. However, this decline has also resulted in reduced opportunities for the development of influenza-specific antibodies in the community. The levels of antibodies required for protection against recently circulating influenza A viruses in the post-COVID-19 era remain unclear. METHODS: This study involved the analysis of paired plasma samples collected from 479 healthy adults in Hong Kong in 2021 and 2022. The neutralizing titers of plasma against influenza A (H1N1) and (H3N2) viruses circulating before and after the COVID-19 outbreak were determined using a microneutralization assay. RESULTS: The H1N1 and H3N2 vaccine strains selected for the 2022/23 season were found to be closely related to the recently circulating viruses. However, in the samples collected in 2022, only 14.61% and 0.42% showed a neutralization titer (MN50) ≥1:20 against H1N1 A/Wisconsin/588/2019 (H1/Wis19) and H3N2 A/Darwin/6/2021 (H3/Dar21), respectively. Notably, participants who reported receiving annual flu vaccinations exhibited a higher seropositive rate for H1/Wis19 compared to those who had never received the flu vaccine (28.06% vs. 5.30%). CONCLUSION: Our results indicate that adults in Hong Kong generally lack neutralizing antibodies against circulating influenza A viruses, particularly H3N2. These findings underscore the importance of promoting flu vaccination in the post-COVID-19 era.

Omicron BA.1-specific T-cell responses in adults vaccinated with CoronaVac or BNT162b2 in Hong Kong: an observational cohort study.

BACKGROUND: The primary aim of using vaccines in public health responses to SARS-CoV-2 variants of concern is to reduce incidence of severe disease, for which T-cell responses are essential. There is a paucity of data on vaccine-induced T-cell immunity to omicron (B.1.1.529). We aimed to compare SARS-CoV-2 omicron BA.1-specific T-cell responses in adults vaccinated with CoronaVac or BNT162b2. METHODS: For this observational cohort, we recruited adults (aged ≥18 years) from three vaccination centres in Hong Kong. We included participants from four cohorts (cohort 1: participants who received two doses of either BNT162b2 or CoronaVac, cohort 2: participants who received two doses and a booster, cohort 3: participants who received two doses and a booster and had a breakthrough omicron infection, and cohort 4: participants who had a previous non-omicron infection and subsequently received one dose of vaccine). People with confirmed history of COVID-19 at recruitment were excluded from cohort 1 and cohort 2. We collected blood samples before vaccination (for cohort 1 and 2), 1-month following vaccination (for all cohorts), and during convalescence for cohort 3 and 4) and determined the proportion of IFNγ+CD4+ and IFNγ+CD8+ T cells in peripheral blood against SARS-CoV-2 using flow cytometry with peptide pools of SARS-CoV-2 wild type or omicron BA.1. The primary outcome was proportion of CD4+ and CD8+ T cells against SARS-CoV-2 1 month after exposure (ie, vaccination or breakthrough infection). FINDINGS: Overall, between May 21, 2020, and Aug 31, 2021, we recruited 659 participants (231 [35%] men and 428 [65%] women). Of these participants, 428 were included in cohort 1 (214 [50%] received BNT162b2 and 214 [50%] received CoronaVac); 127 in cohort 2 (48 [38%] received all BNT162b2, 40 [31%] received all CoronaVac, and 39 [31%] received two CoronaVac and a booster with BNT162b2); 58 in cohort 3, and 46 in cohort 4 (16 [35%] received CoronaVac and 30 [65%] received BNT162b2). Vaccine-induced T-cell responses to the wild-type and omicron BA.1 variants were generally similar in adults receiving two doses of either CoronaVac (CD4+ cells p=0·33; CD8+ cells p=0·70) or BNT162b2 (CD4+ cells p=0·28; CD8+ cells p=1·0). Using a peptide pool of all structural proteins for stimulation, BNT162b2 induced a higher median frequency of omicron-specific CD4+ T cells in adults younger than 60 years (CD4+ cells 0·012% vs 0·010%, p=0·031; CD8+ cells 0·003% vs 0·000%, p=0·055) and omicron-specific CD8+ T cells in people aged 60 years or older (CD4+ cells 0·015% vs 0·006%, p=0·0070; CD8+ cells 0·007% vs 0·000%, p=0·035). A booster dose of either BNT162b2 or CoronaVac after two doses of CoronaVac boosted waning T-cell responses, but T-cell responses did not exceed those at 1 month after the second dose (CoronaVac CD4+ p=0·41, CD8+ p=0·79; BNT162b2 CD4+ p=0·70 CD8+ p=0·80). INTERPRETATION: The evidence that mRNA and inactivated vaccines based on the ancestral SARS-CoV-2 virus elicited T-cell responses to SARS-CoV-2 omicron variants might explain the high observed vaccine effectiveness against severe COVID-19 shown by both types of vaccine, despite great differences in neutralising antibody responses. The use of either vaccine can be considered if the primary aim is to reduce severity and death caused by the new omicron subvariants; however, BNT162b2 is preferable for adults older than 60 years. FUNDING: The Health and Medical Research Fund Commissioned Research on the Novel Coronavirus Disease and S H Ho Foundation.

