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Background: The mucosa serves as the first defence against pathogens and facilitates the surveillance and elimination of symbiotic bacteria by mucosal immunity. Recently, the mRNA vaccine against SARS-CoV-2 has been demonstrated to induce secretory antibodies in the oral and nasal cavities in addition to a systemic immune response. However, the mechanism of induced immune stimulation effect on mucosal immunity and commensal bacteria profile remains unclear. Methods: Here, we longitudinally analysed the changing nasal microbiota and both systemic and nasal immune response upon SARS-CoV-2 mRNA vaccination, and evaluated how mRNA vaccination influenced nasal microbiota in 18 healthy participants who had received the third BNT162b. Results: The nasal S-RBD IgG level correlated significantly with plasma IgG levels until 1 month and the levels were sustained for 3 months post-vaccination. In contrast, nasal S-RBD IgA induction peaked at 1 month, albeit slightly, and correlated only with plasma IgA, but the induction level decreased markedly at 3 months post-vaccination. 16 S rRNA sequencing of the nasal microbiota post-vaccination revealed not an overall change, but a decrease in certain opportunistic bacteria, mainly Fusobacterium. The decrease in these bacteria was more pronounced in those who exhibited nasal S-RBD IgA induction, and those with higher S-RBD IgA induction had lower relative amounts of potentially pathogenic bacteria such as Pseudomonas pre-vaccination. In addition, plasma and mucosal S-RBD IgG levels correlated with decreased commensal pathogens such as Finegoldia. Conclusions: These findings suggest that the third dose of SARS-CoV-2 mRNA vaccination induced S-RBD antibodies in the nasal mucosa and may have stimulated mucosal immunity against opportunistic bacterial pathogens. This effect, albeit probably secondary, may be considered one of the benefits of mRNA vaccination. Furthermore, our data suggest that a cooperative function of mucosal and systemic immunity in the reduction of bacteria and provides a better understanding of the symbiotic relationship between the host and bacteria in the nasal mucosa. [ABSTRACT FROM AUTHOR]

Nirmatrelvir, an oral antiviral agent that targets a SARS-CoV-2 main protease (3CLpro), is clinically useful against infection with SARS-CoV-2 including its omicron variants. Since most omicron subvariants have reduced sensitivity to many monoclonal antibody therapies, potential SARS-CoV-2 resistance to nirmatrelvir is a major public health concern. Several amino acid substitutions have been identified as being responsible for reduced susceptibility to nirmatrelvir. Among them, we selected L50F/E166V and L50F/E166A/L167F in the 3CLpro because these combinations of substitutions are unlikely to affect virus fitness. We prepared and characterized delta variants possessing Nsp5-L50F/E166V and Nsp5-L50F/E166A/L167F. Both mutant viruses showed decreased susceptibility to nirmatrelvir and their growth in VeroE6/TMPRSS2 cells was delayed. Both mutant viruses showed attenuated phenotypes in a male hamster infection model, maintained airborne transmissibility, and were outcompeted by wild-type virus in co-infection experiments in the absence of nirmatrelvir, but less so in the presence of the drug. These results suggest that viruses possessing Nsp5-L50F/E166V and Nsp5-L50F/E166A/L167F do not become dominant in nature. However, it is important to closely monitor the emergence of nirmatrelvir-resistant SARS-CoV-2 variants because resistant viruses with additional compensatory mutations could emerge, outcompete the wild-type virus, and become dominant. Resistance to nirmatrelvir, an oral antiviral agent that targets SARS-CoV-2 and is clinically useful against infection with Omicron variants, is currently not well understood. In this study, the authors characterize mutant viruses with reduced sensitivity to nirmatrelvir in vitro and in vivo. [ABSTRACT FROM AUTHOR]

