Comparative analysis of the efficacy of vaccines using structural protein subunits of the severe fever with thrombocytopenia syndrome virus.

The severe fever with thrombocytopenia syndrome virus (SFTSV) represents a significant emerging health threat as a tick-borne pathogen that causes SFTS, with mortality rates ranging between 10 and 30%. Despite the considerable risk presented by SFTSV, an effective vaccine has yet to be developed. Our study assessed the efficacy of recombinant protein vaccines, focusing on the purified nucleocapsid protein (NP) and surface glycoproteins (Gn and Gc), against SFTSV in both singular and combined formulations. Individual vaccinations with NP or Gn subunits yielded partial protection in type I interferon receptor-knockout (IFNAR-KO) mice, with survival rates of 66.7 and 16.7%, respectively, whereas Gc vaccination did not confer significant protection, resulting in 100% mortality similar to that of the unvaccinated control group. Notably, NP vaccination substantially enhanced antigen-specific T cell responses, and Gc vaccination exhibited strong neutralizing activity against SFTSV. Among the combined recombinant protein formulations (Gn + NP, Gc + NP, and Gn + Gc + NP) tested, the Gc + NP combination provided the highest survival rate (85.7%) following challenge with a lethal dose of SFTSV, highlighting its potential as a vaccine candidate. Longitudinal studies showed that antibody levels in both wild type C57BL/6 and IFNAR-KO mice peaked between 2 and 3 months post-vaccination and declined over time. A notable decrease in NP-specific CD8+ T cell responses was observed 6 months post-vaccination in C57BL/6 mice, while NP-specific CD4+ T cell responses persisted up to 12 months. By 12 months post-vaccination, all IFNAR-KO mice vaccinated with single subunit antigens succumbed to the virus, suggesting that effective protection against SFTS may rely on antibody responses to subunit antigens and/or CD8+ T cell activity. These findings underscore the necessity of an optimized SFTS vaccine that combines protective antigens with an adjuvant system to ensure durable humoral and cellular immunity.

Rise in broadly cross-reactive adaptive immunity against human ß-coronaviruses in MERS-recovered patients during the COVID-19 pandemic.

To develop a universal coronavirus (CoV) vaccine, long-term immunity against multiple CoVs, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, Middle East respiratory syndrome (MERS)-CoV, and future CoV strains, is crucial. Following the 2015 Korean MERS outbreak, we conducted a long-term follow-up study and found that although neutralizing antibodies and memory T cells against MERS-CoV declined over 5 years, some recovered patients exhibited increased antibody levels during the COVID-19 pandemic. This likely resulted from cross-reactive immunity induced by SARS-CoV-2 vaccines or infections. A significant correlation in antibody responses across various CoVs indicates shared immunogenic epitopes. Two epitopes-the spike protein's stem helix and intracellular domain-were highly immunogenic after MERS-CoV infection and after SARS-CoV-2 vaccination or infection. In addition, memory T cell responses, especially polyfunctional CD4+ T cells, were enhanced during the pandemic, correlating significantly with MERS-CoV spike-specific antibodies and neutralizing activity. Therefore, incorporating these cross-reactive and immunogenic epitopes into pan-CoV vaccine formulations may facilitate effective vaccine development.

High-resolution phylogeographical surveillance of Hantaan orthohantavirus using rapid amplicon-based Flongle sequencing, Republic of Korea.

Orthohantaviruses, etiological agents of hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome, pose a critical public health threat worldwide. Hantaan orthohantavirus (HTNV) outbreaks are particularly endemic in Gyeonggi Province in northern area of the Republic of Korea (ROK). Small mammals were collected from three regions in the Gyeonggi Province during 2017 and 2018. Serological and molecular prevalence of HTNV was 25/201 (12.4%) and 10/25 (40%), respectively. A novel nanopore-based diagnostic assay using a cost-efficient Flongle chip was developed to rapidly and sensitively detect HTNV infection in rodent specimens within 3 h. A rapid phylogeographical surveillance of HTNV at high-resolution phylogeny was established using the amplicon-based Flongle sequencing. In total, seven whole-genome sequences of HTNV were newly obtained from wild rodents collected in Paju-si (Gaekhyeon-ri) and Yeoncheon-gun (Hyeonga-ri and Wangnim-ri), Gyeonggi Province. Phylogenetic analyses revealed well-supported evolutionary divergence and genetic diversity, enhancing the resolution of the phylogeographic map of orthohantaviruses in the ROK. Incongruences in phylogenetic patterns were identified among HTNV tripartite genomes, suggesting differential evolution for each segment. These findings provide crucial insights into on-site diagnostics, genome-based surveillance, and the evolutionary dynamics of orthohantaviruses to mitigate hantaviral outbreaks in HFRS-endemic areas in the ROK.

mRNA vaccine encoding Gn provides protection against severe fever with thrombocytopenia syndrome virus in mice.

