PR8 Strain Research Articles

SARS‐CoV‐2 evades immune detection in alveolar macrophages

Respiratory infections, like the current COVID‐19 pandemic, target epithelial cells in the respiratory tract. Alveolar macrophages (AMs) are tissue‐resident macrophages located within the lung. They play a key role in the early phases of an immune response to respiratory viruses. AMs are likely the first immune cells to encounter SARS‐CoV‐2 during an infection, and their reaction to the virus will have a profound impact on the outcome of the infection. Interferons (IFNs) are antiviral cytokines and among the first cytokines produced upon viral infection. In this study, AMs from non‐infectious donors are challenged with SARS‐CoV‐2. We demonstrate that challenged AMs are incapable of sensing SARS‐CoV‐2 and of producing an IFN response in contrast to other respiratory viruses, like influenza A virus and Sendai virus, which trigger a robust IFN response. The absence of IFN production in AMs upon challenge with SARS‐CoV‐2 could explain the initial asymptotic phase observed during COVID‐19 and argues against AMs being the sources of pro‐inflammatory cytokines later during infection.

A vaccine combination of lipid nanoparticles and a cholera toxin adjuvant derivative greatly improves lung protection against influenza virus infection.

This is a proof-of-principle study demonstrating that the combination of a cholera toxin derived adjuvant, CTA1-DD, and lipid nanoparticles (LNP) can significantly improve the immunogenicity and protective capacity of an intranasal vaccine. We explored the self-adjuvanted universal influenza vaccine candidate, CTA1-3M2e-DD (FPM2e), linked to LNPs. We found that the combined vector greatly enhanced survival against a highly virulent PR8 strain of influenza virus as compared to when mice were immunized with FPM2e alone. The combined vaccine vector enhanced early endosomal processing and peptide presentation in dendritic cells and upregulated co-stimulation. The augmenting effect was CTA1-enzyme dependent. Whereas systemic anti-M2e antibody and CD4+ T-cell responses were comparable to those of the soluble protein, the local respiratory tract IgA and the specific Th1 and Th17 responses were strongly enhanced. Surprisingly, the lung tissue did not exhibit gross pathology upon recovery from infection and M2e-specific lung resident CD4+ T cells were threefold higher than in FPM2e-immunized mice. This study conveys optimism as to the protective ability of a combination vaccine based on LNPs and various forms of the CTA1-DD adjuvant platform, in general, and, more specifically, an important way forward to develop a universal vaccine against influenza.

A Self-Adjuvanted, Modular, Antigenic VLP for Rapid Response to Influenza Virus Variability.

The continuous evolution of influenza A virus (IAV) requires the influenza vaccine formulations to be updated annually to provide adequate protection. Recombinant protein-based vaccines provide safer, faster, and a more scalable alternative to the conventional embryonated egg approach for developing vaccines. However, these vaccines are typically poorer in immunogenicity than the vaccines containing inactivated or attenuated influenza viruses and require administration of a large antigen dosage together with potent adjuvants. The presentation of protein antigens on the surface of virus-like particles (VLP) provides an attractive strategy to rapidly induce stronger antigen-specific immune responses. Here we have examined the immunogenic potential and protective efficacy of P22 VLPs conjugated with multiple copies of the globular head domain of the hemagglutinin (HA) protein from the PR8 strain of IAV in a murine model of influenza pathogenesis. Using a covalent attachment strategy (SpyTag/SpyCatcher), we conjugated the HA globular head, which was recombinantly expressed in a genetically modified E.coli strain and found to refold as a monomer, to preassembled P22 VLPs. Immunization of mice with this P22-HAhead conjugate provided full protection from morbidity and mortality following infection with a homologous IAV strain. Moreover, the P22-HAhead conjugate also elicited an accelerated and enhanced HA head specific IgG response, which was significantly higher than the soluble HA head, or the admixture of P22 and HA head without the need for adjuvants. Thus, our results show that the HA head can be easily prepared by in vitro refolding in a modified E.coli strain, maintaining its intact structure and enabling the induction of a strong immune response when conjugated to P22 VLPs, even when presented as a monomer. These results also demonstrate that the P22 VLPs can be rapidly modified in a modular fashion, resulting in an effective vaccine construct that can generate protective immunity without the need for additional adjuvants.