We previously reported that regulatory T (Treg) cells expressing CTLA‐4 on the cell surface are abundant in head and neck squamous cell carcinoma (HNSCC). The role of expanded Treg cells in the tumor microenvironment of HNSCC remains unclear. In this study, we reveal that the tumor microenvironment of HNSCC is characterized by the high expression of genes related to Treg cells, dendritic cells (DCs), and interleukin (IL)‐17‐related molecules. Increased expression of IL17A, IL17F, or IL23A contributes to a favorable prognosis of HNSCC. In the tumor microenvironment of HNSCC, IL23A and IL12B are expressed in mature dendritic cells enriched in regulatory molecules (mregDCs). The mregDCs in HNSCC are a migratory and mature phenotype; their signature genes strongly correlate with Treg signature genes in HNSCC. We also observed that IL17A was highly expressed in Th17 cells and exhausted CD8+ T cells in HNSCC. These data suggest that mregDCs in HNSCC may contribute to the prognosis by balancing Treg cells and effector T cells that produce IL‐17. Targeting mregDCs may be a novel strategy for developing new immune therapies against HNSCC. [ABSTRACT FROM AUTHOR]

Regulatory T (Treg) cells, which constitute about 5–10% of CD4+T cells expressing Foxp3 transcription factor and CD25(IL-2 receptor α chain), are key regulators in controlling immunological self-tolerance and various immune responses. However, how Treg cells control antigen-specific immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains unclear. In this study, we examined the effect of transient breakdown of the immunological tolerance induced by Treg-cell depletion on adaptive immune responses against administered SARS-CoV-2 antigen, spike protein 1 (S1). Notably, without the use of adjuvants, transient Treg-cell depletion in mice induced anti-S1 antibodies that neutralized authentic SARS-CoV-2, follicular helper T cell formation and S1-binding germinal center B cell responses, but prevented the onset of developing autoimmune diseases. To further clarify the mechanisms, we investigated maturation of dendritic cells (DCs), which is essential to initiate antigen-specific immunity. We found that the transient Treg-cell depletion resulted in maturation of both migratory and resident DCs in draining lymph nodes that captured S1-antigen. Moreover, we observed S1-specific CD4+ T cells and CD8+ T cells with interferon-γ production. Thus, captured S1 was successfully presented by DCs, including cross-presentation to CD8+ T cells. These data indicate that transient Treg-cell depletion in the absence of adjuvants induces maturation of antigen-presenting DCs and succeeds in generating antigen-specific humoral and cellular immunity against emerging SARS-CoV-2 antigens. Finally, we showed that SARS-CoV-2 antigen-specific immune responses induced by transient Treg-cell depletion in the absence of adjuvants were compatible with those induced with an effective adjuvant, polyriboinosinic:polyribocytidyl acid (poly IC) and that the combination of transient Treg-cell depletion with poly IC induced potent responses. These findings highlight the capacity for manipulating Treg cells to induce protective adaptive immunity to SARS-CoV-2 with activating antigen-presenting DCs, which may improve the efficacy of ongoing vaccine therapies and help enhance responses to emerging SARS-CoV-2 variants. Author summary: To challenge SARS-CoV-2 and emerging antigens, it is important to explore innovative approaches to induce effective adaptive immunity to SARS-CoV-2. To induce antigen-specific immunity, vaccines generally need adjuvants to activate dendritic cells (DCs) to present antigens to naïve T cells. In this study, we focused on regulatory T (Treg) cells, which play a key role in maintaining self-tolerance and suppress important immune responses, and found that transient Treg-cell depletion without adjuvants induced protective SARS-CoV-2 antigen-specific immunity. Upon transient Treg-cell depletion in mice, a single administration of SARS-CoV-2 spike protein 1 (S1) induced neutralizing antibodies against live SARS-CoV-2 and evaded the onset of autoimmune diseases. Transient Treg-cell depletion initiated maturation of S1-captured DCs in draining lymph nodes, which is a crucial step to initiate antigen-specific immunity. S1-specific CD4+ and CD8+T cells that produced interferon-γ were also induced. Furthermore, we showed that transient Treg-cell depletion induced antigen-specific immune responses similar to vaccination with an adjuvant and that the combination induced a heightened effect. Thus, transient breakdown of the immunological tolerance induced by Treg-cell manipulation stimulates an adaptive response by activating DCs, providing an innovative approach to the design of vaccines for SARS-CoV-2 and emerging variants. [ABSTRACT FROM AUTHOR]