Mouse models susceptible to HCoV-229E and HCoV-NL63 and cross protection from challenge with SARS-CoV-2.

Human coronavirus 229E (HCoV-229E) and NL63 (HCoV-NL63) are endemic causes of upper respiratory infections such as the "common cold" but may occasionally cause severe lower respiratory tract disease in the elderly and immunocompromised patients. There are no approved antiviral drugs or vaccines for these common cold coronaviruses (CCCoV). The recent emergence of COVID-19 and the possible cross-reactive antibody and T cell responses between these CCCoV and SARS-CoV-2 emphasize the need to develop experimental animal models for CCCoV. Mice are an ideal experimental animal model for such studies, but are resistant to HCoV-229E and HCoV-NL63 infections. Here, we generated 229E and NL63 mouse models by exogenous delivery of their receptors, human hAPN and hACE2 using replication-deficient adenoviruses (Ad5-hAPN and Ad5-hACE2), respectively. Ad5-hAPN- and Ad5-hACE2-sensitized IFNAR-/- and STAT1-/- mice developed pneumonia characterized by inflammatory cell infiltration with virus clearance occurring 7 d post infection. Ad5-hAPN- and Ad5-hACE2-sensitized mice generated virus-specific T cells and neutralizing antibodies after 229E or NL63 infection, respectively. Remdesivir and a vaccine candidate targeting spike protein of 229E and NL63 accelerated viral clearance of virus in these mice. 229E- and NL63-infected mice were partially protected from SARS-CoV-2 infection, likely mediated by cross-reactive T cell responses. Ad5-hAPN- and Ad5-hACE2-transduced mice are useful for studying pathogenesis and immune responses induced by HCoV-229E and HCoV-NL63 infections and for validation of broadly protective vaccines, antibodies, and therapeutics against human respiratory coronaviruses including SARS-CoV-2.

Age-related seroprevalence trajectories of seasonal coronaviruses in children including neonates in Guangzhou, China.

OBJECTIVES: Four seasonal coronaviruses, including human coronavirus (HCoV)-229E and HCoV-OC43, HCoV-NL63, and HCoV-HKU1 cause approximately 15-30% of common colds in adults. However, the full landscape of the immune trajectory to these viruses that covers the whole childhood period is still not well understood. METHODS: We evaluated the serological responses against the four seasonal coronaviruses in 1886 children aged under 18 years by using enzyme-linked immunosorbent assay. The optical density values against each HCoV were determined from each sample. Generalized additive models were constructed to determine the relationship between age and seroprevalence throughout the whole childhood period. The specific antibody levels against the four seasonal coronaviruses were also tested from the plasma samples of 485 pairs of postpartum women and their newborn babies. RESULTS: The immunoglobulin (Ig) G levels of the four seasonal coronaviruses in the mother and the newborn babies were highly correlated (229E: r = 0.63; OC43: r = 0.65; NL63: r = 0.69; HKU1: r = 0.63). The seroprevalences in children showed a similar trajectory in that the levels of IgG in the neonates dropped significantly and reached the lowest level after the age of around 1 year (229E: 1.18 years; OC43: 0.97 years; NL63: 1.01 years; HKU1: 1.02 years) and then resurgence in the children who aged older than 1 year. Using the lowest level from the generalized additive models as our cutoff, the seroprevalences for HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1 were 98.11%, 96.23%, 96.23% and 94.34% at the age of 16-18 years. CONCLUSION: Mothers share HCoV-specific IgGs with their newborn babies and the level of maternal IgGs waned at around 1 year after birth. The resurgence of the HCoV-specific IgGs was found thereafter with the increase in age suggesting repeated infection occurred in children.

