Potential of HMGB-inhibitory oligodeoxynucleotide ISM ODN to neutrophil recruitment in mouse model of hepatitis.

High-mobility group box 1 (HMGB1) is a nucleotide-binding chromatin protein that has also been characterized as a prototypical damage-associate molecular pattern. It triggers inflammatory responses upon release from damaged or dying cells. In fact, HMGB1 has been linked to the induction of many inflammatory diseases through immune cell activation including neutrophil recruitment. In this study, we examined the impact of HMGB1-binding inhibitory oligodeoxynucleotide (ISM ODN) on the development of hepatitis using a murine model of the disease. Our results indicate that ISM ODN effectively suppresses pathological features of hepatitis, including neutrophil accumulation. This study therefore may offer clinical insight into the treatment of hepatitis and possibly other inflammatory diseases.

Characterization of a new SARS-CoV-2 variant that emerged in Brazil.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a key role in viral infectivity. It is also the major antigen stimulating the host's protective immune response, specifically, the production of neutralizing antibodies. Recently, a new variant of SARS-CoV-2 possessing multiple mutations in the S protein, designated P.1, emerged in Brazil. Here, we characterized a P.1 variant isolated in Japan by using Syrian hamsters, a well-established small animal model for the study of SARS-CoV-2 disease (COVID-19). In hamsters, the variant showed replicative abilities and pathogenicity similar to those of early and contemporary strains (i.e., SARS-CoV-2 bearing aspartic acid [D] or glycine [G] at position 614 of the S protein). Sera and/or plasma from convalescent patients and BNT162b2 messenger RNA vaccinees showed comparable neutralization titers across the P.1 variant, S-614D, and S-614G strains. In contrast, the S-614D and S-614G strains were less well recognized than the P.1 variant by serum from a P.1-infected patient. Prior infection with S-614D or S-614G strains efficiently prevented the replication of the P.1 variant in the lower respiratory tract of hamsters upon reinfection. In addition, passive transfer of neutralizing antibodies to hamsters infected with the P.1 variant or the S-614G strain led to reduced virus replication in the lower respiratory tract. However, the effect was less pronounced against the P.1 variant than the S-614G strain. These findings suggest that the P.1 variant may be somewhat antigenically different from the early and contemporary strains of SARS-CoV-2.

Transmission of SARS-CoV-2 in domestic cats imposes a narrow bottleneck.

The evolutionary mechanisms by which SARS-CoV-2 viruses adapt to mammalian hosts and, potentially, undergo antigenic evolution depend on the ways genetic variation is generated and selected within and between individual hosts. Using domestic cats as a model, we show that SARS-CoV-2 consensus sequences remain largely unchanged over time within hosts, while dynamic sub-consensus diversity reveals processes of genetic drift and weak purifying selection. We further identify a notable variant at amino acid position 655 in Spike (H655Y), which was previously shown to confer escape from human monoclonal antibodies. This variant arises rapidly and persists at intermediate frequencies in index cats. It also becomes fixed following transmission in two of three pairs. These dynamics suggest this site may be under positive selection in this system and illustrate how a variant can quickly arise and become fixed in parallel across multiple transmission pairs. Transmission of SARS-CoV-2 in cats involved a narrow bottleneck, with new infections founded by fewer than ten viruses. In RNA virus evolution, stochastic processes like narrow transmission bottlenecks and genetic drift typically act to constrain the overall pace of adaptive evolution. Our data suggest that here, positive selection in index cats followed by a narrow transmission bottleneck may have instead accelerated the fixation of S H655Y, a potentially beneficial SARS-CoV-2 variant. Overall, our study suggests species- and context-specific adaptations are likely to continue to emerge. This underscores the importance of continued genomic surveillance for new SARS-CoV-2 variants as well as heightened scrutiny for signatures of SARS-CoV-2 positive selection in humans and mammalian model systems.

Syrian hamsters as a small animal model for SARS-CoV-2 infection and countermeasure development.

