Influenza Challenge In Mice Research Articles

3-Fucosyllactose-mediated modulation of immune response against virus infection

Viral pathogens, particularly influenza and SARS-CoV-2, pose a significant global health challenge. Given the immunomodulatory properties of human milk oligosaccharides, in particular 2′-fucosyllactose and 3-fucosyllactose (3-FL), we investigated their dietary supplementation effects on antiviral responses in mouse models. This study revealed distinct immune modulations induced by 3-FL. RNA-sequencing data showed that 3-FL increased the expression of interferon receptors, such as Interferon Alpha and Beta Receptor (IFNAR) and Interferon Gamma Receptor (IFNGR), while simultaneously downregulating interferons and interferon-stimulated genes, an effect not observed with 2′-fucosyllactose supplementation. Such modulation enhanced antiviral responses in both cell culture and animal models while attenuating pre-emptive inflammatory responses. Nitric oxide concentrations in 3-FL-supplemented A549 cells and mouse lung tissues were elevated exclusively upon infection, reaching 5.8- and 1.9-fold increases over control groups, respectively. In addition, 3-FL promoted leukocyte infiltration into the site of infection upon viral challenge. 3-FL supplementation provided protective efficacy against lethal influenza challenge in mice. The demonstrated antiviral efficacy spanned multiple influenza strains and extended to SARS-CoV-2. In conclusion, 3-FL is a unique immunomodulator that helps protect the host from viral infection while suppressing inflammation prior to infection.

A single-shot adenoviral vaccine provides hemagglutinin stalk-mediated protection against heterosubtypic influenza challenge in mice

Conventional influenza vaccines fail to confer broad protection against diverse influenza A viruses with pandemic potential. Efforts to develop a universal influenza virus vaccine include refocusing immunity towards the highly conserved stalk domain of the influenza virus surface glycoprotein, hemagglutinin (HA). We constructed a non-replicating adenoviral (Ad) vector, encoding a secreted form of H1 HA, to evaluate HA stalk-focused immunity. The Ad5_H1 vaccine was tested in mice for its ability to elicit broad, cross-reactive protection against homologous, heterologous, and heterosubtypic lethal challenge in a single-shot immunization regimen. Ad5_H1 elicited hemagglutination inhibition (HI+) active antibodies (Abs), which conferred 100% sterilizing protection from homologous H1N1 challenge. Furthermore, Ad5_H1 rapidly induced H1-stalk-specific Abs with Fc-mediated effector function activity, in addition to stimulating both CD4+ and CD8+ stalk-specific Tcell responses. This phenotype of immunity provided 100% protection from lethal challenge with a head-mismatched, reassortant influenza virus bearing a chimeric HA, cH6/1, in a stalk-mediated manner. Most importantly, 100% protection from mortality following lethal challenge with a heterosubtypic avian influenza virus, H5N1, was observed following a single immunization with Ad5_H1. In conclusion, Ad-based influenza vaccines can elicit significant breadth of protection in naive animals and could be considered for pandemic preparedness and stockpiling.

Glucan and Vitamin D supplementation showed synergy in improvements of the immune response against an influenza challenge in mice

Glucan and Vitamin D supplementation showed synergy in improvements of the immune response against an influenza challenge in mice

Influenza vaccines using liposomal formulations of toll-like receptor (TLR) 7/8 and 4 agonists as adjuvants

