Enhanced potency of an IgM-like nanobody targeting conserved epitope in SARS-CoV-2 spike N-terminal domain.

Almost all the neutralizing antibodies targeting the receptor-binding domain (RBD) of spike (S) protein show weakened or lost efficacy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged or emerging variants, such as Omicron and its sub-variants. This suggests that highly conserved epitopes are crucial for the development of neutralizing antibodies. Here, we present one nanobody, N235, displaying broad neutralization against the SARS-CoV-2 prototype and multiple variants, including the newly emerged Omicron and its sub-variants. Cryo-electron microscopy demonstrates N235 binds a novel, conserved, cryptic epitope in the N-terminal domain (NTD) of the S protein, which interferes with the RBD in the neighboring S protein. The neutralization mechanism interpreted via flow cytometry and Western blot shows that N235 appears to induce the S1 subunit shedding from the trimeric S complex. Furthermore, a nano-IgM construct (MN235), engineered by fusing N235 with the human IgM Fc region, displays prevention via inducing S1 shedding and cross-linking virus particles. Compared to N235, MN235 exhibits varied enhancement in neutralization against pseudotyped and authentic viruses in vitro. The intranasal administration of MN235 in low doses can effectively prevent the infection of Omicron sub-variant BA.1 and XBB in vivo, suggesting that it can be developed as a promising prophylactic antibody to cope with the ongoing and future infection.

Long-lasting humoral and cellular memory immunity to vaccinia virus Tiantan provides pre-existing immunity against mpox virus in Chinese population.

Investigating immune memory to vaccinia virus and pre-existing immunity to mpox virus (MPXV) among the population is crucial for the global response to this ongoing mpox epidemic. Blood was sampled from vaccinees inoculated with vaccinia virus Tiantan (VTT) strain born before 1981 and unvaccinated control subjects born since 1982. After at least 40 years of the inoculation, 60% or 5% VTT vaccinees possess neutralizing antibodies (NAbs) to VTT or MPXV, with at least 50% having T cell memory to VTT protein antigens. Notably, 46.7% vaccinees show pre-existing T cell responses to MPXV. Broad pre-existing CD8+ T cell reactivities to MPXV are detected not only against conserved epitopes but also against variant epitopes between VTT and MPXV. Persistent NAbs and T cell memory to VTT among vaccinees, along with pre-existing T cells to MPXV among both vaccinees and the unvaccinated population, indicate a particular immune barrier to mpox.

Broad protective RBD heterotrimer vaccines neutralize SARS-CoV-2 including Omicron sub-variants XBB/BQ.1.1/BF.7.

SARS-CoV-2 variants with severe immune evasion are a major challenge for COVID-19 prevention, especially the circulating Omicron XBB/BQ.1.1/BF.7 strains. Thus, the next-generation of broad-spectrum vaccines are urgently needed. Previously, we developed a COVID-19 protein subunit vaccine, ZF2001, based on the RBD-homodimer as the immunogen. To adapt SARS-CoV-2 variants, we developed chimeric RBD-heterodimers to induce broad immune responses. In this study, we further explored the concept of tandem RBD homotrimer and heterotrimer. Prototype SARS-CoV-2 RBD-homotrimer, prototype-Delta-BA.1 (PDO) RBD-heterotrimer and Delta-BA.2-BA.5 (DBA2BA5) RBD-heterotrimer were designed. Biochemical and cryo-EM structural characterization demonstrated total epitope exposure of the RBD-trimers. In mouse experiments, PDO and DBA2BA5 elicited broad SARS-CoV-2 neutralization. Potent protection against SARS-CoV-2 variants was observed in challenge assays and was correlated with neutralizing antibody titer. This study validated the design strategy of tandem RBD-heterotrimers as multivalent immunogens and presented a promising vaccine candidate, DBA2BA5, eliciting broad-spectrum immune responses, including against the circulating XBB/BF.7/BQ.1.1.

A pan-coronavirus peptide inhibitor prevents SARS-CoV-2 infection in mice by intranasal delivery.