At the end of 2019, a novel coronavirus (severe acute respiratory syndrome coronavirus 2; SARS-CoV-2) was detected in Wuhan, China, that spread rapidly around the world, with severe consequences for human health and the global economy. Here, we assessed the replicative ability and pathogenesis of SARS-CoV-2 isolates in Syrian hamsters. SARS-CoV-2 isolates replicated efficiently in the lungs of hamsters, causing severe pathological lung lesions following intranasal infection. In addition, microcomputed tomographic imaging revealed severe lung injury that shared characteristics with SARS-CoV-2-infected human lung, including severe, bilateral, peripherally distributed, multilobular ground glass opacity, and regions of lung consolidation. SARS-CoV-2-infected hamsters mounted neutralizing antibody responses and were protected against subsequent rechallenge with SARS-CoV-2. Moreover, passive transfer of convalescent serum to naïve hamsters efficiently suppressed the replication of the virus in the lungs even when the serum was administrated 2 d postinfection of the serum-treated hamsters. Collectively, these findings demonstrate that this Syrian hamster model will be useful for understanding SARS-CoV-2 pathogenesis and testing vaccines and antiviral drugs.

SARS-CoV-2 Interference of Influenza Virus Replication in Syrian Hamsters.

In hamsters, SARS-CoV-2 infection at the same time as or before H3N2 influenza virus infection resulted in significantly reduced influenza virus titers in the lungs and nasal turbinates. This interference may be correlated with SARS-CoV-2-induced expression of MX1.

Age-Stratified Seroprevalence of SARS-CoV-2 Antibodies before and during the Vaccination Era, Japan, February 2020-March 2022.

Japan has reported a relatively small number of COVID-19 cases. Because not all infected persons receive diagnostic tests for COVID-19, the reported number must be lower than the actual number of infections. We assessed SARS-CoV-2 seroprevalence by analyzing >60,000 samples collected in Japan (Tokyo Metropolitan Area and Hokkaido Prefecture) during February 2020-March 2022. The results showed that ≈3.8% of the population had become seropositive by January 2021. The seroprevalence increased with the administration of vaccinations; however, among the elderly, seroprevalence was not as high as the vaccination rate. Among children, who were not eligible for vaccination, infection was spread during the epidemic waves caused by the SARS-CoV-2 Delta and Omicron variants. Nevertheless, seroprevalence for unvaccinated children <5 years of age was as low as 10% as of March 2022. Our study underscores the low incidence of SARS-CoV-2 infection in Japan and the effects of vaccination on immunity at the population level.

Characterizing Emerging Canine H3 Influenza Viruses.

The continual emergence of novel influenza A strains from non-human hosts requires constant vigilance and the need for ongoing research to identify strains that may pose a human public health risk. Since 1999, canine H3 influenza A viruses (CIVs) have caused many thousands or millions of respiratory infections in dogs in the United States. While no human infections with CIVs have been reported to date, these viruses could pose a zoonotic risk. In these studies, the National Institutes of Allergy and Infectious Diseases (NIAID) Centers of Excellence for Influenza Research and Surveillance (CEIRS) network collaboratively demonstrated that CIVs replicated in some primary human cells and transmitted effectively in mammalian models. While people born after 1970 had little or no pre-existing humoral immunity against CIVs, the viruses were sensitive to existing antivirals and we identified a panel of H3 cross-reactive human monoclonal antibodies (hmAbs) that could have prophylactic and/or therapeutic value. Our data predict these CIVs posed a low risk to humans. Importantly, we showed that the CEIRS network could work together to provide basic research information important for characterizing emerging influenza viruses, although there were valuable lessons learned.

Pulmonary lesions induced by SARS-CoV-2 infection in domestic cats.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019, which ranges from fatal disease in some to mild or subclinical in most affected individuals. Many recovered human patients report persistent respiratory signs; however, lung disease in post-acute infection is poorly understood. Our objective was to describe histologic lung lesions and viral loads following experimental SARS-CoV-2 infection in 11 cats. Microscopic evaluation at 3, 6, 10, or 28 days postinoculation (DPI) identified mild to moderate patchy interstitial pneumonia, bronchiolar epithelial damage, and occlusive histiocytic bronchiolitis. Based on immunohistochemistry, alveolar septal thickening was due to CD204-positive macrophages, fewer B and T lymphocytes, type II pneumocytes, and capillary proliferation with a relative dearth of fibrosis. In blood vessel endothelium, there was reactive hypertrophy or vacuolar degeneration and increased MHC II expression at all time points. Unexpectedly, one cat from the 28 DPI group had severe subacute regionally extensive lymphohistiocytic pneumonia with multifocal consolidation, vasculitis, and alveolar fibrin. Reverse transcriptase-quantitative polymerase chain reaction identified SARS-CoV-2 RNA within the lung at 3 and 6 DPI, and viral RNA was below the limit of detection at 10 and 28 DPI, suggesting that pulmonary lesions persist beyond detection of viral RNA. These findings clarify our comparative understanding of disease induced by SARS-CoV-2 and suggest that cats can serve as an informative model to study post-acute pulmonary sequelae.

