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Recombinant vesicular stomatitis virus (rVSV) is a promising viral vaccine vector for addressing the COVID-19 pandemic. Inducing mucosal immunity via the intranasal route is an ideal strategy for rVSV-based vaccines, but it requires extremely stringent safety standards. In this study, we constructed two rVSV variants with amino acid mutations in their M protein: rVSV-M2 with M33A/M51R mutations and rVSV-M4 with M33A/M51R/V221F/S226R mutations, and developed COVID-19 vaccines based on these attenuated vectors. By comparing viral replication capacity, intranasal immunization, intracranial injection, and blood cell counts, we demonstrated that the M protein mutation variants exhibit significant attenuation effects both in vitro and in vivo. Moreover, preliminary investigations into the mechanisms of virus attenuation revealed that these attenuated viruses can induce a stronger type I interferon response while reducing inflammation compared to the wild-type rVSV. We developed three candidate vaccines against SARS-CoV-2 using the wildtype VSV backbone with either wild-type M (rVSV-JN.1) and two M mutant variants (rVSV-M2-JN.1 and rVSV-M4-JN.1). Our results confirmed that rVSV-M2-JN.1 and rVSV-M4-JN.1 retain strong immunogenicity while enhancing safety in hamsters. In summary, the rVSV variants with M protein mutations represent promising candidate vectors for mucosal vaccines and warrant further investigation.

The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants and “anatomical escape” characteristics threaten the effectiveness of current coronavirus disease 2019 (COVID-19) vaccines. There is an urgent need to understand the immunological mechanism of broad-spectrum respiratory tract protection to guide broader vaccines development. Here we investigate immune responses induced by an NS1-deleted influenza virus vectored intranasal COVID-19 vaccine (dNS1-RBD) which provides broad-spectrum protection against SARS-CoV-2 variants in hamsters. Intranasal delivery of dNS1-RBD induces innate immunity, trained immunity and tissue-resident memory T cells covering the upper and lower respiratory tract. It restrains the inflammatory response by suppressing early phase viral load post SARS-CoV-2 challenge and attenuating pro-inflammatory cytokine ( Il6 , Il1b , and Ifng ) levels, thereby reducing excess immune-induced tissue injury compared with the control group. By inducing local cellular immunity and trained immunity, intranasal delivery of NS1-deleted influenza virus vectored vaccine represents a broad-spectrum COVID-19 vaccine strategy to reduce disease burden. Understanding the utility of SARS -CoV-2 vaccination platforms and strategies through the emerging pandemic and beyond are critical to understanding the efficacy of such interventions. Here the authors assess nasal delivery of an influenza virus based viral vector for vaccination against SARS-CoV-2 in a hamsters model and assess the induced immunity.

The emergence of Rocahepevirus ratti genotype 1 (rat hepatitis E virus; rat HEV) in humans presents an unprecedented threat; however, the risk of rat HEV transmission to humans is not well understood. Here, we report the “Distinguishing Antibody Response Elicitation (DARE)” method, which distinguishes exposure to rat HEV. We use four study sets from China for large-scale population analysis: set 1 (hospital visit) and set 3 (ALT abnormality) from Yunnan province, a biodiversity hotspot, and set 2 (received physical examination) and set 4 (ALT abnormality) from Jiangsu province, a non-hotspot control region. rat HEV exposure risk is significantly higher in Yunnan, with 21.97% (190 of 865) in set 1 and 13.97% (70 of 501) in set 3, compared to 0.75% (9 of 1196) in Jiangsu’s set 2. Six spillover infections for rat HEV are identified in set 1, with one case of abnormal ALT. The rat-1d strains carried by rats are closely related to those human infections. Our study reveals the substantial spillover burden posed by rat HEV in biodiversity hotspots and highlights the utility of DARE method for proactive surveillance of public health emergencies. Rat hepatitis E virus (HEV) can infect humans, but the extent of spillover isn’t well studied. Here the authors develop a serological test that distinguishes exposure to rat HEV from other HEV infection and show substantial spillover in a biodiversity hotspot in China. The method can support surveillance of rat HEV.