EG.5.1 is a subvariant of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron XBB variant that is rapidly increasing in prevalence worldwide. However, the pathogenicity, transmissibility, and immune evasion properties of isolates of EG.5.1 are largely unknown. Here, we show that there are no obvious differences in growth ability and pathogenicity between EG.5.1 and XBB.1.5 in hamsters. We also demonstrate that, like XBB.1.5, EG.5.1 is transmitted more efficiently between hamsters compared to its predecessor, BA.2. In contrast, unlike XBB.1.5, we detect EG.5.1 in the lungs of four of six exposed hamsters, suggesting that the virus properties of EG.5.1 are different from those of XBB.1.5. Finally, we find that the neutralizing activity of plasma from convalescent individuals against EG.5.1 was slightly, but significantly, lower than that against XBB.1.5 or XBB.1.9.2. Our data suggest that the different virus properties after transmission and the altered antigenicity of EG.5.1 may be driving its increasing prevalence over XBB.1.5 in humans. [Display omitted] • EG.5.1 and XBB.1.5 show similar growth ability and pathogenicity in hamsters • Unlike XBB.1.5, infectious EG.5.1 is detected in the lungs of some EG.5.1-exposed hamsters • Immune-evading properties of EG.5.1 are significantly enhanced relative to those of XBB.1.5 Uraki et al. find infectious EG.5.1, but not XBB.1.5, in the lungs of some variant-exposed hamsters in airborne transmission studies, despite similar growth and pathogenicity between EG.5.1 and XBB.1.5. EG.5.1 also exhibits superior immune-evading properties compared to XBB.1.5. [ABSTRACT FROM AUTHOR]

Saliva from the mosquito vector of flaviviruses is capable of changing the local immune environment, leading to an increase of flavivirus-susceptible cells at the infected bite site. Additionally, an antibody response towards specific salivary gland (SG) components changes the pathogenesis of flavivirus in human populations. To investigate if antigenic SG proteins are capable of enhancing Zika virus (ZIKV) infection, a re-emerging flavivirus primarily transmitted by the Aedes aegypti mosquito, we screened for antigenic SG proteins using a yeast display library, and demonstrate a previously undescribed SG protein, we term neutrophil stimulating factor 1 (NeSt1), activates primary mouse neutrophils ex vivo. Passive immunization against NeSt1 decreases pro-IL-1β and CXCL2 expression, prevents macrophages from infiltrating into the bite site, protects susceptible IFNAR-/-IFNGR-/- (AG129) mice from early ZIKV replication, and ameliorates viral-induced pathogenesis. These findings indicate that NeSt1 stimulates neutrophils at the mosquito bite site to change the immune microenviroment, allowing higher early viral replication and enhancing ZIKV pathogenesis. (156 Words) [ABSTRACT FROM AUTHOR]

Few animal models of Zika virus (ZIKV) infection have incorporated arthropod-borne transmission. Here, we establish an Aedes aegypti mosquito model of ZIKV infection of mice, and demonstrate altered vector competency among three strains, (Orlando, ORL, Ho Chi Minh, HCM, and Patilas, PAT). All strains acquired ZIKV in their midguts after a blood meal from infected mice, but ZIKV transmission only occurred in mice fed upon by HCM, and to a lesser extent PAT, but not ORL, mosquitoes. This defect in transmission from ORL or PAT mosquitoes was overcome by intrathoracic injection of ZIKV into mosquito. Genetic analysis revealed significant diversity among these strains, suggesting a genetic basis for differences in ability for mosquito strains to transmit ZIKV. The intrathoracic injection mosquito-mouse transmission model is critical to understanding the influence of mosquitoes on ZIKV transmission, infectivity and pathogenesis in the vertebrate host, and represents a natural transmission route for testing vaccines and therapeutics. (152 words) [ABSTRACT FROM AUTHOR]

Objectives: Broadly reactive human monoclonal antibodies against the HA stem of influenza A virus are being developed as therapeutic agents as well as to understand the epitopes that are essential for a universal influenza virus vaccine.Methods: We isolated and characterized two hetero-reactive human monoclonal antibodies from an H3N2 virus-infected human.Results: These antibodies, which are predominantly bound to the HA stem of group 2 HAs, used IGHV3-66 and IGHV4-38-2 germline genes, respectively. They possessed in vitro neutralizing ability, and in vivo protective efficacy against lethal infection with H3N2 or H7N9 virus. Escape mutations revealed that one of the protective antibodies recognized the α-helix A of HA2, and the other recognized the C-terminal portion of the fusion peptide and the β-sheet that precedes the α-helix A of HA2.Conclusions: Of many human protective monoclonal antibodies against the HA stem, two human protective monoclonal antibodies were isolated in this study that predominantly recognize epitopes on the HA stem of group 2 and use unique IGHV3-66 and IGHV4-38-2 germline genes. [ABSTRACT FROM AUTHOR]