Comparative Metabolomics Revealed the Potential Antitumor Characteristics of Four Endophytic Fungi of Brassica rapa L.

Endophytic fungi of medicinal plants have attracted wide attention due to their various active biochemical substances that are similar to those of the host plants and can be easily fermented and cultured. As a traditional medicine and food homologous plant in Xinjiang, Brassica rapa L. has a long history of applications. Recently, it has been shown that B. rapa L. has hypoglycemic, antimicrobial, immunomodulatory, and antioxidant properties. However, there are no studies on the function and diversity of enophytic fungi of B. rapa L. Four endophytic fungus (pr6, pr7, pr8, and pr10) strains were isolated from B. rapa L. in our laboratory. The metabolic extracts from pr10 have significant effects in terms of antitumor activity. In this study, in terms of types and contents, compared with those of the other three endophytic fungi, the dominant metabolites of pr10 were determined by comparative metabolomics analysis. The results of metabolomics analysis indicated that the metabolites of pr10 are rich in amino acids and sugar derivatives such as trehalose, whose ability to inhibit the A549 cell line has been proved. This study provides a theoretical basis for the development and utilization of B. rapa L. and its endophytic fungi to form antitumor agents.

Aptamer Profiling of A549 Cells Infected with Low-Pathogenicity and High-Pathogenicity Influenza Viruses.

Influenza A viruses (IAVs) are important animal and human emerging and re-emerging pathogens that are responsible for yearly seasonal epidemics and sporadic pandemics. IAVs cause a wide range of clinical illnesses, from relatively mild infections by seasonal strains, to acute respiratory distress during infections with highly pathogenic avian IAVs (HPAI). For this study, we infected A549 human lung cells with lab prototype A/PR/8/34 (H1N1) (PR8), a seasonal H1N1 (RV733), the 2009 pandemic H1N1 (pdm09), or with two avian strains, an H5N1 HPAI strain or an H7N9 strain that has low pathogenicity in birds but high pathogenicity in humans. We used a newly-developed aptamer-based multiplexed technique (SOMAscan®) to examine >1300 human lung cell proteins affected by the different IAV strains, and identified more than 500 significantly dysregulated cellular proteins. Our analyses indicated that the avian strains induced more profound changes in the A549 global proteome compared to all tested low-pathogenicity H1N1 strains. The PR8 strain induced a general activation, primarily by upregulating many immune molecules, the seasonal RV733 and pdm09 strains had minimal effect upon assayed molecules, and the avian strains induced significant downregulation, primarily in antimicrobial response, cardiovascular and post-translational modification systems.

Transcriptome Profiling Reveals Differential Effect of Interleukin-17A Upon Influenza Virus Infection in Human Cells.

Influenza A virus (IAV) has developed elegant strategies to utilize cellular proteins and pathways to promote replication and evade the host antiviral response. Identification of these sabotaged host factors could increase the number of potential antiviral drug targets. Here, IAV A/PR/8/34 (PR8)- and A/California/04/2009-infected A549 and 293T cells displayed differential virus replication. To determine the host cellular responses of A549 and 293T cells to IAV infection, RNA-seq was used to identify differentially expressed genes. Our data revealed that IAV-infected A549 cells activated stronger virus-sensing signals and highly expressed cytokines, which play significant roles in initiating the innate immune and inflammatory responses. In addition, IAV-infected 293T cells displayed weak immune signaling and cytokine production. Remarkably, IL-17A and associated genes were highly enriched in IAV-infected 293T cells. Furthermore, IL-17A can partially facilitate A549 cell infection by the PR8 strain and PR8-infected IL-17A knock-out mice consistently exhibited decreased weight loss and reduced lung immunopathology, as compared to controls. This work uncovered the differential responses of cells infected with two H1N1 IAV strains and the potential roles of IL-17A in modulating virus infection.

Characterization of influenza virus PR8 strain cultured in embryonated eggs by cryo-electron tomography

Influenza A viruses, as causative agents of seasonal epidemics and periodic worldwide pandemics, cause enormous mortality loss globally. The PR8 strain cultured in chicken eggs is widely used for scientific research and the production of influenza vaccines. Here, based on Cryo-electron Tomography (CET), we analyzed the morphological and structural characteristics of the influenza virus PR8 strain at different pHs. We found that a large number of defective virions were propagated in embryonated eggs. By comparing virions with/without the matrix layer, it was revealed that the matrix layer played an essential role in the structural integrity of virions and RNPs encapsulation during the influenza virus life cycle. We also utilized hemagglutinin receptor-containing liposomes to mimic the membrane fusion process. Several potential intermediates of HA during membrane fusion were observed at acidic pH. Our observations afford insight into the architecture and function of influenza virus.

