krn-7000 and Orthomyxoviridae-Infections

Influenza virus infects the host and transmits through the respiratory tract (i.e., the mouth and nose); therefore, the development of intranasal influenza vaccines that mimic the natural infection, coupled with an efficient mucosal adjuvant, is an attractive alternative to current parenteral vaccines. However, with the withdrawal of cholera toxin and Escherichia coli heat-labile endotoxin from clinical use due to side effects, there are no approved adjuvants for intranasal vaccines. Therefore, safe and effective mucosal adjuvants are urgently needed. Previously, we reported that one derivative of α-Galactosylceramide (α-GalCer), 7DW8-5, could enhance the protective efficacy of split influenza vaccine by injection administration. However, the mucosal adjuvanticity of 7DW8-5 is still unclear. In this study, we found that 7DW8-5 promotes the production of secret IgA antibodies and IgG antibodies and enhances the protective efficacy of the split influenza vaccine by intranasal administration. Furthermore, co-administration of 7DW8-5 with the split influenza vaccine significantly reduces the virus shedding in the upper and lower respiratory tract after lethal challenge. Our results demonstrate that 7DW8-5 is a novel mucosal adjuvant for the split influenza vaccine.

Topics: Adjuvants, Immunologic; Administration, Intranasal; Animals; Antibodies, Viral; Galactosylceramides; Glycolipids; Humans; Immunity, Mucosal; Influenza Vaccines; Influenza, Human; Mice; Mice, Inbred BALB C; Orthomyxoviridae Infections; Vaccines, Inactivated

Influenza A viruses (IAV) are a major cause of respiratory diseases in pigs. Invariant natural killer T (iNKT) cells are an innate-like T cell subset that contribute significantly to IAV resistance in mice. In the current work, we explored whether expanding and activating iNKT cells with the iNKT cell superagonist α-galactosylceramide (α-GalCer) would change the course of an IAV infection in pigs. In one study, α-GalCer was administered to pigs intramuscularly (i.m.) 9 days before infection, which systemically expanded iNKT cells. In another study, α-GalCer was administered intranasally (i.n.) 2 days before virus infection to activate mucosal iNKT cells. Despite a synergistic increase in iNKT cells when α-GalCer i.m. treated pigs were infected with IAV, neither approach reduced disease signs, lung pathology, or virus replication. Our results indicate that prophylactic use of iNKT cell agonists to prevent IAV infection is ineffective in pigs. This is significant because this type of approach has been considered for humans whose iNKT cell levels and IAV infections are more similar to those of pigs than mice.

Topics: Animals; Galactosylceramides; Humans; Influenza A virus; Influenza, Human; Injections, Intramuscular; Lung; Lymphocyte Activation; Mice; Nasal Mucosa; Natural Killer T-Cells; Orthomyxoviridae Infections; Swine; Vaccine Efficacy; Virus Replication

The development of a universal vaccine for influenza A virus (IAV) that does not require seasonal modification is a long-standing health goal, particularly in the context of the increasing threat of new global pandemics. Vaccines that specifically induce T cell responses are of considerable interest because they can target viral proteins that are more likely to be shared between different virus strains and subtypes and hence provide effective cross-reactive IAV immunity. From a practical perspective, such vaccines should induce T cell responses with long-lasting memory, while also being simple to manufacture and cost-effective. Here we describe the synthesis and evaluation of a vaccine platform based on solid phase peptide synthesis and bio-orthogonal conjugation methodologies. The chemical approach involves covalently attaching synthetic long peptides from a virus-associated protein to a powerful adjuvant molecule, α-galactosylceramide (α-GalCer). Strain-promoted azide-alkyne cycloaddition is used as a simple and efficient method for conjugation, and pseudoproline methodology is used to increase the efficiency of the peptide synthesis. α-GalCer is a glycolipid that stimulates NKT cells, a population of lymphoid-resident immune cells that can provide potent stimulatory signals to antigen-presenting cells engaged in driving proliferation and differentiation of peptide-specific T cells. When used in mice, the vaccine induced T cell responses that provided effective prophylactic protection against IAV infection, with the speed of viral clearance greater than that seen from previous viral exposure. These findings are significant because the vaccines are highly defined, quick to synthesize, and easily characterized and are therefore appropriate for large scale affordable manufacture.