Abstract Influenza virus infection is a global health concern and causes severe illness and thousands of deaths each year. While there are currently licensed vaccines available for both seasonal and pandemic influenza viruses there are several issues to consider; 1) current vaccines do not provide supra-seasonal or universal protection against influenza subtypes, 2) antigenic mismatch between the vaccine and circulating influenza strains impact vaccine efficacy, 3) and the efficacy of influenza seasonal vaccines could be improved in the elderly and immune compromised populations. Therefore, vaccine adjuvants capable of inducing more robust and broadly cross-protective immune responses to various subtypes of influenza virus is a critical unmet need. To address this challenge, we developed a synthetic dual TLR adjuvant combination system (TRAC-478) to improve influenza vaccine immunity. Preliminary data in human peripheral blood mononuclear cells demonstrate that combining our synthetic TLR7/8 and TLR4 agonists results in a synergistic innate immune responses. Influenza vaccine immunogenicity studies in both murine and pig models demonstrate the safety and efficacy of this new dual TLR adjuvant system. Using a co-encapsulated TRAC-478 liposome delivery system with a split-flu vaccine resulted in strong anti-influenza humoral, Th1 cell mediated immunity, and protection from a heterologous influenza challenge in mice. We hypothesize that using the TRAC-478 adjuvant system in combination with current influenza vaccines will induce a more robust and broadly cross-protective immune responses to circulating or pandemic influenza virus.

Novel Lipidated Imidazoquinoline TLR7/8 Adjuvants Elicit Influenza-Specific Th1 Immune Responses and Protect Against Heterologous H3N2 Influenza Challenge in Mice.

Most licensed seasonal influenza vaccines are non-adjuvanted and rely primarily on vaccine-induced antibody titers for protection. As such, seasonal antigenic drift and suboptimal vaccine strain selection often results in reduced vaccine efficacy. Further, seasonal H3N2 influenza vaccines demonstrate poor efficacy compared to H1N1 and influenza type B vaccines. New vaccines, adjuvants, or delivery technologies that can induce broader or cross-seasonal protection against drifted influenza virus strains, likely through induction of protective T cell responses, are urgently needed. Here, we report novel lipidated TLR7/8 ligands that act as strong adjuvants to promote influenza-virus specific Th1-and Th17-polarized T cell responses and humoral responses in mice with no observable toxicity. Further, the adjuvanted influenza vaccine provided protection against a heterologous H3N2 influenza challenge in mice. These responses were further enhanced when combined with a synthetic TLR4 ligand adjuvant. Despite differences between human and mouse TLR7/8, these novel lipidated imidazoquinolines induced the production of cytokines required to polarize a Th1 and Th17 immune response in human PBMCs providing additional support for further development of these compounds as novel adjuvants for the induction of broad supra-seasonal protection from influenza virus.

Vaccination with three tandem repeats of M2 extracellular domain fused to Leismania major HSP70 protects mice against influenza A virus challenge

Influenza viruses are globally important respiratory pathogens with high degree of morbidity and mortality during annual epidemics. Influenza A vaccination has been challenged due to genetic evolution. M2 protein is the surface antigen of the virus and facilitates virus entry into the host cells. N-terminal 24 amino acid residues that constitute the extracellular domain of M2 protein (M2e) show remarkable conservation among all subtypes of influenza A viruses. The aim of the present study was to investigate the effects of using HSP70 as an adjuvant fused to three tandem repeats of M2e (3M2e) to enhance the immune responses against influenza A challenge.The mice were immunized three times by intradermal inoculations of 3M2e alone or in combination with Alum adjuvant or in fused form to HSP70. The specific immune responses were evaluated by measuring the serum antibody titers, lymphocyte proliferation, as well as Th1 and Th2 cytokines.The results showed that, although 3M2e with no adjuvant could induce secretion of specific antibodies, significantly higher humoral immune responses were stimulated in combination with Alum adjuvant (p < 0.05). Moreover, analysis of specific immune responses revealed that the 3M2e-HSP70 chimer protein mainly stimulated IgG2a and IFN-γ responses indicating aTh1 bias which shows the ability of HSP as a powerful adjuvant for activation of cellular immune responses. This was supported by a higher IgG2a/IgG1 ratio, significantly increased IL-4 production and lymphocyte proliferation (P ˂ 0.001) compared with mice vaccinated with 3M2e alone or supplemented with Alum, suggesting a mixture of Th1 and Th2 type cellular immune response with a Th1 bias.The findings of this study indicated that 3M2e-HSP70 enhances humoral and cellular immune responses and improves immune protection against influenza challenge in mice. Thus, it has the potential to be used as a promising vaccine candidate.