Identification and in vivo Efficacy Assessment of Approved Orally Bioavailable Human Host Protein-Targeting Drugs With Broad Anti-influenza A Activity.

The high genetic variability of influenza A viruses poses a continual challenge to seasonal and pandemic vaccine development, leaving antiviral drugs as the first line of defense against antigenically different strains or new subtypes. As resistance against drugs targeting viral proteins emerges rapidly, we assessed the antiviral activity of already approved drugs that target cellular proteins involved in the viral life cycle and were orally bioavailable. Out of 15 candidate compounds, four were able to inhibit infection by 10- to 100-fold without causing toxicity, in vitro. Two of the drugs, dextromethorphan and ketotifen, displayed a 50% effective dose between 5 and 50 μM, not only for the classic H1N1 PR8 strain, but also for a pandemic H1N1 and a seasonal H3N2 strain. Efficacy assessment in mice revealed that dextromethorphan consistently resulted in a significant reduction of viral lung titers and also enhanced the efficacy of oseltamivir. Dextromethorphan treatment of ferrets infected with a pandemic H1N1 strain led to a reduction in clinical disease severity, but no effect on viral titer was observed. In addition to identifying dextromethorphan as a potential influenza treatment option, our study illustrates the feasibility of a bioinformatics-driven rational approach for repurposing approved drugs against infectious diseases.

PA-X antagonises MAVS-dependent accumulation of early type I interferon messenger RNAs during influenza A virus infection

The sensing of viral nucleic acids by the innate immune system activates a potent antiviral response in the infected cell, a key component of which is the expression of genes encoding type I interferons (IFNs). Many viruses counteract this response by blocking the activation of host nucleic acid sensors. The evolutionarily conserved influenza A virus (IAV) protein PA-X has been implicated in suppressing the host response to infection, including the expression of type I IFNs. Here, we characterise this further using a PA-X-deficient virus of the mouse-adapted PR8 strain to study activation of the innate immune response in a mouse model of the early response to viral infection. We show that levels of Ifna4 and Ifnb1 mRNAs in the lungs of infected mice were elevated in the absence of PA-X and that this was completely dependent on MAVS. This therefore suggests a role for PA-X in preventing the accumulation of early type I IFN mRNAs in the lung during IAV infection.

Screening of Polyvalent Phage-Resistant Escherichia coli Strains Based on Phage Receptor Analysis.

Bacteria-based biotechnology processes are constantly under threat from bacteriophage infection, with phage contamination being a non-neglectable problem for microbial fermentation. The essence of this problem is the complex co-evolutionary relationship between phages and bacteria. The development of phage control strategies requires further knowledge about phage-host interactions, while the widespread use of Escherichia coli strain BL21 (DE3) in biotechnological processes makes the study of phage receptors in this strain particularly important. Here, eight phages infecting E. coli BL21 (DE3) via different receptors were isolated and subsequently identified as members of the genera T4virus, Js98virus, Felix01virus, T1virus, and Rtpvirus. Phage receptors were identified by whole-genome sequencing of phage-resistant E. coli strains and sequence comparison with wild-type BL21 (DE3). Results showed that the receptors for the isolated phages, designated vB_EcoS_IME18, vB_EcoS_IME253, vB_EcoM_IME281, vB_EcoM_IME338, vB_EcoM_IME339, vB_EcoM_IME340, vB_EcoM_IME341, and vB_EcoS_IME347 were FhuA, FepA, OmpF, lipopolysaccharide, Tsx, OmpA, FadL, and YncD, respectively. A polyvalent phage-resistant BL21 (DE3)-derived strain, designated PR8, was then identified by screening with a phage cocktail consisting of the eight phages. Strain PR8 is resistant to 23 of 32 tested phages including Myoviridae and Siphoviridae phages. Strains BL21 (DE3) and PR8 showed similar expression levels of enhanced green fluorescent protein. Thus, PR8 may be used as a phage resistant strain for fermentation processes. The findings of this study contribute significantly to our knowledge of phage-host interactions and may help prevent phage contamination in fermentation.