Topics: Adjuvants, Immunologic; Animals; CD8-Positive T-Lymphocytes; Cycloaddition Reaction; Female; Galactosylceramides; Humans; Influenza A virus; Influenza Vaccines; Influenza, Human; Mice, Inbred C57BL; Natural Killer T-Cells; Orthomyxoviridae Infections; Peptides; Solid-Phase Synthesis Techniques

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.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Viral; Cell Line; Cell Proliferation; Chlorocebus aethiops; COS Cells; Dogs; Enzyme-Linked Immunosorbent Assay; Female; Galactosylceramides; Immunogenicity, Vaccine; Immunoglobulin G; Influenza A Virus, H1N1 Subtype; Influenza Vaccines; Interferon-gamma; Interleukin-12 Subunit p35; Interleukin-4; Madin Darby Canine Kidney Cells; Mice; Mice, Inbred BALB C; Natural Killer T-Cells; Orthomyxoviridae Infections; Th1 Cells; Th2 Cells; Vaccination; Vaccines, DNA; Viral Matrix Proteins

Natural killer T (NKT) -cells activated with the glycolipid ligand α-galactosylceramide (α-GalCer) stimulate a wide array of immune responses with many promising immunotherapeutic applications, including the enhancement of vaccines against infectious diseases and cancer. In the current study, we evaluated whether α-GalCer generates protective immunity against a swine influenza (SI) virus infection when applied as an intramuscular vaccine adjuvant. Immunization of newly weaned piglets with UV-killed pandemic H1N1 A/California/04/2009 (kCA04) SI virus and α-GalCer induced high titers of anti-hemagglutinin antibodies and generated virus-specific T cells that localized in intrapulmonary airways and in alveolar walls. Vaccination with α-GalCer resulted in a systemic increase in NKT-cell concentrations, including in the respiratory tract, which was associated with complete inhibition of viral replication in the upper and lower respiratory tract and much reduced viral shedding. These results indicate that NKT-cell agonists could be used to improve swine vaccine formulations in order to reduce the clinical signs of SI infection and limit the spread of influenza viruses amongst commercial pigs.

Topics: Administration, Intranasal; Animals; Antibodies, Viral; Galactosylceramides; Influenza A Virus, H1N1 Subtype; Influenza Vaccines; Injections, Intramuscular; Natural Killer T-Cells; Orthomyxoviridae Infections; Respiratory System; Swine; Virus Replication

Pigs are considered as the source of some of the emerging human flu viruses. Inactivated swine influenza virus (SwIV) vaccine has been in use in the US swine herds, but it failed to control the flu outbreaks. The main reason has been attributed to lack of induction of strong local mucosal immunity in the respiratory tract. Invariant natural killer T (iNKT) cell is a unique T cell subset, and activation of iNKT cell using its ligand α-Galactosylceramide (α-GalCer) has been shown to potentiate the cross-protective immunity to inactivated influenza virus vaccine candidates in mice. Recently, we discovered iNKT cell in pig and demonstrated its activation using α-GalCer. In this study, we evaluated the efficacy of an inactivated H1N1 SwIV coadministered with α-GalCer intranasally against a homologous viral challenge. Our results demonstrated the potent adjuvant effects of α-GalCer in potentiating both innate and adaptive immune responses to SwIV Ags in the lungs of pigs, which resulted in reduction in the lung viral load by 3 logs compared to without adjuvant. Immunologically, in the lungs of pigs vaccinated with α-GalCer an increased virus specific IgA response, IFN-α secretion and NK cell-cytotoxicity was observed. In addition, iNKT cell-stimulation enhanced the secretion of Th1 cytokines (IFN-γ and IL-12) and reduced the production of immunosuppressive cytokines (IL-10 and TGF-β) in the lungs of pigs⋅ In conclusion, we demonstrated for the first time iNKT cell adjuvant effects in pigs to SwIV Ags through augmenting the innate and adaptive immune responses in the respiratory tract.

Topics: Adaptive Immunity; Adjuvants, Immunologic; Animals; Cytokines; Galactosylceramides; Immunity, Innate; Immunoglobulin A; Influenza A Virus, H1N1 Subtype; Influenza Vaccines; Lung; Natural Killer T-Cells; Orthomyxoviridae Infections; Random Allocation; Swine; Swine Diseases; Vaccines, Inactivated; Viral Load; Weight Gain

Despite the application of vaccination programs and antiviral drugs, influenza A virus (IAV) infection is responsible for widespread morbidity and mortality (500,000 deaths/year). Influenza infections can also result in sporadic pandemics that can be devastating: the 1918 pandemic led to the death of 50 million people. Severe bacterial infections are commonly associated with influenza and are significant contributors to the excess morbidity and mortality of influenza. Today's treatments of secondary bacterial (pneumococcal) infections are still not effective enough, and antibiotic resistance is a major issue. Hence, there is an urgent need for novel therapies. In the present study, we set out to evaluate the efficacy of α-galactosylceramide (α-GalCer)-a potent agonist of invariant NKT cells that is currently in clinical development-in a mouse model of postinfluenza, highly invasive pneumococcal pneumonia. Our data indicate that treatment with α-GalCer reduces susceptibility to superinfections and, when combined with the corticosteroid dexamethasone, reduces viral-bacterial pneumonia.