Adjuvant use of the NKT cell agonist alpha-galactosylceramide leads to enhancement of M2-based DNA vaccine immunogenicity and protective immunity against influenza A virus.

DNA vaccines can induce both humoral and cellular immune responses in animals. However, DNA vaccines suffer from limited vaccine potency due to low immunogenicity. Therefore, different strategies are required for significant improvement of DNA vaccine efficacy such as inclusion of strong adjuvants. The aim of the present study was to investigate the effects of using α-Galactosylceramide (α-GalCer) as an adjuvant to enhance the immune responses induced by a DNA vaccine, encoding influenza A virus matrix protein 2 (M2), against influenza A challenge. BALB/c mice were immunized three times by intramuscular inoculations of DNA vaccine encoding M2 alone or in combination with α-GalCer adjuvant. The adjuvant effect was evaluated by measuring the serum antibody titers, using ELISA, lymphocyte proliferation, using MTT assay as well as Th1 (IFN-γ and IL-12) and Th2 (IL-4) cytokines. The results showed that co-administration of α-GalCer with the vaccine exert protective effects by influencing the magnitude and quality of humoral responses. Adjuvanted DNA-vaccinated mice revealed a higher IgG titer and IgG2a/IgG1 ratio than mice vaccinated with DNA alone. Furthermore, analysis of M2-specific responses revealed that the DNA vaccine triggered predominately IgG1 and IL-4 responses indicating a Th2 bias. The data also showed that α-GalCer is a potent adjuvant for activation of cellular immune responses to DNA vaccine. This was supported by a higher IgG2a/IgG1 ratio, significantly increased IFN-γ and IL-4 production and CD4+ proliferation, compared with mice receiving the DNA vaccine alone, suggesting a mixed Th1/Th2-type cellular immune response with a Th1 bias. The findings of this study indicate that α-GalCer has the potential to be used as a potent adjuvant for a DNA vaccine encoding M2, since it enhances humoral and cellular immune response and improves immune protection against influenza challenge in mice.

A Novel Glucan-Sulforaphane Combination Stimulates Immune Response to Influenza in Mouse Model

Influenza remains a serious health problem and causes approximately 500,000 deaths world-wide. With current available treatments offering neither dependable protection nor a rapid cure, many have focused their attention to alternative treatments. The aim of this study was to evaluate the possible effect of a novel maitake glucan-sulforaphane combination on immune response against influenza challenge in mice. We evaluated the effects of a glucan-sulforaphane combination on basic immune reactions, virus titre and overall survival after influenza infection. Results: We found 2 weeks supplementation with this glucan-sulforaphane combination significantly improved immunosuppression caused by the viral infection. Based on these results, we conclude that the significant immunostimulation caused by this combination helps to overcome virus-dependent suppression of defense reactions and that addition of sulforaphane to glucan can improve already established biological effects of glucan.

Novel synthetic plasmid and Doggybone™ DNA vaccines induce neutralizing antibodies and provide protection from lethal influenza challenge in mice

Nucleic acid-based vaccines (NAVs) are a promising alternative to conventional influenza vaccines with the potential to increase influenza vaccine availability due to their simplicity in design and rapid speed of production. NAVs can also target multiple influenza antigens and control flu variants. Traditionally NAVs have been DNA plasmids however, we are continuing to explore new methods that may enhance vaccine efficacy. Recently new focus has been on RNA cassettes as NAVs. RNA vaccines combine conceptual advantages in that they focus on delivery of only the coding cassette. However, RNA vaccines have a short half-life and cause interferon-induced fevers. Here we describe a new NAV approach where we study delivery of a linear DNA cassette [Doggybone™ linear closed DNA [(dbDNA™)] produced by an enzymatic process that yields an antigen expression cassette comprising a promoter, DNA antigen, poly A tail, and telomeric ends. This focused approach has many of the advantages of plasmid DNA as well as a minimal cassette size similar to RNA strategies. For this study, we characterized the specific CD4+ and CD8+ T cell responses and determined the hemagglutination inhibition (HI) titers induced by dbDNA™ and compared the responses with those of an optimized plasmid DNA (pDNA) vaccine encoding the same H1N1 influenza A/PR/8/34 HA gene. Immunizations with the constructs resulted in similar humoral and cellular immune responses. Both constructs induced high-titer HI antibodies and fully protected animals from lethal viral challenge. The data obtained from this study provides important validation for further development of novel vector approaches.