Plaque-neutralizing antibody to BA.2.12.1, BA.4 and BA.5 in individuals with three doses of BioNTech or CoronaVac vaccines, natural infection and breakthrough infection.

BACKGROUND: BA.2.12.1, BA.4 and BA.5 subvariants of SARS-CoV-2 variant-of-concern (VOC) Omicron (B.1.1.529) are spreading globally. They demonstrate higher transmissibility and immune escape. OBJECTIVES: Determine BA.2.12.1, BA.4 and BA.5 virus plaque reduction neutralization test (PRNT) antibody titres in individuals recently vaccinated with BNT162b2 (n = 20) or CoronaVac (n = 20) vaccines or those convalescent from ancestral wild- type (WT) SARS-CoV-2 (n = 20) or BA.2 infections with (n = 17) or without (n = 7) prior vaccination. RESULTS: Relative to neutralization of the WT virus, those vaccinated with BNT162b2 had 4.8, 3.4, 4.6, 11.3 and 15.5-fold reductions of geometric mean antibody titres (GMT) to BA.1, BA.2, BA.2.12.1, BA.4 and BA.5 viruses, respectively. Similarly, those vaccinated with CoronaVac had 8.0, 7.0, 11.8, 12.0 and 12.0 fold GMT reductions and those with two doses of CoronaVac boosted by BNT162b2 had 6.1, 6.7, 6,3, 13.0 and 21.2 fold GMT reductions to these viruses, respectively. Vaccinated individuals with BA.2 breakthrough infections had higher GMT antibody levels vs. BA.4 (36.9) and BA.5 (36.9) than unvaccinated individuals with BA.2 infections (BA.4 GMT 8.2; BA.5 GMT 11.0). CONCLUSIONS: BA.4 and BA.5 subvariants were less susceptible to BNT162b2 or CoronaVac vaccine elicited antibody neutralization than subvariants BA.1, BA.2 and BA.2.12.1. Nevertheless, three doses BNT162b2 or booster of BNT162b2 following two doses of CoronaVac elicited detectable BA.4 and BA.5 neutralizing antibody responses while those vaccinated with three doses of CoronaVac largely fail to do so. BA.2 infections in vaccinated individuals led to higher levels of BA.4 or BA.5 neutralizing antibody compared to those who were vaccine-naive.

SARS-CoV-2 Omicron variant BA.2 neutralisation in sera of people with Comirnaty or CoronaVac vaccination, infection or breakthrough infection, Hong Kong, 2020 to 2022.

BackgroundOmicron subvariant BA.2 circulation is rapidly increasing globally.AimWe evaluated the neutralising antibody response from vaccination or prior SARS-CoV-2 infection against symptomatic infection by BA.2 or other variants.MethodsUsing 50% plaque reduction neutralisation tests (PRNT50), we assessed neutralising antibody titres to BA.2, wild type (WT) SARS-CoV-2 and other variants in Comirnaty or CoronaVac vaccinees, with or without prior WT-SARS-CoV-2 infection. Titres were also measured for non-vaccinees convalescing from a WT-SARS-CoV-2 infection. Neutralising antibodies in BA.2 and BA.1 breakthrough infections and in BA.2 infections affecting non-vaccinees were additionally studied.ResultsIn vaccinees or prior WT-SARS-CoV-2-infected people, BA.2 and BA.1 PRNT50 titres were comparable but significantly (p < 10 - 5) lower than WT. In each group of 20 vaccinees with (i) three-doses of Comirnaty, (ii) two CoronaVac followed by one Comirnaty dose, or (iii) one dose of either vaccine after a WT-SARS-CoV-2 infection, ≥ 19 individuals developed detectable (PRNT50 titre ≥ 10) antibodies to BA.2, while only 15 of 20 vaccinated with three doses of CoronaVac did. Comirnaty vaccination elicited higher titres to BA.2 than CoronaVac. In people convalescing from a WT-SARS-CoV-2 infection, a single vaccine dose induced higher BA.2 titres than three Comirnaty (p = 0.02) or CoronaVac (p = 0.00001) doses in infection-naïve individuals. BA.2 infections in previously uninfected and unvaccinated individuals elicited low (PRNT50 titre ≤ 80) responses with little cross-neutralisation of other variants. However, vaccinees with BA.1 or BA.2 breakthrough infections had broad cross-neutralising antibodies to WT viruses, and BA.1, BA.2, Beta and Delta variants.ConclusionsExisting vaccines can be of help against the BA.2 subvariant.