Mutation of Influenza A Virus PA-X Decreases Pathogenicity in Chicken Embryos and Can Increase the Yield of Reassortant Candidate Vaccine Viruses.

The PA-X protein of influenza A virus has roles in host cell shutoff and viral pathogenesis. While most strains are predicted to encode PA-X, strain-dependent variations in activity have been noted. We found that PA-X protein from the A/PR/8/34 (PR8) strain had significantly lower repressive activity against cellular gene expression than PA-X proteins from the avian strains A/turkey/England/50-92/91 (H5N1) (T/E) and A/chicken/Rostock/34 (H7N1). Loss of normal PA-X expression, either by mutation of the frameshift site or by truncating the X open reading frame (ORF), had little effect on the infectious virus titer of PR8 or PR8 7:1 reassortants with T/E segment 3 grown in embryonated hens' eggs. However, in both virus backgrounds, mutation of PA-X led to decreased embryo mortality and lower overall pathology, effects that were more pronounced in the PR8 strain than in the T/E reassortant, despite the low shutoff activity of the PR8 PA-X. Purified PA-X mutant virus particles displayed an increased ratio of hemagglutinin (HA) to nucleoprotein (NP) and M1 compared to values for their wild-type (WT) counterparts, suggesting altered virion composition. When the PA-X gene was mutated in the background of poorly growing PR8 6:2 vaccine reassortant analogues containing the HA and neuraminidase (NA) segments from H1N1 2009 pandemic viruses or from an avian H7N3 strain, HA yield increased up to 2-fold. This suggests that the PR8 PA-X protein may harbor a function unrelated to host cell shutoff and that disruption of the PA-X gene has the potential to improve the HA yield of vaccine viruses.IMPORTANCE Influenza A virus is a widespread pathogen that affects both humans and a variety of animal species, causing regular epidemics and sporadic pandemics, with major public health and economic consequences. A better understanding of virus biology is therefore important. The primary control measure is vaccination, which for humans mostly relies on antigens produced in eggs from PR8-based viruses bearing the glycoprotein genes of interest. However, not all reassortants replicate well enough to supply sufficient virus antigen for demand. The significance of our research lies in identifying that mutation of the PA-X gene in the PR8 strain of virus can improve antigen yield, potentially by decreasing the pathogenicity of the virus in embryonated eggs.

Global proteomic analyses of murine lungs during earlier and later phases of influenza pneumonia

Background: Influenza infection is among the most common and important infectious diseases in the world. We previously showed that specific microRNAs (miRNAs) and mRNAs were significantly differentially expressed (DE) during different phases of influenza pneumonia. Since proteins are the major effectors of biological pathways, we thus proceeded to investigate the global protein changes in the lungs during influenza pneumonia. Methods & Materials: BALB/c mice were infected with a sub-lethal dose of influenza H1N1 virus (PR8 strain) to induce viral pneumonia. At 7 and 15 days post-infection (dpi), mouse lungs were harvested and subjected to relative protein quantification via protein mass spectrometry. Mock-infected mice served as controls. Significant protein hits were identified using the Z-statistics threshold that controlled the false discovery rate at 5%. Downstream pathways implicated by the global protein changes were identified through Gene Ontology (GO) and pathway analysis (KEGG and IPA). Results: In terms of global protein expression changes at 7 dpi, a total of 719 proteins (366 downregulated, 353 upregulated) were DE, with the top 5 downregulated proteins being FGF1, CBR2, FABP1, COL1A1 and SFTPA1, and the top 5 upregulated proteins being MT2, IFI44, GBP5, IFIT3, and DOK3. At 15 dpi, a total of 842 proteins (464 downregulated, 378 upregulated) were significantly DE. The top 5 downregulated proteins were PGAP2, CBR2, NEB, TPPP3 and H1F0, whereas the top 5 upregulated proteins were ALOX5AP, GBP5, IGH-1A, IGFALS and RETNLA. Via GO analysis, overexpressed proteins at both 7 and 15 dpi were associated with inflammation such as “acute-phase response” and “innate immune response”. Pathway analysis also revealed that metabolic and infection pathways were the top hits at both 7 and 15 dpi, suggesting the persistence of inflammatory pathways for clearance of infection. Comparison of the miRNA and protein global expression changes revealed that only 23% (on average) of predicted target proteins were DE when their corresponding miRNAs were DE. Conclusion: Our data from the proteomic analyses of murine lungs during earlier and later phases of influenza pneumonia have elucidated the relevant proteins and pathways that play important roles in mediating the persistent state of inflammation for influenza viral clearance.