Diverse heterologous primary infections radically alter immunodominance hierarchies and clinical outcomes following H7N9 influenza challenge in mice.

The recent emergence of a novel H7N9 influenza A virus (IAV) causing severe human infections in China raises concerns about a possible pandemic. The lack of pre-existing neutralizing antibodies in the broader population highlights the potential protective role of IAV-specific CD8+ cytotoxic T lymphocyte (CTL) memory specific for epitopes conserved between H7N9 and previously encountered IAVs. In the present study, the heterosubtypic immunity generated by prior H9N2 or H1N1 infections significantly, but variably, reduced morbidity and mortality, pulmonary virus load and time to clearance in mice challenged with the H7N9 virus. In all cases, the recall of established CTL memory was characterized by earlier, greater airway infiltration of effectors targeting the conserved or cross-reactive H7N9 IAV peptides; though, depending on the priming IAV, each case was accompanied by distinct CTL epitope immunodominance hierarchies for the prominent KbPB1703, DbPA224, and DbNP366 epitopes. While the presence of conserved, variable, or cross-reactive epitopes between the priming H9N2 and H1N1 and the challenge H7N9 IAVs clearly influenced any change in the immunodominance hierarchy, the changing patterns were not tied solely to epitope conservation. Furthermore, the total size of the IAV-specific memory CTL pool after priming was a better predictor of favorable outcomes than the extent of epitope conservation or secondary CTL expansion. Modifying the size of the memory CTL pool significantly altered its subsequent protective efficacy on disease severity or virus clearance, confirming the important role of heterologous priming. These findings establish that both the protective efficacy of heterosubtypic immunity and CTL immunodominance hierarchies are reflective of the immunological history of the host, a finding that has implications for understanding human CTL responses and the rational design of CTL-mediated vaccines.

Protection against H5N1 by multiple immunizations with seasonal influenza vaccine in mice is correlated with H5 cross-reactive antibodies

BackgroundCurrent seasonal influenza vaccines are believed to confer protection against a narrow range of virus strains. However, their protective ability is commonly estimated based on an in vitro correlate of protection that only considers a subset of anti-influenza antibodies that are typically strain specific, i.e., hemagglutination inhibiting antibodies. Here, we evaluate the breadth of protection induced with a seasonal trivalent influenza vaccine (composition H1N1 A/California/07/09, H3N2 A/Victoria/210/08, B/Brisbane/60/08) against influenza challenge in mice. MethodsBalb/c mice were immunized once, twice, or three times with seasonal influenza vaccine to assess protection against heterosubtypic H5N1 influenza challenge, or homologous H1N1 influenza virus as a control. Passive transfer of immune serum was used to determine the contribution of humoral immunity to protection. ResultsMultiple immunizations with seasonal influenza vaccine induced up to 80% protection against heterosubtypic H5N1 influenza challenge in mice without eliciting detectable H5N1 neutralizing antibodies. Comparable levels of protection were reached by passive transfer of immune serum, and protection was correlated with the titer of vaccine-induced, H5 cross-reactive, non-neutralizing antibodies that are at least in part directed against conserved HA epitopes. ConclusionsHere, we demonstrate that seasonal vaccine has the ability to induce broad serum-mediated protection, and that the mechanism of this protection is different from the vaccine-induced homologous protection.

Glucan supplementation enhances the immune response against an influenza challenge in mice.