Topics: Administration, Intranasal; Animals; Disease Models, Animal; Galactosylceramides; Lymphocyte Activation; Mice; Natural Killer T-Cells; Orthomyxoviridae Infections; Pneumococcal Infections; Time Factors; Treatment Outcome

Swine influenza A viruses (IAV) are a major cause of respiratory disease in pigs and humans. Currently approved anti-influenza therapies directly target the virus, but these approaches are losing effectiveness as new viral strains quickly develop drug resistance. To over come this challenge, there is an urgent need for more effective antiviral drugs. Here we tested the anti-influenza efficacy of the invariant natural killer T (NKT) cell superagonist, α-galactosylceramide (α-GalCer), which stimulates a wide array of anti-viral immune responses. We show that intranasal but not systemic administration of α-GalCer to piglets infected with pandemic A/California/04/2009 (CA04) H1N1 IAV ameliorated disease symptoms and resulted in the restoration of weight gain to the level of uninfected pigs. Correspondingly, viral titers in the upper-and lower-respiratory tract were reduced only in piglets that had received intranasal α-GalCer. Most significantly, lung inflammation as a consequence of virus persistence was largely prevented when NKT-cells were targeted via the respiratory route. Thus, targeting mucosal NKT-cells may provide a novel and potent platform for improving the course of disease in swine infected with seasonal and pandemic influenza viruses, and leads to the suggestion that this may also be true in humans and therefore deserves further study.

Topics: Adjuvants, Immunologic; Administration, Intranasal; Animals; Antiviral Agents; Galactosylceramides; Influenza A Virus, H1N1 Subtype; Lymphocyte Activation; Natural Killer T-Cells; Orthomyxoviridae Infections; Swine; Viral Load

α-Galactosylceramide (α-GalCer) is a safe and effective adjuvant for nasal vaccines and induces protective immune responses against tumors and viral infections. In our previous study, the fatty acyl chains of α-GalCer were modified based on the CD1d/glycolipid structure to generate α-GalCer analogues with branched acyl chains. In this study, two α-GalCer analogues, KBC-007 and KBC-009, that have different branched chain lengths were prepared and evaluated for their efficacy as nasal influenza vaccine adjuvants. These analogues displayed improved solubility over α-GalCer and potently stimulated NKT cells in both murine and in vitro human systems. Examination of serum cytokines in vivo revealed that these analogues elicited different cytokine release profiles compared to α-GalCer. KBC-009 induced both Th1/Th2 cytokines, whereas KBC-007 induced a more Th2-polarized cytokine response with diminished IFN-γ production. We found that a single immunization of inactivated influenza virus A/PR/8/34 (PR8) combined with α-GalCer analogues enhanced PR8-specific humoral and cellular immune responses in both systemic and mucosal compartments. Notably, KBC-009 exhibited potent adjuvant effects, inducing significantly higher systemic IgG and mucosal IgA antibody titers and enhancing cytotoxic T lymphocyte generation when compared to immunization with inactivated PR8 alone. In contrast, addition of KBC-007 to inactivated PR8 only marginally increased PR8-specific immune responses. The protective effect of KBC-009 against challenge infection was comparable to the effect produced by α-GalCer. These results suggest that an α-GalCer analogue with a branched acyl chain could be used as an effective mucosal adjuvant for the induction of protective immune responses against influenza virus infection.

Topics: Adjuvants, Immunologic; Administration, Intranasal; Animals; Antibodies, Viral; Cytokines; Female; Galactosylceramides; Humans; Immunity, Mucosal; Immunoglobulin A; Immunoglobulin G; Influenza A virus; Influenza Vaccines; Lymphocyte Subsets; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Orthomyxoviridae Infections; Th1 Cells; Th2 Cells