Coronaviruses (CoVs) have brought serious threats to humans, particularly severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2), which continually evolves into multiple variants. These variants, especially Omicron, reportedly escape therapeutic antibodies and vaccines, indicating an urgent need for new antivirals with pan-SARS-CoV-2 inhibitory activity. We previously reported that a peptide fusion inhibitor, P3, targeting heptad repeated-1 (HR1) of SARS-CoV-2 spike (S) protein, could inhibit viral infections. Here, we further designed multiple derivatives of the P3 based on structural analysis and found that one derivative, the P315V3, showed the most efficient antiviral activity against SARS-CoV-2 variants and several other sarbecoviruses, as well as other human-CoVs (HCoVs). P315V3 also exhibited effective prophylactic efficacy against the SARS-CoV-2 Delta and Omicron variants in mice via intranasal administration. These results suggest that P315V3, which is in Phase II clinical trial, is promising for further development as a nasal pan-SARS-CoV-2 or pan-CoVs inhibitor to prevent or treat CoV diseases.

Rational design of an influenza-COVID-19 chimeric protective vaccine with HA-stalk and S-RBD.

Highly contagious respiratory illnesses like influenza and COVID-19 pose serious risks to public health. A two-in-one vaccine would be ideal to avoid multiple vaccinations for these diseases. Here, we generated a chimeric receptor binding domain of the spike protein (S-RBD) and hemagglutinin (HA)-stalk-based vaccine for both SARS-CoV-2 and influenza viruses. The S-RBD from SARS-CoV-2 Delta was fused to the headless HA from H1N1 (H1Delta), creating a chimera that forms trimers in solution. The cryo-electron microscopy structure of the chimeric protein complexed with the RBD-targeting CB6 and the HA-stalk-targeting CR9114 antibodies shows that the trimeric protein is stable and accessible for neutralizing antibody binding. Immunization with the vaccine elicited high and long-lasting neutralizing antibodies and effectively protected mice against the challenges of lethal H1N1 or heterosubtypic H5N8, as well as the SARS-CoV-2 Delta or Omicron BA.2 variants. Overall, this study offers a two-in-one universal vaccine design to combat infections caused by both SARS-CoV-2 variants of concern and influenza viruses.

Low levels of neutralizing antibodies against XBB Omicron subvariants after BA.5 infection.

The COVID-19 response strategies in Chinese mainland were recently adjusted due to the reduced pathogenicity and enhanced infectivity of Omicron subvariants. In Chengdu, China, an infection wave was predominantly induced by the BA.5 subvariant. It is crucial to determine whether the hybrid anti-SARS-CoV-2 immunity following BA.5 infection, coupled with a variety of immune background, is sufficient to shape the immune responses against newly emerged Omicron subvariants, especially for XBB lineages. To investigate this, we collected serum and nasal swab samples from 108 participants who had been infected in this BA.5 infection wave, and evaluated the neutralization against pseudoviruses. Our results showed that convalescent sera from individuals, regardless of vaccination history, had remarkably compromised neutralization capacities against the newly emerged XBB and XBB.1.5 subvariants. Although post-vaccination with BA.5 breakthrough infection slightly elevated plasma neutralizing antibodies against a part of pseudoviruses, the neutralization activities were remarkably impaired by XBB lineages. Furthermore, we analyzed the impacts of the number of vaccinations, age, and sex on the humoral and cellular immune response after BA.5 infection. Our findings suggest that the neutralization against XBB lineages that elicited by current hybrid immunity after BA.5 infection, are remained at low levels, indicating an urgent need for the development of next-generation of COVID-19 vaccines that designed based on the XBB sub-lineages and other future variants.

Stability of SARS-CoV-2 and persistence of viral nucleic acids on common foods and widely used packaging material surfaces.