Protective Immunity and Persistent Lung Sequelae in Domestic Cats after SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus 2 readily transmits between domestic cats. We found that domestic cats that recover from an initial infection might be protected from reinfection. However, we found long-term persistence of inflammation and other lung lesions after infection, despite a lack of clinical symptoms and limited viral replication in the lungs.

Revisiting the role of IRF3 in inflammation and immunity by conditional and specifically targeted gene ablation in mice.

IFN regulatory factor 3 (IRF3) is a transcription regulator of cellular responses in many cell types that is known to be essential for innate immunity. To confirm IRF3's broad role in immunity and to more fully discern its role in various cellular subsets, we engineered Irf3-floxed mice to allow for the cell type-specific ablation of Irf3 Analysis of these mice confirmed the general requirement of IRF3 for the evocation of type I IFN responses in vitro and in vivo. Furthermore, immune cell ontogeny and frequencies of immune cell types were unaffected when Irf3 was selectively inactivated in either T cells or B cells in the mice. Interestingly, in a model of lipopolysaccharide-induced septic shock, selective Irf3 deficiency in myeloid cells led to reduced levels of type I IFN in the sera and increased survival of these mice, indicating the myeloid-specific, pathogenic role of the Toll-like receptor 4-IRF3 type I IFN axis in this model of sepsis. Thus, Irf3-floxed mice can serve as useful tool for further exploring the cell type-specific functions of this transcription factor.

Effectiveness of Whole, Inactivated, Low Pathogenicity Influenza A(H7N9) Vaccine against Antigenically Distinct, Highly Pathogenic H7N9 Virus.

The recent emergence of highly pathogenic influenza A(H7N9) variants poses a great risk to humans. We show that ferrets vaccinated with low pathogenicity H7N9 virus vaccine do not develop severe symptoms after infection with an antigenically distinct, highly pathogenic H7N9 virus. These results demonstrate the protective benefits of this H7N9 vaccine.

Characterization of a Feline Influenza A(H7N2) Virus.

During December 2016-February 2017, influenza A viruses of the H7N2 subtype infected ≈500 cats in animal shelters in New York, NY, USA, indicating virus transmission among cats. A veterinarian who treated the animals also became infected with feline influenza A(H7N2) virus and experienced respiratory symptoms. To understand the pathogenicity and transmissibility of these feline H7N2 viruses in mammals, we characterized them in vitro and in vivo. Feline H7N2 subtype viruses replicated in the respiratory organs of mice, ferrets, and cats without causing severe lesions. Direct contact transmission of feline H7N2 subtype viruses was detected in ferrets and cats; in cats, exposed animals were also infected via respiratory droplet transmission. These results suggest that the feline H7N2 subtype viruses could spread among cats and also infect humans. Outbreaks of the feline H7N2 viruses could, therefore, pose a risk to public health.

Structural basis for the major role of O-phosphoseryl-tRNA kinase in the UGA-specific encoding of selenocysteine.

The 21(st) amino acid, selenocysteine (Sec), is assigned to the codon UGA and is biosynthesized on the selenocysteine-specific tRNA (tRNA(Sec)) with the corresponding anticodon. In archaea/eukarya, tRNA(Sec) is ligated with serine by seryl-tRNA synthetase (SerRS), the seryl moiety is phosphorylated by O-phosphoseryl-tRNA kinase (PSTK), and the phosphate group is replaced with selenol by Sep-tRNA:Sec-tRNA synthase. PSTK selectively phosphorylates seryl-tRNA(Sec), while SerRS serylates both tRNA(Ser) and tRNA(Sec). In this study, we determined the crystal structures of the archaeal tRNA(Sec).PSTK complex. PSTK consists of two independent linker-connected domains, the N-terminal catalytic domain (NTD) and the C-terminal domain (CTD). The D-arm.CTD binding occurs independently of and much more strongly than the acceptor-arm.NTD binding. PSTK thereby distinguishes the characteristic D arm with the maximal stem and the minimal loop of tRNA(Sec) from the canonical D arm of tRNA(Ser), without interacting with the anticodon. This mechanism is essential for the UGA-specific encoding of selenocysteine.