A new form of influenza A vaccine that can provide broadly cross-protective immunity is central in developing strategies to prepare for the next global flu pandemic. The ectodomain of the M2 protein (M2e) is an attractive target for developing such a kind of vaccine and several approaches have been proposed to overcome its poor immunogenicity nature. Here, we show change to the poor immunogenic characteristic of this antigen. This study demonstrates that α-galactosylceramide, which is an immunomodulatory glycolipid, can greatly enhance the protective immunity induced by M2e peptide absorbed in alum adjuvant. Mice were fully protected against highly pathogenic H5N1 avian influenza virus infection, exhibiting significantly reduced morbidity and lung viral titer after supplementing with α-galactosylceramide. α-Galactosylceramide simultaneously augmented the IgG1 and IgG2a antibody responses. In addition, mice immune sera showed enhanced abilities in binding to native M2 proteins on virus infected cells. The adjuvant also modulated the cytokine release of mice upon infection, upregulated the expressions of IFN-γ, IL-4 and several proinflammatory cytokines. In conclusion, we believe that M2e-peptide supplemented with α-galactosylceramide in alum adjuvant would be a promising vaccine formulation to combat the next influenza pandemic.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antibodies, Viral; Female; Galactosylceramides; Immunoglobulin G; Influenza A Virus, H5N1 Subtype; Influenza Vaccines; Interferon-gamma; Interleukin-4; Lung; Mice; Mice, Inbred BALB C; Orthomyxoviridae Infections; Viral Load

There is a substantial need to develop better influenza virus vaccines that can protect populations that are not adequately protected by the currently licensed vaccines. While live attenuated influenza virus vaccines induce superior immune responses compared to inactivated vaccines, the manufacturing process of both types of influenza virus vaccines is time consuming and may not be adequate during a pandemic. Adjuvants would be particularly useful if they could enhance the immune response to live attenuated influenza virus vaccines so that the amount of vaccine needed for a protective dose could be reduced. The glycolipid, alpha-galactosylceramide (alpha-GalCer), has recently been shown to have adjuvant activity for both inactivated and replicating recombinant vaccines. The goal of these experiments was to determine whether a derivative of alpha-GalCer, alpha-C-galactosylceramide (alpha-C-GalCer) can enhance the immune response elicited by a live attenuated influenza virus vaccine containing an NS1 protein truncation and reduce the amount of vaccine required to provide protection after challenge. Our results indicated that the adjuvant reduced both morbidity and mortality in BALB/c mice after challenge with wild type influenza virus. The adjuvant also increased the amount of influenza virus specific total IgG, IgG1, and IgG2a antibodies as well as IFN-gamma secreting CD8(+) T cells. By using knockout mice that are not able to generate NKT cells, we were able to demonstrate that the mechanism of adjuvant activity is dependent on NKT cells. Thus, our data indicate that stimulators of NKT cells represent a new avenue of adjuvants to pursue for live attenuated virus vaccines.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Viral; CD8-Positive T-Lymphocytes; Chick Embryo; Female; Galactosylceramides; Influenza Vaccines; Interferon-gamma; Killer Cells, Natural; Male; Mice; Mice, Inbred BALB C; Orthomyxoviridae Infections; Severity of Illness Index; Survival Analysis; Vaccines, Attenuated

Invariant NKT (iNKT) cells have an indubitable role in antiviral immunity, although the mechanisms by which these cells exert their functions are not fully elucidated. With the emerging importance of high-pathogenicity influenza A virus infections in humans, we questioned whether iNKT cells contribute to immune defence against influenza A virus and whether activation of these cells influences outcome. We show that activation of iNKT cells with alpha-galactosylceramide (alpha-GC) during influenza virus infection transiently enhanced early innate immune response without affecting T cell immunity, and reduced early viral titres in lungs of C57BL/6 mice. This is accompanied by a better disease course with improved weight loss profile. Temporal changes in iNKT cells in the liver, blood and lungs suggest activation and migration of iNKT cells from the liver to the lungs in mice that were administered alpha-GC. Improvement in viral titres appears dependent on activation of iNKT cells via the intraperitoneal route since intranasal administration of alpha-GC did not have the same effect. We conclude that activation of iNKT cells enhances early innate immune response in the lungs and contribute to antiviral immunity and improved disease course in influenza A virus infection.

Topics: Animals; Bronchoalveolar Lavage Fluid; Cytokines; Disease Models, Animal; Galactosylceramides; Humans; Immunity, Innate; Inflammation Mediators; Influenza A virus; Influenza A Virus, H1N1 Subtype; Influenza A Virus, H3N2 Subtype; Influenza, Human; Liver; Lung; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Orthomyxoviridae Infections; T-Lymphocyte Subsets; T-Lymphocytes

Invariant natural killer T (iNKT) cells are innate-like lymphocytes recognizing CD1d-restricted glycolipid antigens, such as alpha-galactosylceramide (alphaGC). We assessed whether iNKT cells help B lymphocyte responses and found that mice immunized with proteins and alphaGC develop antibody titers 1-2 logs higher than those induced by proteins alone. Activation of iNKT cells enhances protection against infections such as influenza and elicits higher frequencies of memory B cells and higher antibody responses to booster immunizations. Protein vaccination with alphaGC, but not with conventional adjuvants, elicits IgG responses in mice lacking MHC class II molecules, demonstrating that iNKT cells can substitute for CD4(+) T cell help to B cells. Interestingly, the decay of circulating antibodies is faster in mice lacking iNKT cells. These findings point to a homeostatic role for iNKT cells on critical features of the antibody response such as immunity and B cell memory.