SARS-CoV-2 is still spreading globally. Studies have reported the stability of SARS-CoV-2 in aerosols and on surfaces under different conditions. However, studies on the stability of SARS-CoV-2 and viral nucleic acids on common food and packaging material surfaces are insufficient. The study evaluated the stability of SARS-CoV-2 using TCID50 assays and the persistence of SARS-CoV-2 nucleic acids using droplet digital polymerase chain reaction on various food and packaging material surfaces. Viral nucleic acids were stable on food and material surfaces under different conditions. The viability of SARS-CoV-2 varied among different surfaces. SARS-CoV-2 was inactivated on most food and packaging material surfaces within 1 day at room temperature but was more stable at lower temperatures. Viruses survived for at least 1 week on pork and plastic at 4°C, while no viable viruses were detected on hairtail, orange, or carton after 3 days. There were viable viruses and a slight titer decrease after 8 weeks on pork and plastic, but titers decreased rapidly on hairtail and carton at -20°C. These results highlight the need for targeted preventive and disinfection measures based on different types of foods, packaging materials, and environmental conditions, particularly in the cold-chain food trade, to combat the ongoing pandemic.

Surveillance of SARS-CoV-2 at the Huanan Seafood Market.

SARS-CoV-2, the causative agent of COVID-19, emerged in December 2019. Its origins remain uncertain. It has been reported that a number of the early human cases had a history of contact with the Huanan Seafood Market. Here we present the results of surveillance for SARS-CoV-2 within the market. From January 1st 2020, after closure of the market, 923 samples were collected from the environment. From 18th January, 457 samples were collected from 18 species of animals, comprising of unsold contents of refrigerators and freezers, swabs from stray animals, and the contents of a fish tank. Using RT-qPCR, SARS-CoV-2 was detected in 73 environmental samples, but none of the animal samples. Three live viruses were successfully isolated. The viruses from the market shared nucleotide identity of 99.99% to 100% with the human isolate HCoV-19/Wuhan/IVDC-HB-01/2019. SARS-CoV-2 lineage A (8782T and 28144C) was found in an environmental sample. RNA-seq analysis of SARS-CoV-2 positive and negative environmental samples showed an abundance of different vertebrate genera at the market. In summary, this study provides information about the distribution and prevalence of SARS-CoV-2 in the Huanan Seafood Market during the early stages of the COVID-19 outbreak.

Development of neutralizing antibodies against SARS-CoV-2, using a high-throughput single-B-cell cloning method.

Background: Rapid and efficient strategies are needed to discover neutralizing antibodies (nAbs) from B cells derived from virus-infected patients. Methods: Here, we report a high-throughput single-B-cell cloning method for high-throughput isolation of nAbs targeting diverse epitopes on the SARS-CoV-2-RBD (receptor binding domain) from convalescent COVID-19 patients. This method is simple, fast and highly efficient in generating SARS-CoV-2-neutralizing antibodies from COVID-19 patients' B cells. Results: Using this method, we have developed multiple nAbs against distinct SARS-CoV-2-RBD epitopes. CryoEM and crystallography revealed precisely how they bind RBD. In live virus assay, these nAbs are effective in blocking viral entry to the host cells. Conclusion: This simple and efficient method may be useful in developing human therapeutic antibodies for other diseases and next pandemic.

Immunological and metabolic characteristics of the Omicron variants infection.

The Omicron variants of SARS-CoV-2, primarily authenticated in November 2021 in South Africa, has initiated the 5th wave of global pandemics. Here, we systemically examined immunological and metabolic characteristics of Omicron variants infection. We found Omicron resisted to neutralizing antibody targeting receptor binding domain (RBD) of wildtype SARS-CoV-2. Omicron could hardly be neutralized by sera of Corona Virus Disease 2019 (COVID-19) convalescents infected with the Delta variant. Through mass spectrometry on MHC-bound peptidomes, we found that the spike protein of the Omicron variants could generate additional CD8 + T cell epitopes, compared with Delta. These epitopes could induce robust CD8 + T cell responses. Moreover, we found booster vaccination increased the cross-memory CD8 + T cell responses against Omicron. Metabolic regulome analysis of Omicron-specific T cell showed a metabolic profile that promoted the response of memory T cells. Consistently, a greater fraction of memory CD8 + T cells existed in Omicron stimulated peripheral blood mononuclear cells (PBMCs). In addition, CD147 was also a receptor for the Omicron variants, and CD147 antibody inhibited infection of Omicron. CD147-mediated Omicron infection in a human CD147 transgenic mouse model induced exudative alveolar pneumonia. Taken together, our data suggested that vaccination booster and receptor blocking antibody are two effective strategies against Omicron.