Suppression of immune responses by nonimmunogenic oligodeoxynucleotides with high affinity for high-mobility group box proteins (HMGBs).

The activation of innate immune responses by nucleic acids is central to the generation of host responses against pathogens; however, nucleic acids can also trigger the development and/or exacerbation of pathogenic responses such as autoimmunity. We previously demonstrated that the selective activation of nucleic acid-sensing cytosolic and Toll-like receptors is contingent on the promiscuous sensing of nucleic acids by high-mobility group box proteins (HMGBs). From this, we reasoned that nonimmunogenic nucleotides with high-affinity HMGB binding may function as suppressing agents for HMGB-mediated diseases, particularly those initiated and/or exacerbated by nucleic acids. Here we characterize an array of HMGB-binding, nonimmunogenic oligodeoxynucleotides (ni-ODNs). Interestingly, we find that binding affinity is rather independent of nucleotide sequence, but is instead dependent on length and structure of the deoxyribose backbone. We further show that these ni-ODNs can strongly suppress the activation of innate immune responses induced by both classes of nucleic acid-sensing receptors. We also provide evidence for the suppressive effect of an ni-ODN, termed ISM ODN, on the induction of adaptive immune responses and in mouse models of sepsis and autoimmunity. We discuss our findings in relation to the critical role of HMGBs in initiating immune responses and the possible use of these ni-ODNs in therapeutic interventions.

Characterization of a human H3N8 influenza virus.

BACKGROUND: In 2022 and 2023, novel reassortant H3N8 influenza viruses infected three people, marking the first human infections with viruses of this subtype. METHODS: Here, we generated one of these viruses (A/Henan/4-10CNIC/2022; hereafter called A/Henan/2022 virus) by using reverse genetics and characterized it. FINDINGS: In intranasally infected mice, reverse genetics-generated A/Henan/2022 virus caused weight loss in all five animals (one of which had to be euthanized) and replicated efficiently in the respiratory tract. Intranasal infection of ferrets resulted in minor weight loss and moderate fever but no mortality. Reverse genetics-generated A/Henan/2022 virus replicated efficiently in the upper respiratory tract of ferrets but was not detected in the lungs. Virus transmission via respiratory droplets occurred in one of four pairs of ferrets. Deep-sequencing of nasal swab samples from inoculated and exposed ferrets revealed sequence polymorphisms in the haemagglutinin protein that may affect receptor-binding specificity. We also tested 90 human sera for neutralizing antibodies against reverse genetics-generated A/Henan/2022 virus and found that some of them possessed neutralizing antibody titres, especially sera from older donors with likely exposure to earlier human H3N2 viruses. INTERPRETATION: Our data demonstrate that reverse genetics-generated A/Henan/2022 virus is a low pathogenic influenza virus (of avian influenza virus descent) with some antigenic resemblance to older human H3N2 viruses and limited respiratory droplet transmissibility in ferrets. FUNDING: This work was supported by the Japan Program for Infectious Diseases Research and Infrastructure (JP23wm0125002), and the Japan Initiative for World-leading Vaccine Research and Development Centers (JP233fa627001) from the Japan Agency for Medical Research and Development (AMED).

Influenza H3 hemagglutinin vaccine with scrambled immunodominant epitopes elicits antibodies directed toward immunosubdominant head epitopes.

Vaccination is the most effective countermeasure to reduce the severity of influenza. Current seasonal influenza vaccines mainly elicit humoral immunity targeting hemagglutinin (HA). In particular, the amino acid residues around the receptor-binding site in the HA head domain are predominantly targeted by humoral immunity as "immunodominant" epitopes. However, mutations readily accumulate in the head domain due to high plasticity, resulting in antigenic drift and vaccine mismatch, particularly with influenza A (H3N2) viruses. A vaccine strategy that targets more conserved immunosubdominant epitopes is required to attain a universal vaccine. Here, we designed an H3 HA vaccine antigen with various amino acids at immunodominant epitopes of the HA head domain, termed scrambled HA (scrHA). In ferrets, scrHA vaccination induced lower serum neutralizing antibody levels against homologous virus compared with wild-type (WT) HA vaccination; however, similar levels of moderately neutralizing titers against antigenically distinct H3N2 viruses were observed. Ferrets vaccinated with scrHA twice and then challenged with homologous or heterologous virus showed the same level of reduced virus shedding in nasal swabs as WT HA-vaccinated animals but reduced body temperature increase, whereas WT HA-vaccinated ferrets exhibited body temperature increases similar to those of mock-vaccinated animals. scrHA elicited antibodies against HA immunodominant and -subdominant epitopes at lower and higher levels, respectively, than WT HA vaccination, whereas antistalk antibodies were induced at the same level for both groups, suggesting scrHA-induced redirection from immunodominant to immunosubdominant head epitopes. scrHA vaccination thus induced broader coverage than WT HA vaccination by diluting out the immunodominancy of HA head epitopes. IMPORTANCE Current influenza vaccines mainly elicit antibodies that target the immunodominant head domain, where strain-specific mutations rapidly accumulate, resulting in frequent antigenic drift and vaccine mismatch. Targeting conserved immunosubdominant epitopes is essential to attain a universal vaccine. Our findings with the scrHA developed in this study suggest that designing vaccine antigens that "dilute out" the immunodominancy of the head epitopes may be an effective strategy to induce conserved immunosubdominant epitope-based immune responses.