Gut microbiota composition is associated with SARS-CoV-2 vaccine immunogenicity and adverse events.

OBJECTIVE: The gut microbiota plays a key role in modulating host immune response. We conducted a prospective, observational study to examine gut microbiota composition in association with immune responses and adverse events in adults who have received the inactivated vaccine (CoronaVac; Sinovac) or the mRNA vaccine (BNT162b2; BioNTech; Comirnaty). DESIGN: We performed shotgun metagenomic sequencing in stool samples of 138 COVID-19 vaccinees (37 CoronaVac and 101 BNT162b2 vaccinees) collected at baseline and 1 month after second dose of vaccination. Immune markers were measured by SARS-CoV-2 surrogate virus neutralisation test and spike receptor-binding domain IgG ELISA. RESULTS: We found a significantly lower immune response in recipients of CoronaVac than BNT162b2 vaccines (p<0.05). Bifidobacterium adolescentis was persistently higher in subjects with high neutralising antibodies to CoronaVac vaccine (p=0.023) and their baseline gut microbiome was enriched in pathways related to carbohydrate metabolism (linear discriminant analysis (LDA) scores >2 and p<0.05). Neutralising antibodies in BNT162b2 vaccinees showed a positive correlation with the total abundance of bacteria with flagella and fimbriae including Roseburia faecis (p=0.028). The abundance of Prevotella copri and two Megamonas species were enriched in individuals with fewer adverse events following either of the vaccines indicating that these bacteria may play an anti-inflammatory role in host immune response (LDA scores>3 and p<0.05). CONCLUSION: Our study has identified specific gut microbiota markers in association with improved immune response and reduced adverse events following COVID-19 vaccines. Microbiota-targeted interventions have the potential to complement effectiveness of COVID-19 vaccines.

Neutralizing antibodies against the SARS-CoV-2 Omicron variant BA.1 following homologous and heterologous CoronaVac or BNT162b2 vaccination.

The Omicron variant is rapidly becoming the dominant SARS-CoV-2 virus circulating globally. It is important to define reductions in virus neutralizing activity in the serum of convalescent or vaccinated individuals to understand potential loss of protection against infection by Omicron. We previously established that a 50% plaque reduction neutralization antibody titer (PRNT50) ≥25.6 in our live virus assay corresponded to the threshold for 50% protection from infection against wild-type (WT) SARS-CoV-2. Here we show markedly reduced serum antibody titers against the Omicron variant (geometric mean titer (GMT) < 10) compared to WT virus 3-5 weeks after two doses of BNT162b2 (GMT = 218.8) or CoronaVac vaccine (GMT = 32.5). A BNT162b2 booster dose elicited Omicron PRNT50 titers ≥25.6 in 88% of individuals (22 of 25) who previously received 2 doses of BNT162b2 and 80% of individuals (24 of 30) who previously received CoronaVac. However, few (3%) previously infected individuals (1 of 30) or those vaccinated with three doses of CoronaVac (1 of 30) met this threshold. Our findings suggest that countries primarily using CoronaVac vaccines should consider messenger RNA vaccine boosters in response to the spread of Omicron. Studies evaluating the effectiveness of different vaccines against the Omicron variant are urgently needed.