Summary Tyro3, Axl, and Mertk (TAM) receptors are candidate entry receptors for infection with the Zika virus (ZIKV), an emerging flavivirus of global public health concern. To investigate the requirement of TAM receptors for ZIKV infection, we used several routes of viral inoculation and compared viral replication in wild-type versus Axl −/− , Mertk −/− , Axl −/− Mertk −/− , and Axl −/− Tyro3 −/− mice in various organs. Pregnant and non-pregnant mice treated with interferon-α-receptor (IFNAR)-blocking (MAR1-5A3) antibody and infected subcutaneously with ZIKV showed no reliance on TAMs for infection. In the absence of IFNAR-blocking antibody, adult female mice challenged intravaginally with ZIKV showed no difference in mucosal viral titers. Similarly, in young mice that were infected with ZIKV intracranially or intraperitoneally, ZIKV replication occurred in the absence of TAM receptors, and no differences in cell tropism were observed. These findings indicate that, in mice, TAM receptors are not required for ZIKV entry and infection. [ABSTRACT FROM AUTHOR]

Background: Secondary bacterial infections after influenza can be a serious problem, especially in young children and the elderly, yet the efficacy of current vaccines is limited. Earlier work demonstrated that a replication-incompetent PB2-knockout (PB2-KO) influenza virus possessing a foreign gene in the coding region of its PB2 segment can serve as a platform for a bivalent vaccine.Methods: In the current study, we generated the PB2-KO virus expressing pneumococcal surface protein A (PspA), PB2-KO-PspA virus, the replication of which is restricted to PB2-expressing cells. We then examined the protective efficacy of intranasal immunization with this virus as a bivalent vaccine in a mouse model.Results: High levels of influenza virus-specific and PspA-specific antibodies were induced in the serum and airways of immunized mice. The intranasally immunized mice were protected from lethal doses of influenza virus or Streptococcus pneumoniae. These mice were also completely protected from secondary pneumococcal pneumonia after influenza virus infection.Conclusions: These findings indicate that our recombinant influenza virus serves as a novel and powerful bivalent vaccine against primary and secondary pneumococcal pneumonia as well as influenza. [ABSTRACT FROM AUTHOR]

Vaccination is an effective means to protect against influenza virus. Although inactivated and live-attenuated vaccines are cur-rently available, each vaccine has disadvantages (e.g., immunogenicity and safety issues). To overcome these problems, we previ-ously developed a replication-incompetent PB2-knockout (PB2-KO) influenza virus that replicates only in PB2 protein-express-ing cells. Here, we generated two PB2-KO viruses whose PB2-coding regions were replaced with the HA genes of either A/California/04/2009 (HlNlpdm09) or A/Vietnam/1203/2004 (H5N1). The resultant viruses comparably, or in some cases more efficiently, induced virus-specific antibodies in the serum, nasal wash, and bronchoalveolar lavage fluid of mice relative to a con-ventional formalin-inactivated vaccine. Furthermore, mice immunized with these PB2-KO viruses were protected from lethal challenges with not only the backbone virus strain but also strains from which their foreign HAs originated, indicating that PB2-KO viruses with antigenically different HAs could serve as bivalent influenza vaccines. [ABSTRACT FROM AUTHOR]

Prion diseases are a family of neurodegenerative zoonotic foodborne disorders. Although prions can be transmitted orally, the mechanism by which prions are incorporated into the intestine remains unclear. Our previous studies have shown that an abnormal isoform of prion protein (PrPSc), which is the main component of prions, was efficiently incorporated into the intestine in suckling mice but not in weaned mice. Furthermore, suckling SCID mice lacking maternal antibodies showed decreased uptake of PrPSc into the intestine compared with suckling wild-type mice, while the lack of PrPSc uptake into the intestine of suckling SCID mice was rescued by the oral administration of IgG. These findings raise the possibility that the neonatal Fc receptor (nFcR), which contributes to the uptake of maternal antibodies into the intestine, plays a role in PrPSc incorporation into the intestine. The present immunohistochemical study further showed that the FcR blocker Z-ϵ- aminocaproic acid (ZAA) inhibited PrPSc incorporation into the intestinal villi of suckling mice, supporting the above mentioned concept. Therefore, our findings provide strong evidence that nFcR and maternal antibodies are involved in PrPSc incorporation into the intestine before the weaning period. [ABSTRACT FROM AUTHOR]