Ferret model to mimic the sequential exposure of humans to historical H3N2 influenza viruses.

Mutations accumulate in influenza A virus proteins, especially in the main epitopes on the virus surface glycoprotein hemagglutinin (HA). For influenza A(H3N2) viruses, in particular, the antigenicity of their HA has altered since their emergence in 1968, requiring changes of vaccine strains every few years. Most adults have been exposed to several antigenically divergent H3N2 viruses through infection and/or vaccination, and those exposures affect the immune responses of those individuals. However, animal models reflecting this 'immune history' in humans are lacking and naïve animals are generally used for vaccination and virus challenge studies. Here, we describe a ferret model to mimic the serial exposure of humans to antigenically different historical H3HA proteins. In this model, ferrets were sequentially immunized with adjuvanted recombinant H3HA proteins from two or three different H3HA antigenic clusters in chronological order, and serum neutralizing antibody titers were examined against the homologous virus and viruses from different antigenic clusters. For ferrets immunized with a single HA antigen, serum neutralizing antibody titers were elevated specifically against the homologous virus. However, after immunization with the second or third antigenically distinct HA antigen in chronological order, the ferrets showed an increase in more broadly cross-reactive neutralizing titers against the antigenically distinct viruses and against the homologous virus. Sequentially immunized animals challenged with an antigenically advanced H3N2 virus showed attenuated virus growth and less body temperature increase compared with naïve animals. These results suggest that sequential exposure to antigenically different HAs elicits broader neutralizing activity in sera and enhances immune responses against more antigenically distinct viruses Our findings may partly explain why adults who have been exposed to antigenically divergent HAs are less likely to be infected with influenza virus and have severe symptoms than children.

Recombinant spike protein vaccines coupled with adjuvants that have different modes of action induce protective immunity against SARS-CoV-2.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a glycoprotein, expressed on the virion surface, that mediates infection of host cells by directly interacting with host receptors. As such, it is a reasonable target to neutralize the infectivity of the virus. Here we found that a recombinant S protein vaccine adjuvanted with Alhydrogel or the QS-21-like adjuvant Quil-A effectively induced anti-S receptor binding domain (RBD) serum IgG and neutralizing antibody titers in the Syrian hamster model, resulting in significantly low SARS-CoV-2 replication in respiratory organs and reduced body weight loss upon virus challenge. Severe lung inflammation upon virus challenge was also strongly suppressed by vaccination. We also found that the S protein vaccine adjuvanted with Alhydrogel, Quil-A, or an AS03-like adjuvant elicited significantly higher neutralizing antibody titers in mice than did unadjuvanted vaccine. Although the neutralizing antibody titers against the variant viruses B.1.351 and B.1.617.2 declined markedly in mice immunized with wild-type S protein, the binding antibody levels against the variant S proteins were equivalent to those against wild-type S. When splenocytes from the immunized mice were re-stimulated with the S protein in vitro, the induced Th1 or Th2 cytokine levels were not significantly different upon re-stimulation with wild-type S or variant S, suggesting that the T-cell responses against the variants were the same as those against the wild-type virus. Upon Omicron XBB-challenge in hamsters, wild-type S-vaccination with Alhydrogel or AS03 reduced lung virus titers on Day 3, and the Quil-A adjuvanted group showed less body weight loss, although serum neutralizing antibody titers against XBB were barely detected in vitro. Collectively, recombinant vaccines coupled with different adjuvants may be promising modalities to combat new variant viruses by inducing various arms of the immune response.