We developed a promising mRNA vaccine against severe fever with thrombocytopenia syndrome (SFTS), an infectious disease caused by the SFTS virus that is primarily transmitted through tick bites. Administration of lipid nanoparticle-encapsulated mRNA-Gn successfully induced neutralizing antibodies and T-cell responses in mice. The vaccinated mice were protected against a lethal SFTS virus challenge, suggesting that this mRNA vaccine may be an effective and successful SFTS vaccine candidate.

Dual-Armed Oncolytic Myxoma Virus Encoding IFN-γ and CD47 Promotes Lymphocyte Infiltration and Tumor Suppression of Syngeneic Murine Melanoma.

Myxoma virus (MyxV) is a rabbit-specific poxvirus. However, its ability to selectively target tumor cells has established it as a safe and effective anticancer therapy. To strengthen its preclinical efficacy, transgenes that can prolong cancer cell infection and enhance anti-tumor effector functions are currently being investigated. We engineered MyxV armed with CD47, to turn on a 'do not eat me' signal within infected cells with actively replicating viruses, and with IFN-γ to further activate host immune anticancer responses. Tumor suppressive activities were significantly enhanced by the dual-armed MyxV_CD47/IFN-γ compared to parental MyxV or single-armed MyxV_CD47 or MyxV_IFN-γ. In addition, significant increases in IFN-γ+ CD8+T-cells and CD4+ T-cells populations within tumor-infiltrating lymphocytes (TIL) were observed after MyxV_CD47/IFN-γ treatment. Notably, all groups treated with MyxV showed a marked reduction in Foxp3+ CD4+ regulatory T-cells (Tregs) within TIL. We also show that MyxV infection induces PD-L1 up-regulation in cancer cells, and combinational treatment of MyxV with anti-mouse PD-L1 antibodies (αPD-L1) further controlled tumor burden and increased survival in the syngeneic melanoma model B16F10. Our data demonstrate that a CD47 and IFNγ dual-armed MyxV is an effective oncolytic viral immunotherapeutic. These findings strongly support further preclinical investigations to develop next-generation MyxV-based immunotherapy approaches.

Genome-informed investigation of the molecular evolution and genetic reassortment of severe fever with thrombocytopenia syndrome virus.

BACKGROUND: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a viral pathogen causing significant clinical signs from mild fever with thrombocytopenia to severe hemorrhages. World Health Organization has paid special attention to the dramatic increase in human SFTS cases in China, Japan, and South Korea since the 2010s. The present study investigated the molecular evolution and genetic reassortment of SFTSVs using complete genomic sequences. METHODS/PRINCIPAL FINDING: We collected the complete genome sequences of SFTSVs globally isolated until 2019 (L segment, n = 307; M segment, n = 326; and S segment, n = 564) and evaluated the evolutionary profiles of SFTSVs based on phylogenetic and molecular selection pressure analyses. By employing a time-scaled Bayesian inference method, we found the geographical heterogeneity of dominant SFTSV genotypes in China, Japan, and South Korea around several centuries before and locally spread by tick-born spillover with infrequent long-distance transmission. Purifying selection predominated the molecular evolution of SFTSVs with limited gene reassortment and fixed substitution, but almost all three gene segments appeared to harbor at least one amino acid residue under positive selection. Specifically, the nonstructural protein and glycoprotein (Gn/Gc) genes were preferential selective targets, and the Gn region retained the highest number of positively selected residues. CONCLUSION/SIGNIFICANCE: Here, the large-scale genomic analyses of SFTSVs improved prior knowledge of how this virus emerged and evolved in China, Japan, and South Korea. Our results highlight the importance of SFTSV surveillance in both human and non-human reservoirs at the molecular level to fight against fatal human infection with the virus.

A natural variation in the RNA polymerase of severe fever with thrombocytopenia syndrome virus enhances viral replication and in vivo virulence.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease with high mortality in Eastern Asia. The disease is caused by the SFTS virus (SFTSV), also known as Dabie bandavirus, which has a segmented RNA genome consisting of L, M, and S segments. Previous studies have suggested differential viral virulence depending on the genotypes of SFTSV; however, the critical viral factor involved in the differential viral virulence is unknown. Here, we found a significant difference in viral replication in vitro and virulence in vivo between two Korean isolates belonging to the F and B genotypes, respectively. By generating viral reassortants using the two viral strains, we demonstrated that the L segment, which encodes viral RNA-dependent RNA polymerase (RdRp), is responsible for the enhanced viral replication and virulence. Comparison of amino acid sequences and viral replication rates revealed a point variation, E251K, on the surface of RdRp to be the most significant determinant for the enhanced viral replication rate and in vivo virulence. The effect of the variation was further confirmed using recombinant SFTSV generated by reverse genetic engineering. Therefore, our results indicate that natural variations affecting the viral replicase activity could significantly contribute to the viral virulence of SFTSV.

