Heterosubtypic immunity against human influenza A viruses, including recently emerged avian H5 and H9 viruses, induced by FLU-ISCOM vaccine in mice requires both cytotoxic T-lymphocyte and macrophage function.

Induction of heterosubtypic immunity to influenza viral antigens is of paramount importance to the prevention of epidemics and potential pandemics. The 1997 incidence of avian influenza infections in humans in Hong Kong heightened the need for pandemic preparedness and a search for vaccines and vaccine delivery systems that can confer broad protection. In this report, we demonstrate that the delivery of H1N1 subtype influenza viral antigens as immunostimulating complexes (ISCOM) induces broad cross-protection in mice against challenge with various influenza virus subtypes, including the avian H9 and the H5 strains that were recently responsible for deaths in humans. The ISCOM delivery system induced high and long-lived serum antiviral antibodies and class I-restricted cytotoxic T-lymphocytes (CTL). Studies with perforin, IFN-gamma, and mu-chain gene knock-out mice demonstrated that the heterosubtypic protection required cross-reactive, functional cytotoxic T cells and nonhemagglutination inhibiting serum antibodies. Interferon-gamma, a major player in viral clearance by nonlytic mechanisms, did not appear to play a role in heterosubtypic immunity. Nonformulated H1N1 influenza antigens failed to induce significant CTL or long-lasting antibody responses or to protect mice against challenge with heterosubtypic viruses. Furthermore, while influenza virus infection induced a dominant nucleoprotein (NP)-specific CTL response in H2 mice, the ISCOM delivery system induced a dominant hemagglutinin-specific CTL response. Moreover, non-neutralizing but cross-reactive antibodies played a role in reducing viral titers by macrophages. These results suggest that exogenous delivery of influenza antigens as ISCOM can influence their antigen processing and presentation, their ability to induce/recall CTL specificities, and their capacity to mediate broad cross-protection against influenza virus variants.

Severe impairment of primary but not memory responses to influenza viral antigens in aged mice: costimulation in vivo partially reverses impaired primary immune responses.

Profound alterations in humoral and cellular immune responses are a hallmark of aging, and understanding the immunobiology of aging is key to the success of preventive vaccination strategies. With aging, while recall or memory responses to influenza viral antigens for the most part remained unaltered, primary immune responses are severely impaired. The impaired primary responses are partly due to a lack of costimulation, as providing costimulation at the time of induction of primary immune responses against influenza virus vaccine partially reversed aged-related immune dysfunction and conferred enhanced protection. Inclusion of immunomodulators that up-regulate the expression of costimulatory molecules must be considered to improve the efficacy of vaccination in the elderly, particularly to novel immunogens.

Enhanced immune responses and resistance against infection in aged mice conferred by Flu-ISCOMs vaccine correlate with up-regulation of costimulatory molecule CD86.

Ageing is associated with a decline in immune function and our primary objective is to 'reverse' age-related decline in protective immune responses to vaccination by formulating vaccines in appropriate delivery systems. In this paper, we demonstrate that influenza vaccine formulated as ISCOMs is highly immunogenic and confers protection in aged mice, when compared to current influenza vaccine. The enhanced protection conferred by Flu-ISCOMs in aged mice correlates with the up-regulation of co-stimulatory molecule, CD86 (B7.2) and to a lesser extent, CD80 (B7.1) expression on antigen presenting cells.

Enhanced antibody and cytokine responses to influenza viral antigens in perforin-deficient mice.

Cytotoxic T lymphocytes (CTL) lyse virus-infected target cells by secreting the pore-forming effector molecule, perforin. Perforin-mediated cell death appears to be a major mechanism in viral clearance but its role in regulating immune responses in vivo is unclear. In this report, we show that following immunization with influenza viral antigens, perforin-deficient mice generated about 100-fold greater serum antibody responses than wild-type mice. Further, immune spleen cells from perforin knock-out mice secreted over 10-fold more IFN-gamma following in vitro restimulation than immune spleen cells from control mice. Finally, there were over 10-fold more IFN-gamma-secreting cells in cultures from perforin-deficient mice than those from control mice, suggesting that the enhanced cytokine release by T cells from perforin-deficient mice is due to an increase in the effector cell pool. Collectively, these results suggest that perforin-mediated effector function is required in the down-regulation of the immune response by way of limiting antigen-presenting cell function.

Heterotypic protection against influenza by immunostimulating complexes is associated with the induction of cross-reactive cytotoxic T lymphocytes.

Influenza immunostimulating complexes (flu-ISCOMs) and a monovalent subvirion vaccine prepared with an H1N1 strain of influenza virus were compared in mice for immunogenicity and protection against challenge with homologous and heterotypic influenza viruses. flu-ISCOMs but not subvirion vaccine fully protected mice against homologous virus challenge after one immunization as assessed by measurement of virus lung titers. The improved protection induced by flu-ISCOMs was associated with a 10-fold higher prechallenge serum hemagglutination inhibition titer. Furthermore, only flu-ISCOMs fully protected mice against mortality and reduced morbidity following challenge with an influenza virus of the serologically distinct H2N2 subtype. This cross-protection correlated with the induction of virus cross-reactive cytotoxic T lymphocytes that recognized a known major histocompatibility class I (H2-Kd)-restricted epitope within the hemagglutinin of influenza virus that is conserved among the H1 and H2 influenza virus subtypes. flu-ISCOMs may offer significant advantages over current commercial formulations as an improved influenza vaccine.

Influenza (H1N1)-ISCOMs enhance immune responses and protection in aged mice.

Aging is associated with a decline in immune function and the elderly are therefore more susceptible to infectious disease and less responsive to vaccination. Influenza antigens complexed as immunostimulatory complexes (ISCOMs) generate more potent protective immune responses compared with non-adjuvanted flu antigens in young adult mice. We report on the protective efficacy of flu-ISCOMs compared with the current split flu vaccine in an aged mouse model. DBA/2 mice aged 2 or 18 months were immunized with flu vaccine, ISCOMs or live virus, prior to challenge with the homologous virus. In aged mice, flu-ISCOMs induced significantly higher serum hemagglutination inhibition (HAI) titers compared to vaccine, similar to the levels obtained in young adult mice that received the split vaccine. Flu-ISCOMs but not vaccine induced cytotoxic T lymphocyte (CTL) responses in young and to a lesser degree in aged mice. In aged mice flu-ISCOMs significantly reduced illness and enhanced recovery from viral infection compared with vaccine. Our data suggests that flu-ISCOMs may offer an improved vaccine strategy for protection of elderly humans against the complications of influenza infection.