Generation of a broadly reactive influenza H1 antigen using a consensus HA sequence

H1N1, one of the most prevalent influenza A virus subtypes affecting the human population, can cause infections varying from mild respiratory syndrome to severe pneumonia. The current H1N1 vaccine needs to be updated annually and does not protect against future outbreaks. Here, we downloaded 2,656 HA protein sequences of human H1N1 viruses from the NCBI influenza database (up to the date of Aug. 2012) and constructed a phylogenetic tree of these H1 proteins via the neighbor-joining method using MEGA 5.0 software. A consensus H1 protein (CH1) was generated and was further modified with published conserved T-cell and B-cell epitopes. Interestingly, this CH1 protein is genetically similar to an H1 isolate obtained during the 1980s (A/Memphis/7/1980), indicating that a universal HA antigen may exist in nature. Vaccination with a DNA vaccine expressing CH1 elicited broadly reactive T-cell and B-cell responses to heterologous H1N1 viruses, though this vaccine did not successfully neutralize pdm09 H1N1 viruses. A combination of CH1 and pdm09 HA in a DNA vaccination neutralized pdm09 H1N1 viruses and protected mice from lethal infections by all representative H1N1 viruses. Moreover, a recombinant chimeric PR8-CH1 virus carrying HA sequence of the consensus H1 and all other seven genes from the PR8 strain was highly attenuated in mice, with a lethal dose (LD50) of more than 106 pfu. Vaccination with PR8-CH1 virus provided complete protection against infections by heterologous H1N1 strains. Taken together, a universal H1 antigen, CH1, was developed by constructing a consensus HA sequence, and the PR8-CH1 virus containing this consensus sequence elicited broadly protective immunity against heterologous H1N1 viruses.

Disease manifestations with immune alteration by bacterial neuraminidases in influenza virus infection with Streptococcus pneumoniae superinfection

Abstract Streptococcus pneumoniae often causes superinfection in influenza and exacerbates the disease with more severity and even mortality. In our influenza hemagglutinin (HA) antigen-specific transgenic mouse model, mice suffered from severe disease and died with increased virus titer and pathology in the lungs upon serotype-3 Streptococcus pneumoniae superinfection on day 4- post PR8 strain H1N1 influenza virus infection. Same dose viral or bacterial infection caused mild disease in control mice. Streptococcus pneumoniae supreinfection attenuated anti-influenza T cell immunity in the lungs. There was less clonotypic expansion, T-bet induction and effector cytokines IFN-γ and TNF-α production in HA-specific CD4+ T cells on day 2- and 3- post bacterial superinfection, with impaired viral clearance. Streptococcus pneumoniae strains with different combinations of bacterial neuraminidase (NA) demonstrated varied degrees of T cell immunity attenuation. Streptococcus pneumoniae with NA-B only was the most suppressive with up-regulated active TGF-β and down-regulated IFN-γ expression in HA-specific CD4+ T cells, followed by those with both NA-A and NA-B. Streptococcus pneumoniae with both NA-A and NA-B and inserted NA-C was least suppressive. With in vitro experiments, recombinant bacterial NA-A or NA-B augmented TGF-β and attenuated IFN-γ in HA-specific CD4+ T cells in overnight culture with PR8 virus. Recombinant bacterial NA-C mitigated TGF-β and increased IFN-γ in HA-specific CD4+ T cells. Our results demonestrates distinct immune alteration by different bacterial neuraminidases, which may play a significnt role of disease manifestation in influenza with bacterial superinfection.