Evaluation of humoral immune response in relation to COVID-19 severity over 1 year post-infection: critical cases higher humoral immune response than mild cases.

Introduction: Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2. We investigated the antibody response against SARS-CoV-2 until 1 year after symptom onset. Methods: We collected 314 serum samples from 97 patients with COVID-19. Antibody responses were tested using an indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), and plaque reduction neutralization test (PRNT) to detect specific neutralizing antibodies. Results: The positivity rates for neutralizing antibodies at a 1:10 titer cutoff were 58.1% at 1 week, 97.8% at 4 weeks, and 78% at 1 year after symptom onset (53.8% in asymptomatic patients and 89.3% in symptomatic patients). The IFA and anti-S1 ELISA IgG results significantly correlated with neutralizing antibody titers. Critical/fatal cases showed significantly higher antibody titers than the asymptomatic or mild-to-moderate illness groups. Nonetheless, the median number of days to the seroconversion of neutralizing antibodies was 10 and 15 in asymptomatic and symptomatic patients, respectively. The asymptomatic group had a significantly higher neutralizing potency index than the mild-to-severe illness groups. Conclusions: Neutralizing antibodies corresponded to earlier seroconversion but had a shorter presence in the asymptomatic group than in the symptomatic group and were still present 1 year after symptom onset in critical/fatal cases.

A single-cell atlas of in vitro multiculture systems uncovers the in vivo lineage trajectory and cell state in the human lung.

We present an in-depth single-cell atlas of in vitro multiculture systems on human primary airway epithelium derived from normal and diseased lungs of 27 individual donors. Our large-scale single-cell profiling identified new cell states and differentiation trajectories of rare airway epithelial cell types in human distal lungs. By integrating single-cell datasets of human lung tissues, we discovered immune-primed subsets enriched in lungs and organoids derived from patients with chronic respiratory disease. To demonstrate the full potential of our platform, we further illustrate transcriptomic responses to various respiratory virus infections in vitro airway models. Our work constitutes a single-cell roadmap for the cellular and molecular characteristics of human primary lung cells in vitro and their relevance to human tissues in vivo.

SARS-CoV-2 Infection Induces HMGB1 Secretion Through Post-Translational Modification and PANoptosis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induces excessive pro-inflammatory cytokine release and cell death, leading to organ damage and mortality. High-mobility group box 1 (HMGB1) is one of the damage-associated molecular patterns that can be secreted by pro-inflammatory stimuli, including viral infections, and its excessive secretion levels are related to a variety of inflammatory diseases. Here, the aim of the study was to show that SARS-CoV-2 infection induced HMGB1 secretion via active and passive release. Active HMGB1 secretion was mediated by post-translational modifications, such as acetylation, phosphorylation, and oxidation in HEK293E/ACE2-C-GFP and Calu-3 cells during SARS-CoV-2 infection. Passive release of HMGB1 has been linked to various types of cell death; however, we demonstrated for the first time that PANoptosis, which integrates other cell death pathways, including pyroptosis, apoptosis, and necroptosis, is related to passive HMGB1 release during SARS-CoV-2 infection. In addition, cytoplasmic translocation and extracellular secretion or release of HMGB1 were confirmed via immunohistochemistry and immunofluorescence in the lung tissues of humans and angiotensin-converting enzyme 2-overexpressing mice infected with SARS-CoV-2.

Enhancing immune protection against MERS-CoV: the synergistic effect of proteolytic cleavage sites and the fusion peptide and RBD domain targeting VLP immunization.