Neutralizing antibody titres to SARS-CoV-2 Omicron variant and wild-type virus in those with past infection or vaccinated or boosted with mRNA BNT162b2 or inactivated CoronaVac vaccines.

Omicron, a novel SARS-CoV-2 variant has emerged and is rapidly becoming the dominant SARS-CoV-2 virus circulating globally. It is important to define reductions in virus neutralizing activity in serum of convalescent or vaccinated individuals to understand potential loss of protection from infection or re-infection. Two doses of BNT162b2 or CoronaVac vaccines provided little 50% plaque reduction neutralization test (PRNT 50 ) antibody immunity against the Omicron variant, even at one-month post vaccination. Booster doses with BNT162b2 in those with two doses of either BNT162b2 or CoronaVac provided acceptable neutralizing immunity against Omicron variant at 1-month post-booster dose. However, three doses of BNT162b2 elicited higher levels of PRNT 50 antibody to Omicron variant suggesting longer duration of protection. Convalescent from SARS-CoV-2 infection did not have protective PRNT 50 antibody levels to Omicron, but a single dose of BNT162b2 vaccine provided protective immunity. Field vaccine-efficacy studies against Omicron variant against different vaccines are urgently needed.

Comparison of the immunogenicity of BNT162b2 and CoronaVac COVID-19 vaccines in Hong Kong.

BACKGROUND AND OBJECTIVE: Few head-to-head evaluations of immune responses to different vaccines have been reported. METHODS: Surrogate virus neutralization test (sVNT) antibody levels of adults receiving either two doses of BNT162b2 (n = 366) or CoronaVac (n = 360) vaccines in Hong Kong were determined. An age-matched subgroup (BNT162b2 [n = 49] vs. CoronaVac [n = 49]) was tested for plaque reduction neutralization (PRNT) and spike-binding antibody and T-cell reactivity in peripheral blood mononuclear cells. RESULTS: One month after the second dose of vaccine, BNT162b2 elicited significantly higher PRNT50 , PRNT90 , sVNT, spike receptor binding, spike N-terminal domain binding, spike S2 domain binding, spike FcR binding and antibody avidity levels than CoronaVac. The geometric mean PRNT50 titres in those vaccinated with BNT162b2 and CoronaVac vaccines were 251.6 and 69.45, while PRNT90 titres were 98.91 and 16.57, respectively. All of those vaccinated with BNT162b2 and 45 (91.8%) of 49 vaccinated with CoronaVac achieved the 50% protection threshold for PRNT90. Allowing for an expected seven-fold waning of antibody titres over 6 months for those receiving CoronaVac, only 16.3% would meet the 50% protection threshold versus 79.6% of BNT162b2 vaccinees. Age was negatively correlated with PRNT90 antibody titres. Both vaccines induced SARS-CoV-2-specific CD4+ and CD8+ T-cell responses at 1 month post-vaccination but CoronaVac elicited significantly higher structural protein-specific CD4+ and CD8+ T-cell responses. CONCLUSION: Vaccination with BNT162b2 induces stronger humoral responses than CoronaVac. CoronaVac induces higher CD4+ and CD8+ T-cell responses to the structural protein than BNT162b2.