Two pan-SARS-CoV-2 nanobodies and their multivalent derivatives effectively prevent Omicron infections in mice.

With the widespread vaccinations against coronavirus disease 2019 (COVID-19), we are witnessing gradually waning neutralizing antibodies and increasing cases of breakthrough infections, necessitating the development of drugs aside from vaccines, particularly ones that can be administered outside of hospitals. Here, we present two cross-reactive nanobodies (R14 and S43) and their multivalent derivatives, including decameric ones (fused to the immunoglobulin M [IgM] Fc) that maintain potent neutralizing activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) after aerosolization and display not only pan-SARS-CoV-2 but also varied pan-sarbecovirus activities. Through respiratory administration to mice, monovalent and decameric R14 significantly reduce the lung viral RNAs at low dose and display potent pre- and post-exposure protection. Furthermore, structural studies reveal the neutralizing mechanisms of R14 and S43 and the multiple inhibition effects that the multivalent derivatives exert. Our work demonstrates promising convenient drug candidates via respiratory administration against SARS-CoV-2 infection, which can contribute to containing the COVID-19 pandemic.

Immune response and homeostasis mechanism following administration of BBIBP-CorV SARS-CoV-2 inactivated vaccine.

The BBIBP-CorV severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inactivated vaccine has been authorized for emergency use and widely distributed. We used single-cell transcriptome sequencing to characterize the dynamics of immune responses to the BBIBP-CorV inactivated vaccine. In addition to the expected induction of humoral immunity, we found that the inactivated vaccine induced multiple, comprehensive immune responses, including significantly increased proportions of CD16+ monocytes and activation of monocyte antigen presentation pathways; T cell activation pathway upregulation in CD8+ T cells, along with increased activation of CD4+ T cells; significant enhancement of cell-cell communications between innate and adaptive immunity; and the induction of regulatory CD4+ T cells and co-inhibitory interactions to maintain immune homeostasis after vaccination. Additionally, comparative analysis revealed higher neutralizing antibody levels, distinct expansion of naive T cells, a shared increased proportion of regulatory CD4+ T cells, and upregulated expression of functional genes in booster dose recipients with a longer interval after the second vaccination. Our research will support a comprehensive understanding of the systemic immune responses elicited by the BBIBP-CorV inactivated vaccine, which will facilitate the formulation of better vaccination strategies and the design of new vaccines.

Isolation and Characterization of Monkeypox Virus from the First Case of Monkeypox - Chongqing Municipality, China, 2022.

Introduction: The first imported case of monkeypox (MPX) from the mainland of China was reported in September 2022. Herein, the study reports the isolation and characterization of MPX virus (MPXV) in this case. Methods: Clinical specimens including skin blister fluid, oropharyngeal and nasopharyngeal swabs, and blood were collected and inoculated onto Vero cells. The isolated virus was identified as MPXV using quantitative polymerase chain reaction (qPCR), cytopathic effects (CPEs), immunofluorescence assay (IFA) and transmission electron microscopy (TEM). Plaque assays were employed to quantify infectious plaque-forming units (PFUs). The plaque reduction neutralization test (PRNT) was developed to determine the neutralizing antibody (nAb) against MPXV. Results: MPXV replication was confirmed with qPCR. Typical CPEs were observed 48 h post-incubation. The isolated virus was named MPXV-B.1-China-C-Tan-CQ01. IFA showed that MPXV reacted with serum of MPX case. Orthopoxvirus morphology was observed using TEM. The virus titer increased to >106 PFU/mL after three passages. The serum PRNT 50% neutralization titer (NT50) was 35 for the MPX patient 6 days after symptom onset. Discussion: The study successfully isolated the first MPXV strain in the mainland of China, MPXV-B.1-China-C-Tan-CQ01. Infectious titration and PRNT methods have been developed. The study provides key resources and technical platforms for further research as well as anti-viral drug and vaccine development against MPX.