Introduction: The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a zoonotic infectious virus that has caused significant outbreaks in the Middle East and beyond. Due to a highly mortality rate, easy transmission, and rapid spread of the MERS-CoV, it remains as a significant public health treat. There is currently no licensed vaccine available to protect against MERS-CoV. Methods: In this study, we investigated whether the proteolytic cleavage sites and fusion peptide domain of the MERS-CoV spike (S) protein could be a vaccine target to elicit the MERS-CoV S protein-specific antibody responses and confer immune protection against MERS-CoV infection. Our results demonstrate that immunization of the proteolytic cleavage sites and the fusion peptide domain using virus-like particle (VLP) induced the MERS-CoV S protein-specific IgG antibodies with capacity to neutralize pseudotyped MERS-CoV infection in vitro. Moreover, proteolytic cleavage sites and the fusion peptide VLP immunization showed a synergistic effect on the immune protection against MERS-CoV infection elicited by immunization with VLP expressing the receptor binding domain (RBD) of the S protein. Additionally, immune evasion of MERS-CoV RBD variants from anti-RBD sera was significantly controlled by anti-proteolytic cleavage sites and the fusion peptide sera. Conclusion and discussion: Our study demonstrates the potential of VLP immunization targeting the proteolytic cleavage sites and the fusion peptide and RBD domains of the MERS-CoV S protein for the development of effective treatments and vaccines against MERS-CoV and related variants.

Fatal outcome of severe fever with thrombocytopenia syndrome (SFTS) and severe and critical COVID-19 is associated with the hyperproduction of IL-10 and IL-6 and the low production of TGF-ß.

Severe fever with thrombocytopenia syndrome virus (SFTSV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause the hyperproduction of inflammatory cytokines, which have pathological effects in patient including severe or fatal cytokine storms. To characterize the effect of SFTSV and SARS-CoV-2 infection on the production of cytokines in severe fever with thrombocytopenia syndrome (SFTS) and COVID-19 patients, we performed an analysis of cytokines in SFTS and COVID-19 patients and also investigated the role of interleukin-10 (IL-10) in vitro studies: lipopolysaccharide-induced THP-1-derived macrophages, SFTSV infection of THP-1 cells, and SARS-CoV-2 infection of THP-1 cells. In this study, we found that levels of both IL-10 and IL-6 were significantly elevated, the level of transforming growth factor-ß (TGF-ß) was significantly decreased and IL-10 was elevated earlier than IL-6 in severe and critical COVID-19 and fatal SFTS patients, and inhibition of IL-10 signaling decreased the production of IL-6 and elevated that of TGF-ß. Therefore, the hyperproduction of IL-10 and IL-6 and the low production of TGF-ß have been linked to cytokine storm-induced mortality in fatal SFTS and severe and critically ill COVID-19 patients and that IL-10 can play an important role in the host immune response to severe and critical SARS-CoV-2 and fatal SFTSV infection.

Heterologous vaccination utilizing viral vector and protein platforms confers complete protection against SFTSV.

Severe fever with thrombocytopenia syndrome virus was first discovered in 2009 as the causative agent of severe fever with thrombocytopenia syndrome. Despite its potential threat to public health, no prophylactic vaccine is yet available. This study developed a heterologous prime-boost strategy comprising priming with recombinant replication-deficient human adenovirus type 5 (rAd5) expressing the surface glycoprotein, Gn, and boosting with Gn protein. This vaccination regimen induced balanced Th1/Th2 immune responses and resulted in potent humoral and T cell-mediated responses in mice. It elicited high neutralizing antibody titers in both mice and non-human primates. Transcriptome analysis revealed that rAd5 and Gn proteins induced adaptive and innate immune pathways, respectively. This study provides immunological and mechanistic insight into this heterologous regimen and paves the way for future strategies against emerging infectious diseases.

Corrigendum: Acute surge of atypical memory and plasma B-cell subsets driven by an extrafollicular response in severe COVID-19.

[This corrects the article DOI: 10.3389/fcimb.2022.909218.].

Corrigendum: Differential association of viral dynamics with disease severity depending on patients' age group in COVID-19.

[This corrects the article DOI: 10.3389/fmicb.2021.712260.].

Corrigendum: Elevated IFNA1 and suppressed IL12p40 associated with persistent hyperinflammation in COVID-19 pneumonia.

[This corrects the article DOI: 10.3389/fimmu.2023.1101808.].

Elevated IFNA1 and suppressed IL12p40 associated with persistent hyperinflammation in COVID-19 pneumonia.

Introduction: Despite of massive endeavors to characterize inflammation in COVID-19 patients, the core network of inflammatory mediators responsible for severe pneumonia stillremain remains elusive. Methods: Here, we performed quantitative and kinetic analysis of 191 inflammatory factors in 955 plasma samples from 80 normal controls (sample n = 80) and 347 confirmed COVID-19 pneumonia patients (sample n = 875), including 8 deceased patients. Results: Differential expression analysis showed that 76% of plasmaproteins (145 factors) were upregulated in severe COVID-19 patients comparedwith moderate patients, confirming overt inflammatory responses in severe COVID-19 pneumonia patients. Global correlation analysis of the plasma factorsrevealed two core inflammatory modules, core I and II, comprising mainly myeloid cell and lymphoid cell compartments, respectively, with enhanced impact in a severity-dependent manner. We observed elevated IFNA1 and suppressed IL12p40, presenting a robust inverse correlation in severe patients, which was strongly associated with persistent hyperinflammation in 8.3% of moderate pneumonia patients and 59.4% of severe patients. Discussion: Aberrant persistence of pulmonary and systemic inflammation might be associated with long COVID-19 sequelae. Our comprehensive analysis of inflammatory mediators in plasmarevealed the complexity of pneumonic inflammation in COVID-19 patients anddefined critical modules responsible for severe pneumonic progression.