Topics: Animals; Antigens, CD1; Antigens, CD1d; B-Lymphocytes; CD4-Positive T-Lymphocytes; Galactosylceramides; Immune System; Immunologic Memory; Killer Cells, Natural; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Transgenic; Orthomyxoviridae Infections; Time Factors

alpha-Galactosylceramide (alpha-GalCer), originally isolated from a marine sponge, was known to activate natural killer T (NKT) cells through CD1d-mediated Ag presentation and induce Th1 and/or Th2 immunity. In this study, we evaluated the nasal adjuvanticity of alpha-GalCer when co-administered with formalin-inactivated influenza virus A/PR/8/34 (PR8) in BALB/c mice. A single nasal immunization of inactivated PR8 and alpha-GalCer induced brisk levels of PR8-specific IgG and IgA Abs in serum and lung washes. Antigen-specific Ab responses lasted for 3 months, providing protective immunity against challenge with live PR8. In addition, mice given alpha-GalCer also exhibited cellular immune responses including cytotoxic T lymphocyte (CTL) generation. Because it did not redirect Ags into brain, alpha-GalCer would likely pose no risk if administered as a nasal adjuvant. These results suggest for the first time that a single nasal immunization of inactivated virus and alpha-GalCer is a safe and effective means of preventing influenza infection.

Topics: Administration, Intranasal; Animals; Antibody Formation; Central Nervous System; Cytokines; Cytotoxicity, Immunologic; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Female; Galactosylceramides; Immunity, Cellular; Immunization; Influenza Vaccines; Kaplan-Meier Estimate; Killer Cells, Natural; Lymphocytes; Mice; Mice, Inbred BALB C; Orthomyxoviridae; Orthomyxoviridae Infections; Th2 Cells; Time Factors; Vaccines, Inactivated

alpha-Galactosylceramide (alpha-GalCer) is a ligand of invariant Valpha14+ NKT cells and is presented by CD1d molecule on APC. NKT cells produce a large amount of Th1 and Th2 cytokines in response to alpha-GalCer-presented APC. In this study, we assessed whether alpha-GalCer could act as an effective nasal vaccine adjuvant for mucosal vaccine that would be capable of inducing systemic as well as mucosal immune responses. When alpha-GalCer was administered with OVA via the intranasal route to C57BL/6 and BALB/c mice, significant OVA-specific mucosal secretory IgA, systemic IgG, and CTL responses were induced with mixed Th1 and Th2 cytokine profiles seen in both strains of mice. Interestingly, as BALB/c mice were intranasally immunized with PR8 hemagglutinin Ag isolated from influenza virus A/PR/8/34 together with alpha-GalCer, significant protection was afforded against influenza viral infection. When alpha-GalCer was coimmunized with a replication-deficient live adenovirus to BALB/c mice, it significantly induced both humoral and cellular immune responses. In addition, intranasal administration of OVA with alpha-GalCer showed complete protection against EG7 tumor challenge in C57BL/6. The adjuvant effects induced by intranasal coadministration with alpha-GalCer were blocked in CD1d-/- mice, indicating that the immune responses were exclusively mediated by CD1d molecule on APC. Most interestingly, intranasally coadministered alpha-GalCer activated naive T cells and triggered them to differentiate into functional effector T cells when CFSE-labeled OT-1 cells were adoptively transferred into syngeneic mice. Overall, our results are the first to show that alpha-GalCer can act as a nasal vaccine adjuvant inducing protective immune responses against viral infections and tumors.

Topics: Adjuvants, Immunologic; Administration, Intranasal; Animals; Antigens, CD1; Antigens, CD1d; Cancer Vaccines; Cytokines; Defective Viruses; Female; Galactosylceramides; Immunoglobulin A, Secretory; Immunoglobulin G; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasms, Experimental; Orthomyxoviridae Infections; Ovalbumin; T-Lymphocytes; T-Lymphocytes, Cytotoxic; Viral Vaccines