The CD8+ and CD4+ T Cell Immunogen Atlas of Zika Virus Reveals E, NS1 and NS4 Proteins as the Vaccine Targets.

Zika virus (ZIKV)-specific T cells are activated by different peptides derived from virus structural and nonstructural proteins, and contributed to the viral clearance or protective immunity. Herein, we have depicted the profile of CD8+ and CD4+ T cell immunogenicity of ZIKV proteins in C57BL/6 (H-2b) and BALB/c (H-2d) mice, and found that featured cellular immunity antigens were variant among different murine alleles. In H-2b mice, the proteins E, NS2, NS3 and NS5 are recognized as immunodominant antigens by CD8+ T cells, while NS4 is dominantly recognized by CD4+ T cells. In contrast, in H-2d mice, NS1 and NS4 are the dominant CD8+ T cell antigen and NS4 as the dominant CD4+ T cell antigen, respectively. Among the synthesized 364 overlapping polypeptides spanning the whole proteome of ZIKV, we mapped 91 and 39 polypeptides which can induce ZIKV-specific T cell responses in H-2b and H-2d mice, respectively. Through the identification of CD8+ T cell epitopes, we found that immunodominant regions E294-302 and NS42351-2360 are hotspots epitopes with a distinct immunodominance hierarchy present in H-2b and H-2d mice, respectively. Our data characterized an overall landscape of the immunogenic spectrum of the ZIKV polyprotein, and provide useful insight into the vaccine development.

Integrated System for On-Site Rapid and Safe Screening of COVID-19.

Since the outbreak of coronavirus disease 2019 (COVID-19), the epidemic has been spreading around the world for more than 2 years. Rapid, safe, and on-site detection methods of COVID-19 are in urgent demand for the control of the epidemic. Here, we established an integrated system, which incorporates a machine-learning-based Fourier transform infrared spectroscopy technique for rapid COVID-19 screening and air-plasma-based disinfection modules to prevent potential secondary infections. A partial least-squares discrimination analysis and a convolutional neural network model were built using the collected infrared spectral dataset containing 857 training serum samples. Furthermore, the sensitivity, specificity, and prediction accuracy could all reach over 94% from the results of the field test regarding 968 blind testing samples. Additionally, the disinfection modules achieved an inactivation efficiency of 99.9% for surface and airborne tested bacteria. The proposed system is conducive and promising for point-of-care and on-site COVID-19 screening in the mass population.

Impact of the COVID-19 epidemic anxiety on college students' employment confidence and employment situation perception in China.

The psychological problems and employment problems of college students have always been the focus of attention of all sectors of society. The COVID-19 epidemic has a great impact on the mental health and employment of Chinese college students. Under this background, this study discusses how epidemic anxiety affects the employment confidence and perception of employment situation of Chinese college students. Through the online questionnaire survey of 1,132 college students nationwide, and the ordinal logistic regression analysis of the survey data using Stata 16.0 software, the results show that: (1) Epidemic anxiety negatively affects Chinese college students' employment confidence and employment situation perception, and has a significant impact on employment confidence. The three control variables of employment guidance, older age and higher education have a significant positive impact on college students' employment confidence and employment situation perception. College students in the eastern region have stronger employment confidence and more optimistic employment situation perception. But the expected monthly salary is negatively correlated with employment confidence. (2) Male college students and Science and Engineering students' epidemic anxiety have a stronger negative impact on employment confidence and employment situation perception. (3) Employment guidance has a moderating effect on the relationship between epidemic anxiety, employment confidence and employment situation perception. Employment guidance can enhance college students' employment confidence and reduce their sense of employment crisis by alleviating epidemic anxiety. Combined with the research conclusions, it is proposed that the state and schools should pay attention to the psychological counseling of college students, strengthen the employment guidance of colleges and universities, vigorously support the development of small, medium-sized and micro enterprises, and improve the employment and entrepreneurship service system of college students, so as to promote the employment of college students.