An Alternative Splicing Variant of the Mixed-Lineage Leukemia 5 Protein Is a Cellular Adhesion Receptor for ScaA of Orientia tsutsugamushi.

Scrub typhus is a mite-borne disease caused by the obligately intracellular bacterium Orientia tsutsugamushi. We previously demonstrated that ScaA, an autotransporter membrane protein of O. tsutsugamushi, is commonly shared in various genotypes and involved in adherence to host cells. Here, we identified a mixed-lineage leukemia 5 (MLL5) mammalian trithorax group protein as a host receptor that interacts with ScaA. MLL5, identified by yeast two-hybrid screening, is an alternative splicing variant of MLL5 (vMLL5) which contains 13 exons with additional intron sequences encoding a tentative transmembrane domain. Indeed, vMLL5 is expressed on the plasma membrane as well as in intracellular compartments in eukaryotic cells and colocalized with adherent O. tsutsugamushi. In addition, ScaA-expressing Escherichia coli showed significantly increased adherence to vMLL5-overexpressing cells compared with vector control cells. We mapped the C-terminal region of the passenger domain of ScaA as a ligand for vMLL5 and determined that the Su(var)3-9, Enhancer of zeste, Trithorax (SET) domain of MLL5 is an essential and sufficient motif for ScaA binding. We observed significant and specific inhibition of bacterial adhesion to host cells in competitive inhibition assays using the C-terminal fragment of ScaA or the SET domain of vMLL5. Moreover, immunization with the C-terminal fragment of ScaA provided neutralizing activity and protective immunity against lethal challenge with O. tsutsugamushi as efficiently as vaccination with the whole passenger domain of ScaA. These results indicate that vMLL5 is a novel cellular receptor for ScaA-mediated adhesion of O. tsutsugamushi and facilitates bacterial adhesion to host cells, thereby enhancing bacterial infection. IMPORTANCE O. tsutsugamushi is a mite-borne pathogen that causes scrub typhus. As an obligately intracellular pathogen, its adhesion to and invasion of host cells are critical steps for bacterial growth. However, the molecular basis of the bacterial ligand and host receptor interaction is poorly defined. Here, we identified a splicing variant of MLL5 (vMLL5) as a cellular adhesion receptor of ScaA, an outer membrane autotransporter protein of O. tsutsugamushi. We mapped the interacting domains in the bacterial ligand and host receptor and confirmed their functional interaction. In addition, immunization with the C-terminal region of ScaA, which involves an interaction with the SET domain of vMLL5, not only induces enhanced neutralizing antibodies but also provides protective immunity against lethal challenge with O. tsutsugamushi.

Serological Evidence of Severe Fever with Thrombocytopenia Syndrome Virus and IgM Positivity Were Identified in Healthy Residents in Vietnam.

Severe fever with thrombocytopenia syndrome (SFTS), an emerging tick-borne viral disease, is prevalent in East Asia and has also been reported in Southeast Asia since 2019. SFTS patients in Vietnam were first reported in 2019. However, the seroprevalence of severe fever with thrombocytopenia syndrome virus (SFTSV) in Vietnam has not been reported. To investigate the seroprevalence of SFTSV in Vietnam, we collected serum samples from 714 healthy residents in Thua Thien Hue and Quang Nam Province, Vietnam, and the seroprevalence of SFTSV was assessed using immunofluorescence antibody assay (IFA), Enzyme-Linked Immunosorbent Assays (ELISAs) and the 50% focus reduction neutralization test (FRNT50) assay. The seroprevalence of anti-SFTSV IgM or IgG was observed to be 3.64% (26/714), high IgM positivity was >80 (0.28%, 2/714) and the titer of neutralizing antibodies against SFTSV ranged from 15.5 to 55.9. In Pakistan, SFTSV infection confirmed using a microneutralization test (MNT) assay (prevalence is 2.5%) and ELISAs showed a high seroprevalence (46.7%) of SFTSV. Hence, the seroprevalence rate in Vietnam is similar to that in Pakistan and the number of SFTS patients could increase in Vietnam.