GM-CSF overexpression after influenza a virus infection prevents mortality and moderates M1-like airway monocyte/macrophage polarization

BackgroundInfluenza A viruses cause life-threatening pneumonia and lung injury in the lower respiratory tract. Application of high GM-CSF levels prior to infection has been shown to reduce morbidity and mortality from pathogenic influenza infection in mice, but the mechanisms of protection and treatment efficacy have not been established.MethodsMice were infected intranasally with influenza A virus (PR8 strain). Supra-physiologic levels of GM-CSF were induced in the airways using the double transgenic GM-CSF (DTGM) or littermate control mice starting on 3 days post-infection (dpi). Assessment of respiratory mechanical parameters was performed using the flexiVent rodent ventilator. RNA sequence analysis was performed on FACS-sorted airway macrophage subsets at 8 dpi.ResultsSupra-physiologic levels of GM-CSF conferred a survival benefit, arrested the deterioration of lung mechanics, and reduced the abundance of protein exudates in bronchoalveolar (BAL) fluid to near baseline levels. Transcriptome analysis, and subsequent validation ELISA assays, revealed that excess GM-CSF re-directs macrophages from an “M1-like” to a more “M2-like” activation state as revealed by alterations in the ratios of CXCL9 and CCL17 in BAL fluid, respectively. Ingenuity pathway analysis predicted that GM-CSF surplus during IAV infection elicits expression of anti-inflammatory mediators and moderates M1 macrophage pro-inflammatory signaling by Type II interferon (IFN-γ).ConclusionsOur data indicate that application of high levels of GM-CSF in the lung after influenza A virus infection alters pathogenic “M1-like” macrophage inflammation. These results indicate a possible therapeutic strategy for respiratory virus-associated pneumonia and acute lung injury.

Efficient control of chronic LCMV infection by CD4 T cell epitope-based heterologous prime-boost vaccination in a murine model

Abstract CD4+ T cells are essential for sustaining CD8+ T cell responses during chronic infection. The adoptive transfer of virus-specific CD4+ T cells has been shown to efficiently rescue exhausted CD8+ T cells. However, the question of whether endogenous virus-specific CD4+ T cell responses can be enhanced by certain vaccination strategies and subsequently reinvigorate exhausted CD8+ T cells remains unexplored. In this study, we developed a CD4+ T cell epitope-based heterologous prime-boost immunization strategy and examined the efficacy of this strategy using a mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection. We primed chronically LCMV-infected mice with the Listeria monocytogenes vector expressing the LCMV glycoprotein-specific I-Ab-restricted CD4+ T cell epitope GP61-80 (LM-GP61) and subsequently boosted the primed mice with an influenza virus A (PR8 strain) vector expressing the same CD4+ T cell epitope (IAV-GP61). This heterologous prime-boost vaccination strategy elicited strong anti-viral CD4+ T cell responses, which further improved both the quantity and quality of the virus-specific CD8+ T cells and led to better control of viral loads. The combination of this strategy and the blockade of the programmed cell death-1 (PD-1) inhibitory pathway further enhanced anti-viral CD8+ T cell responses and viral clearance. Therefore, heterologous prime-boost immunization that selectively induces virus-specific CD4+ T cell responses in conjunction with inhibitory pathway blockade may represent a promising therapeutic approach to treating patients with chronic viral infection.

Pre-existing matched antigen exacerbates the disease of influenza virus infection by attenuating antigen-specific T cell immunity

Abstract Influenza virus infection causes mild and self-limiting illness in most of the healthy individuals, but some healthy people develops complications as a result of the infection and suffers from severe disease and eventually dies. Here we report that pre-existing matched antigens attenuate antigen-specific immunity to the infection. High virus load and associated increase of pathogenic innate response in the lungs exacerbates the disease. The C3-HAHigh and C3-HALow transgenic mice are with inherent expression of Hemagglutinin (HA) antigen from PR8 strain of H1N1 influenza virus. Compared to wild type mice, these mice suffered from severe disease and higher mortality upon infection of wild type PR8 virus but not upon infection of reverse genetically raised PR8 virus with replaced HA from H3N2 influenza virus. Adoptively transferred PR8 virus HA-specific 6.5 CD4+ and Clone-4 CD8+ T cells responded minimally to PR8 virus infection in C3-HA mice. There was less clonotypic expansion, T-bet induction and effector cytokines IFN-γ and TNF-α production of both 6.5 CD4+ and Clone-4 CD8+ T cells. Virus neutralizing antibodies were less as well. The attenuation was associated with increased LAG-3+ but not Foxp-3+ population of HA-specific T cells in the lungs. In contrast to the instigated weak antigen-specific immunity, PR8 virus infection caused neutrophils more efficient for inflammatory IL-6 secretion and robust bronchus infiltration in C3-HA mice. These results suggest that a host factor cross-reactive to the antigens of influenza virus may exacerbate the disease with similar attenuated antigen-specific adaptive immunity accompanied by augmented innate immunity to influenza virus infection.