Mosquito-borne Zika virus (ZIKV) can cause congenital microcephaly and Guillain–Barré syndrome, among other symptoms. Specific treatments and vaccines for ZIKV are not currently available. To further understand the host factors that support ZIKV replication, we used mass spectrometry to characterize mammalian proteins that associate with the ZIKV NS1 protein and identified the TRiC/CCT complex as an interacting partner. Furthermore, the suppression of CCT2, one of the critical components of the TRiC/CCT complex, inhibited ZIKV replication in both mammalian cells and mosquitoes. These results highlight an important role for the TRiC/CCT complex in ZIKV infection, suggesting that the TRiC/CCT complex may be a promising therapeutic target. [ABSTRACT FROM AUTHOR]

Highly pathogenic avian H5 influenza viruses persist among poultry and wild birds throughout the world. They sometimes cause interspecies transmission between avian and mammalian hosts. H5 viruses possessing the HA of subclade 2.3.4.4, 2.3.2.1, 2.2.1, or 7.2 were detected between 2015 and 2018. To understand the neutralizing epitopes of H5-HA, we characterized 15 human monoclonal antibodies (mAbs) against the HA of H5 viruses, which were obtained from volunteers who received the H5N1 vaccine that contains a subclade 2.2.1 or 2.1.3.2 virus as an antigen. Twelve mAbs were specific for the HA of subclade 2.2.1, two mAbs were specific for the HA of subclade 2.1.3.2, and one mAb was specific for the HA of both. Of the 15 mAbs analyzed, nine, which were specific for the HA of subclade 2.2.1, and shared the VH and VL genes, possessed hemagglutination inhibition and neutralizing activities, whereas the others did not. A single amino acid substitution or insertion at positions 144–147 in antigenic site A conferred resistance against these nine mAbs to the subclade 2.2.1 viruses. The amino acids at positions 144–147 are highly conserved among subclade 2.2.1, but differ from those of other subclades. These results show that the neutralizing epitope including amino acids at positions 144–147 is targeted by human antibodies, and plays a role in the antigenic difference between subclade 2.2.1 and other subclades. [ABSTRACT FROM AUTHOR]

We tested the biological significance of two amino acid mutations in the PB2 protein (glutamic acid to lysine at position 627 and aspartic acid to asparagine at position 701) of A(H7N9) viruses for mammalian adaptation. Mutants were assessed for their viral polymerase activities, growth kinetics in mammalian and avian cells, and pathogenicity in mice. We found that lysine at position 627 and asparagine at position 701 in PB2 are essential for mammalian adaptation of A(H7N9) viruses. [ABSTRACT FROM AUTHOR]

Highlights: [•] PB2-KO virus bearing HN of HPIV3 did not replicate in normal cells. [•] PB2-KO virus bearing HN of HPIV3 elicited mucosal and systemic immunity. [•] Hamsters immunized with PB2-KO virus bearing HN of HPIV3 were protected from both influenza virus and parainfluenza virus challenge. [ABSTRACT FROM AUTHOR]

Abstract: Reactive oxygen species (ROS) play an important role in diverse vital functions including host defense via anti-viral and anti-bacterial effects, but ROS also lead to peroxynitrite and hydroxyl radical production, which are powerful mediators of brain injury in brain inflammation. It is known that NADPH oxidases (NOX) are the major source of ROS. In the present study, NOX2 expression and distribution were examined after intracranial encephalomyocarditis virus B variant (EMCV-B) infection, which causes encephalitis. The reverse transcriptase (RT)-polymerase chain reaction (PCR) and immunohistochemistry showed that the expression and distribution of NOX2 were significantly up-regulated after EMCV-B infection in microglial cells, which invaded into the surrounding regions where neurons were subjected to oxidative stress. These findings suggest that the oxidative stress generated by NOX2 in activated microglial cells damages neurons and that this is an important process in the pathogenesis of EMCV-B infection. [Copyright &y& Elsevier]