Coxsackievirus A6 (CVA6) has emerged as a major pathogen causing hand, foot and mouth disease (HFMD) outbreaks worldwide. The CVA6 epidemic poses a new challenge in HFMD control since there is currently no vaccine available against CVA6 infections. The Vero cell line has been widely used in vaccine production, particularly in the preparation of viral vaccines, including poliovirus vaccines and EV71 vaccines. Unfortunately, most CVA6 strains failed to propagate effectively on Vero cells. The expression level of virus-specific receptors on the cell membrane significantly influences viral infection. Here, a Vero cell line with stable over-expressing of KREMEN1 (KRM1), a crucial receptor for CVA6, was constructed using the lentivirus system. The cloned cell line, called Vero-KRM1_#11, could efficiently support most CVA6 infections. The propagation of CVA6-TW00141 strain on Vero-KRM1_#11 was equal to that on RD cells. After four passages, the virus batch was obtained with a titer of about 10 7 TCID 50 /mL. Moreover, the purified CVA6 particles produced from Vero-KRM1_#11 or RD cells both could induce high and comparable levels of IgG and neutralizing antibodies. Importantly, passive transfer of the antisera from CVA6-vaccined mice showed 100% preventive efficacy against CVA6 infection in mice. Therefore, KRM1-expressing cells have the potential to serve as a valuable tool for the development and production of CVA6 or polyvalent HFMD vaccines.

ObjectivesWe aimed to evaluate the duration and breadth of antibodies elicited by inactivated COVID-19 vaccinations in healthy blood donors.MethodsWe performed serological tests on 1,417 samples from 658 blood donors who received two (n=357), or three (n=301) doses of COVID-19 inactivated vaccine. We also accessed the change in antibody response before and after booster vaccination in 94 participants and their neutralization breadth to the current variants after the booster.ResultsFollowing vaccination, for either the 2- or 3-dose, the neutralizing antibodies (nAbs) peaked with about 97% seropositivity approximately within one month but subsequently decreased over time. Of plasmas collected 6-8 months after the last immunization, the nAb seropositivities were 37% and 85% in populations with 2-dose and 3-dose vaccinations, respectively. The nAbs of plasma samples (collected between 2-6 weeks after the 3rd dose) from triple-vaccinated donors (n=94) showed a geometric mean titer of 145.3 (95% CI: 117.2 to 180.1) against the ancestral B.1, slightly reduced by 1.7-fold against Delta variant, but markedly decreased by 4-6 fold in neutralizing Omicron variants, including the sub-lineages of BA.1 (5.6-fold), BA.1.1 (6.0-fold), BA.2 (4.2-fold), B.2.12.1 (6.2-fold) and BA.4/5 (6.5-fold).ConclusionThese findings suggested that the 3rd dose of inactivated COVID-19 vaccine prolongs the antibody duration in healthy populations, but the elicited-nAbs are less efficient in neutralizing circulating Omicron variants.

Chronic hepatitis E virus (HEV) infection occurs mainly in immunosuppressed populations. We describe an investigation of chronic HEV infection of genotype 3a in an individual without evidence for immune deficiency who presented hepatitis with significant HEV viremia and viral shedding. We monitored HEV RNA in plasma and stools, and assessed anti-HEV specific immune responses. The patient was without apparent immunodeficiency based on quantified results of white blood cell, lymphocyte, neutrophilic granulocyte, CD3+ T cell, CD4+ T cell, and CD8+ T cell counts and CD4/CD8 ratio, as well as total serum IgG, IgM, and IgA, which were in the normal range. Despite HEV specific cellular response and strong humoral immunity being observed, viral shedding persisted up to 10 9 IU/mL. After treatment with ribavirin combined with interferon, the indicators of liver function in the patient returned to normal, accompanied by complete suppression and clearance of HEV. These results indicate that HEV chronicity can also occur in individuals without evidence of immunodeficiency.

Introduction: The effective and persistent suppression of hepatitis B surface antigen (HBsAg) in patients with chronic HBV infection (CHB) is considered to be a promising approach to achieve a functional cure of hepatitis B. In our previous study, we found that the antibody E6F6 can clear HBsAg through FcγR-mediated phagocytosis, and its humanized form (huE6F6 antibody) is expected to be a new tool for the treatment of CHB. Previous studies have shown that the glycosylation of Fc segments affects the binding of antibodies to FcγR and thus affects the biological activity of antibodies in vivo . Methods: To further improve the therapeutic potential of huE6F6, in this study, we defucosylated huE6F6 (huE6F6-fuc-), preliminarily explored the developability of this molecule, and studied the therapeutic potential of this molecule and its underlying mechanism in vitro and in vivo models. Results: huE6F6-fuc- has desirable physicochemical properties. Compared with huE6F6-wt, huE6F6-fuc- administration resulted in a stronger viral clearance in vivo . Meanwhile, huE6F6-fuc- keep a similar neutralization activity and binding activity to huE6F6-wt in vitro . Immunological analyses suggested that huE6F6-fuc- exhibited enhanced binding to hCD32b and hCD16b, which mainly contributed to its enhanced therapeutic activity in vivo . Conclusions: In summary, the huE6F6-fuc- molecule that was developed in this study, which has desirable developability, can clear HBsAg more efficiently in vivo , providing a promising treatment for CHB patients. Our study provides new guidance for antibody engineering in other disease fields.