3D visualization of sex differences in the immune response to influenza infection in the mouse lung

Abstract Recent developments of imaging techniques, including histo-cytometry and a new tissue clearing and staining protocol called Clearing-enhanced 3D (Ce3D), allow for high-resolution, 3D multiplex quantitative imaging and analysis of cell populations in intact organ material. We have established a panel of 10–14 markers for use in Ce3D-cleared 250μm-thick sections of lung tissue to visualize both innate and adaptive aspects of the immune response as comprehensively as possible. This panel, combined with quantitative analysis provided by histo-cytometry, allows for the collection of larger, more informative imaging datasets than typically generated by conventional histochemistry methods. In our study, we applied these techniques to the mouse model of influenza A virus (IAV) infection to investigate the patterns of immune cell distribution and viral spread in the lungs of male and female mice. In C57BL/6J mice, we observed increased mortality in female mice compared to males when infected with the same dose of the lethal PR8 strain of IAV. Literature research as well as data from the infection of different transgenic mouse lines suggest that higher levels of innate inflammation in female mice may lead to the observed difference in mortality. Ce3D multiplex images show multiple differences in the distribution of both innate immune cells and infected areas in lungs from male and female mice. This study will contribute to a better understanding of both the immune response to IAV infection and sex differences in immunity in general, potentially leading to better design of tailored vaccines and treatments in the future.

Efficient control of chronic LCMV infection by a CD4 T cell epitope-based heterologous prime-boost vaccination in a murine model.

CD4+ T cells are essential for sustaining CD8+ T cell responses during a chronic infection. The adoptive transfer of virus-specific CD4+ T cells has been shown to efficiently rescue exhausted CD8+ T cells. However, the question of whether endogenous virus-specific CD4+ T cell responses can be enhanced by certain vaccination strategies and subsequently reinvigorate exhausted CD8+ T cells remains unexplored. In this study, we developed a CD4+ T cell epitope-based heterologous prime-boost immunization strategy and examined the efficacy of this strategy using a mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection. We primed chronically LCMV-infected mice with a Listeria monocytogenes vector that expressed the LCMV glycoprotein-specific I-Ab-restricted CD4+ T cell epitope GP61-80 (LM-GP61) and subsequently boosted the primed mice with an influenza virus A (PR8 strain) vector that expressed the same CD4+ T cell epitope (IAV-GP61). This heterologous prime-boost vaccination strategy elicited strong anti-viral CD4+ T cell responses, which further improved both the quantity and quality of the virus-specific CD8+ T cells and led to better control of the viral loads. The combination of this strategy and the blockade of the programmed cell death-1 (PD-1) inhibitory pathway further enhanced the anti-viral CD8+ T cell responses and viral clearance. Thus, a heterologous prime-boost immunization that selectively induces virus-specific CD4+ T cell responses in conjunction with blockade of the inhibitory pathway may represent a promising therapeutic approach to treating patients with chronic viral infections.

In vitro Anti-Influenza Virus Activity of Agaricus brasiliensis KA21.

Agaricus is known to have immunostimulatory and anti-tumor effects. However, the antiviral effects of Agaricus have not yet been examined. In the present study, the antiviral effects of an extract of Agaricus brasiliensis KA21 (AE) on the H1N1 influenza virus (PR8 strain) were investigated. The anti-influenza virus effects of AE were examined by using the plaque formation inhibition test. AE inhibited the plaque formation of PR8 in a dose-dependent manner: 98 and 50% (IC50) inhibition at 2.5 and 0.99 mg/mL, respectively. To elucidate the mechanisms of AE, the direct actions and adsorption and invasion inhibition of AE were examined, and were found to have no inhibitory effect on PR8 infection. Thus, in vitro antiviral effects may somehow inhibit PR8 after the viral invasion of cells. These results demonstrated that it is expected that AE can effectively prevent the spread of the influenza virus.