The N-Terminal Region of Middle East Respiratory Syndrome Coronavirus Accessory Protein 8b Is Essential for Enhanced Virulence of an Attenuated Murine Coronavirus.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a beta coronavirus that emerged in 2012, causing severe pneumonia and renal failure. MERS-CoV encodes five accessory proteins. Some of them have been shown to interfere with host antiviral immune response. However, the roles of protein 8b in innate immunity and viral virulence was rarely studied. Here, we introduced individual MERS-CoV accessory protein genes into the genome of an attenuated murine coronavirus (Mouse hepatitis virus, MHV), respectively, and found accessory protein 8b could enhance viral replication in vivo and in vitro and increase the lethality of infected mice. RNA-seq analysis revealed that protein 8b could significantly inhibit type I interferon production (IFN-I) and innate immune response in mice infected with MHV expressing protein 8b. We also found that MERS-CoV protein 8b could initiate from multiple internal methionine sites and at least three protein variants were identified. Residues 1-23 of protein 8b was demonstrated to be responsible for increased virulence in vivo. In addition, the inhibitory effect on IFN-I of protein 8b might not contribute to its virulence enhancement as aa1-23 deletion did not affect IFN-I production in vitro and in vivo. Next, we also found that protein 8b was localized to the endoplasmic reticulum (ER)/Golgi membrane in infected cells, which was disrupted by C-terminal region aa 88-112 deletion. This study will provide new insight into the pathogenesis of MERS-CoV infection. IMPORTANCE Multiple coronaviruses (CoV) cause severe respiratory infections and become global public health threats such as SARS-CoV, MERS-CoV, and SARS-CoV-2. Each coronavirus contains different numbers of accessory proteins which show high variability among different CoVs. Accessory proteins are demonstrated to play essential roles in pathogenesis of CoVs. MERS-CoV contains 5 accessory proteins (protein 3, 4a, 4b, 5, 8b), and deletion of all four accessory proteins (protein 3, 4a, 4b, 5), significantly affects MERS-CoV replication and pathogenesis. However, whether ORF8b also regulates MERS-CoV infection is unknown. Here, we constructed mouse hepatitis virus (MHV) recombinant virus expressing MERS-CoV protein 8b and demonstrated protein 8b could significantly enhance the virulence of MHV, which is mediated by N-terminal domain of protein 8b. This study will shed light on the understanding of pathogenesis of MERS-CoV infection.

Tracking the Transcription Kinetic of SARS-CoV-2 in Human Cells by Reverse Transcription-Droplet Digital PCR.

Viral transcription is an essential step of SARS-CoV-2 infection after invasion into the target cells. Antiviral drugs such as remdesivir, which is used to treat COVID-19 patients, targets the viral RNA synthesis. Understanding the mechanism of viral transcription may help to develop new therapeutic treatment by perturbing virus replication. In this study, we established 28 ddPCR assays and designed specific primers/probe sets to detect the RNA levels of 15 NSP, 9 ORF, and 4 structural genes of SARS-CoV-2. The transcriptional kinetics of these viral genes were determined longitudinally from the beginning of infection to 12 h postinfection in Caco-2 cells. We found that SARS-CoV-2 takes around 6 h to hijack the cells before the initiation of viral transcription process in human cells. Our results may contribute to a deeper understanding of the mechanisms of SARS-CoV-2 infection.

Multi-platform omics analysis reveals molecular signature for COVID-19 pathogenesis, prognosis and drug target discovery.

Disease progression prediction and therapeutic drug target discovery for Coronavirus disease 2019 (COVID-19) are particularly important, as there is still no effective strategy for severe COVID-19 patient treatment. Herein, we performed multi-platform omics analysis of serial plasma and urine samples collected from patients during the course of COVID-19. Integrative analyses of these omics data revealed several potential therapeutic targets, such as ANXA1 and CLEC3B. Molecular changes in plasma indicated dysregulation of macrophage and suppression of T cell functions in severe patients compared to those in non-severe patients. Further, we chose 25 important molecular signatures as potential biomarkers for the prediction of disease severity. The prediction power was validated using corresponding urine samples and plasma samples from new COVID-19 patient cohort, with AUC reached to 0.904 and 0.988, respectively. In conclusion, our omics data proposed not only potential therapeutic targets, but also biomarkers for understanding the pathogenesis of severe COVID-19.

Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment.

COVID-19, caused by SARS-CoV-2, is a virulent pneumonia, with >4,000,000 confirmed cases worldwide and >290,000 deaths as of May 15, 2020. It is critical that vaccines and therapeutics be developed very rapidly. Mice, the ideal animal for assessing such interventions, are resistant to SARS-CoV-2. Here, we overcome this difficulty by exogenous delivery of human ACE2 with a replication-deficient adenovirus (Ad5-hACE2). Ad5-hACE2-sensitized mice developed pneumonia characterized by weight loss, severe pulmonary pathology, and high-titer virus replication in lungs. Type I interferon, T cells, and, most importantly, signal transducer and activator of transcription 1 (STAT1) are critical for virus clearance and disease resolution in these mice. Ad5-hACE2-transduced mice enabled rapid assessments of a vaccine candidate, of human convalescent plasma, and of two antiviral therapies (poly I:C and remdesivir). In summary, we describe a murine model of broad and immediate utility to investigate COVID-19 pathogenesis and to evaluate new therapies and vaccines.