Hepatitis E virus (HEV) is an important public health burden worldwide, causing approximately 20 million infections and 70,000 deaths annually. The viral capsid protein is encoded by open reading frame 2 (ORF2) of the HEV genome. Most ORF2 protein present in body fluids is the glycosylated secreted form of the protein (ORF2S). A recent study suggested that ORF2S is not necessary for the HEV life cycle. A previously reported efficient HEV cell culture system can be used to understand the origin and life cycle of ORF2S but is not sufficient for functional research. A more rapid and productive method for yielding ORF2S could help to study its antigenicity and immunogenicity. In this study, the ORF2S (tPA) expression construct was designed as a candidate tool. A set of representative anti-HEV monoclonal antibodies was further used to map the functional antigenic sites in the candidates. ORF2S (tPA) was used to study antigenicity and immunogenicity. Indirect ELISA revealed that ORF2S (tPA) was not antigenically identical to HEV 239 antigen (p239). The ORF2S-specific antibodies were successfully induced in one-dose-vaccinated BALB/c mice. The ORF2S-specific antibody response was detected in plasma from HEV-infected patients. Recombinant ORF2S (tPA) can act as a decoy to against B cells. Altogether, our study presents a design strategy for ORF2S expression and indicates that ORF2S (tPA) can be used for functional and structural studies of the HEV life cycle.

Background: Although immune checkpoint inhibitors (ICIs) have demonstrated efficacy in treating advanced cancers, their therapeutic success remains limited for many patients, with initial responders often experiencing resistance and relapse. Interleukin-12 (IL-12) is a powerful cytokine for antitumor immunotherapy, enhancing both lymphocyte recruitment into tumors and immune cell activation. Methods: In this study, we successfully produced mouse interleukin-12 (mIL12) through eukaryotic recombinant expression. In vivo, mIL12 exhibited significant control of tumor immunity in ICI-resistant and aggressive tumor models. Further mechanistic analysis indicated that treatment with mIL12 led to a substantial increase in tumor-infiltrating CD4+ T, CD8+ T, cDC1, and CD103+ cDC1 cells. Results: Our data underscore the potential of a combined therapeutic strategy involving IL-12 with PD-1 and CTLA-4 blockade to elicit a potent antitumor immune response. Notably, the co-administration of mIL12 and PD-1 blockade significantly enhanced the presence of central memory T cells (TCM) within tumors. Conclusions: This study is the first to provide evidence that the combination of mIL12 and PD-1 blockers promotes the generation of TCM, potentially contributing to a robust and durable antitumor effect.

Asparaginyl ligases have been extensively utilized as valuable tools for site-specific bioconjugation or surface-modification. However, the application is hindered by the laborious and poorly reproducible preparation processes, unstable activity and ambiguous substrate requirements. To address these limitations, this study employed a structure-based rational approach to obtain a high-yield and high-activity protein ligase called OaAEP1-C247A-aa55-351. It was observed that OaAEP1-C247A-aa55-351 exhibits appreciable catalytic activities across a wide pH range, and the addition of the Fe 3+ metal ion effectively enhances the catalytic power. Importantly, this study provides insight into the recognition and nucleophile peptide profiles of OaAEP1-C247A-aa55-351. The ligase demonstrates a higher recognition ability for the “Asn-Ala-Leu” motif and an N-terminus “Arg-Leu” as nucleophiles, which significantly increases the reaction yield. Consequently, the catalytic activity of OaAEP1-C247A-aa55-351 with highly efficient recognition and nucleophile motif, “Asn-Ala-Leu” and “Arg-Leu” under the buffer containing Fe 3+ is 70-fold and 2-fold higher than previously reported OaAEP1-C247A and the most efficient butelase-1, respectively. Thus, the designed OaAEP1-C247A-aa55-351, with its highly efficient recognition and alternative nucleophile options, holds promising potential for applications in protein engineering, chemo-enzymatic modification, and the development of drugs. Asparaginyl ligases have been utilized as valuable tools for protein engineering, such as through site-specific bioconjugation or surface modification, however, their application is limited due to time-consuming preparation processes and unstable activities. Here, the authors develop a truncated protein ligase OaAEP1-C247A-aa55-351 from an OaAEP1-C247A mutant, which simplifies the preparation steps, tolerates a wider pH range, and enhances the catalytic activities by using efficient recognition and nucleophile motifs.