The strong immunostimulating potential of β-glucans has been well established in numerous diseases. However, the effects on viral infection were less studied. In our study, we focused on possible effects of a special combined glucan formulation on immunosuppression caused by influenza infection. We found that a 2-week oral feeding with glucan mixture significantly reduced the effects of influenza infection in total mortality. Our study was focused on phagocytosis, cytokine levels, antibody response and cytotoxicity assay. Based on our data, we concluded that these effects are caused by stimulation of both cellular and humoral immune reaction resulting in lower viral load.

Alterations in EEG activity and sleep after influenza viral infection in GHRH receptor-deficient mice

Viral infections induce excess non-rapid eye movement sleep (NREMS) in mice. Growth hormone-releasing hormone receptor (GHRH receptor) was previously identified as a candidate gene responsible for NREMS responses to influenza challenge in mice. The dwarf lit/lit mouse with a nonfunctional GHRH receptor was used to assess the role of the GHRH receptor in viral-induced NREMS. After influenza A virus infection the duration and intensity [electroencephalogram (EEG) delta power] of NREMS increased in heterozygous mice with the normal phenotype, whereas NREMS and EEG delta power decreased in homozygous lit/lit mice. Lit/lit mice developed a pathological state with EEG slow waves and enhanced muscle tone. Other influenza-induced responses (decreases in rapid eye movement sleep, changes in the EEG high-frequency bands during the various stages of vigilance, hypothermia, and decreased motor activity) did not differ between the heterozygous and lit/lit mice. GH replacement failed to normalize the NREMS responses in the lit/lit mice after influenza inoculation. Decreases in NREMS paralleled hypothermia in the lit/lit mice. Lung virus levels were similar in the two mouse strains. Lit/lit mice had a higher death rate after influenza challenge than the heterozygotes. In conclusion, GHRH signaling is involved in the NREMS response to influenza infection.

Immunization with recombinant Semliki Forest virus induces protection against influenza challenge in mice

The replicon of Semliki Forest virus (SFV) offers the possibility to direct high-level, transient expression of heterologous proteins in vivo. We initiated studies to determine the possibility of employing the SFV expression system for recombinant vaccine purposes. Mice immunized with recombinant SFV encoding Influenza A nucleoprotein (NP) or E. coli LacZ developed long-lasting antigen-specific IgG levels and induction of cytotoxic T-cell (CTL) memory that persisted for over one year. Predominantly type 1 T-helper cells were induced as shown by IgG subclass ELISA. Humoral and cell-mediated immune responses could be induced upon delivery by several administration routes and mucosal immunizations induced secretory IgA in the respiratory tract. Development of immune responses against the vector itself did not inhibit boost responses by subsequent immunizations with recombinant SFV. Immunization of mice with vectors encoding the Influenza A virus antigens nucleoprotein (NP) and hemagglutinin (HA) resulted in immune responses that were protective against challenge infection with Influenza virus.

Body temperature and nesting behavior following influenza challenge in mice: Effects of age

We studied the interaction of age and influenza on core body temperature (T c) of mice. Following influenza challenge, 2-mo-old female BALB/c mice demonstrated a significant fall in T c. Female BALB/c mice 24 mo of age had lower baseline T c than young mice and a larger fall in T c post influenza challenge. We noted there were marked differences in nesting behavior between the young and aged mice. A nesting score was devised, and we found that at baseline, aged mice had a much lower score than young mice (15.6 ± 7.4 vs. 24.7 ± 0.3, P < 0.0001). Following influenza challenge, nesting behavior of young mice dropped considerably, while no significant change occurred in the behavior of aged mice. When mice were housed without bedding, there were significant decreases in T c of young, but not aged mice. There was a further fall in T c with influenza challenge in young mice. These data imply that nesting is an important mechanism for maintaining T c in young mice, but alternative mechanisms are used by aged mice. The lower body temperatures in the aged mice are similar to studies in aging humans.