Kinetics of viral load and antibody response in relation to COVID-19 severity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent for coronavirus 2019 (COVID-19) pneumonia. Little is known about the kinetics, tissue distribution, cross-reactivity, and neutralization antibody response in patients with COVID-19. Two groups of patients with RT-PCR-confirmed COVID-19 were enrolled in this study: 12 severely ill patients in intensive care units who needed mechanical ventilation and 11 mildly ill patients in isolation wards. Serial clinical samples were collected for laboratory detection. Results showed that most of the severely ill patients had viral shedding in a variety of tissues for 20-40 days after onset of disease (8/12, 66.7%), while the majority of mildly ill patients had viral shedding restricted to the respiratory tract and had no detectable virus RNA 10 days after onset (9/11, 81.8%). Mildly ill patients showed significantly lower IgM response compared with that of the severe group. IgG responses were detected in most patients in both the severe and mild groups at 9 days after onset, and remained at a high level throughout the study. Antibodies cross-reactive to SARS-CoV and SARS-CoV-2 were detected in patients with COVID-19 but not in patients with MERS. High levels of neutralizing antibodies were induced after about 10 days after onset in both severely and mildly ill patients which were higher in the severe group. SARS-CoV-2 pseudotype neutralization test and focus reduction neutralization test with authentic virus showed consistent results. Sera from patients with COVID-19 inhibited SARS-CoV-2 entry. Sera from convalescent patients with SARS or Middle East respiratory syndrome (MERS) did not. Anti-SARS-CoV-2 S and N IgG levels exhibited a moderate correlation with neutralization titers in patients' plasma. This study improves our understanding of immune response in humans after SARS-CoV-2 infection.

Probing the nuclear import signal and nuclear transport molecular determinants of PRV ICP22.

BACKGROUND: Herpes simplex virus 1 (HSV-1) ICP22 is a multifunctional protein and important for HSV-1 replication. Pseudorabies virus (PRV) ICP22 (P-ICP22) is a homologue of HSV-1 ICP22 and is reported to be able to selectively modify the transcription of different kinetic classes of PRV genes, however, the subcellular localization, localization signal and molecular determinants for its transport to execute this function is less well understood. RESULTS: In this study, by utilizing live cells fluorescent microscopy, P-ICP22 fused to enhanced yellow fluorescent protein (EYFP) gene was transient expressed in live cells and shown to exhibit a predominantly nucleus localization in the absence of other viral proteins. By transfection of a series of P-ICP22 deletion mutants fused to EYFP, a bona fide nuclear localization signal (NLS) and its key amino acids (aa) of P-ICP22 was, for the first time, determined and mapped to aa 41-60 (PASTPTPPKRGRYVVEHPEY) and aa 49-50 (KR), respectively. Besides, the P-ICP22 was demonstrated to be targeted to the nucleus via Ran-, importin α1-, and α7-mediated pathway. CONCLUSIONS: Our findings reported herein disclose the NLS and molecular mechanism for nuclear transport of P-ICP22, these results will uncover new avenues for depicting the biological roles of P-ICP22 during PRV infection.

Identification of molecular determinants for the nuclear import of pseudorabies virus UL31.

Herpes simplex virus 1 (HSV-1) UL31 is a multifunctional protein and important for HSV-1 infection. Pseudorabies virus (PRV) UL31 is a late protein homologous to HSV-1 UL31. Previous studies showed that PRV UL31 is predominantly localized to nucleus, however, the molecular determinants for its nuclear import were unclear to date. Here, by utilizing live cells fluorescent microscopy, UL31 fused with enhanced yellow fluorescent protein was transiently expressed in live cells and confirmed to exclusively target to the nucleus in the absence of other viral proteins. Furthermore, the nuclear import of UL31 was found to be dependent on the Ran-, importin α1-, α3-, α5-, α7-, ß1-and transportin-1-mediated pathway. Therefore, these results would open up new avenues for depicting the biological functions of UL31 during PRV infection.