Influenza remains the single most important cause of excess disability and mortality during the winter months. In spite of widespread influenza vaccination programs leading to demonstrated cost-savings in the over 65 population, hospitalization and death rates for acute respiratory illnesses continue to rise. As a person ages, increased serum levels of inflammatory cytokines are commonly recorded (TNF-α, IL-1, IL-6). Termed “inflammaging”, this has been linked to persistent cytomegalovirus (CMV) infection and immune senescence, while increased anti-inflammatory cytokines (IL-10, TGF-β) are possibly associated with more healthy aging. Paradoxically, a shift with aging toward an anti-inflammatory (IL-10) response and decline in the IFN-γ:IL-10 ratio in influenza-challenged peripheral blood mononuclear cells is associated with a decline in the cytolytic capacity of CD8+ T cells responsible for clearing influenza virus from infected lung tissue. Thus, it is seemingly counter intuitive that the immune phenotype of healthy aging predicts a poor cell-mediated immune response and more serious outcomes of influenza. Herein we postulate a mechanistic link between the accumulation of late-stage, potentially terminally-differentiated T cells, many or most of which result from CMV infection, and the immunopathogenesis of influenza infection, mediated by granzyme B in older adults. Further, adjuvanted influenza vaccines that stimulate inflammatory cytokines and suppress the IL-10 response to influenza challenge, would be expected to enhance protection in the 65+ population.

Introduction of a new influenza virus in humans urges quick analysis of its virological and immunological characteristics to determine the impact on public health and to develop protective measures for the human population. At present, however, the necessity of executing pandemic influenza virus research under biosafety level 3 (BSL-3) high-containment conditions severely hampers timely characterization of such viruses. We tested heat, formalin, Triton X-100, and ␤-propiolactone treatments for their potencies in inactivating human influenza A(H3N2) and avian A(H7N3) viruses, as well as seasonal and pandemic A(H1N1) virus isolates, while allowing the specimens to retain their virological and immunological properties. Successful heat inactivation coincided with the loss of hemagglutinin (HA) and neuraminidase (NA) characteristics, and ␤-propiolactone inactivation reduced the hemagglutination titer and NA activity of the human influenza virus 10-fold or more. Although Triton X-100 treatment resulted in inconsistent HA activity, the NA activities in culture supernatants were enhanced consistently. Nonetheless, formalin treatment permitted the best retention of HA and NA properties. Triton X-100 treatment proved to be the easiest-to-use influenza virus inactivation protocol for application in combination with phenotypic NA inhibitor susceptibility assays, while formalin treatment preserved B-cell and T-cell epitope antigenicity, allowing the detection of both humoral and cellular immune responses. In conclusion, we demonstrated successful influenza virus characterization using formalin-and Triton X-100-inactivated virus samples. Application of these inactivation protocols limits work under BSL-3 conditions to virus culture, thus enabling more timely determination of public health impact and development of protective measures when a new influenza virus, e.g., pandemic A(H1N1)v virus, is introduced in humans.

Influenza infections are responsible for significant morbidity and mortality each year, with the highest infection rates found in the elderly population. The main strategy to reduce the impact of influenza infections in the elderly population is vaccination. However, the efficacy of influenza vaccines that are licensed for use in the elderly is relatively low (17-53%). The complex age-related changes that occur in both innate and adaptive immunity are thought to hamper the immune response to influenza immunization and to reduce protection against infection in the elderly. For the development of improved vaccines that overcome the limitations of an aged immune system, it is crucial to understand the mechanisms that lead to immune dysfunction. Here, we review the recent progress in unravelling the mechanisms behind the age-related immune dysfunction in elderly, as well as the recent developments in improving influenza vaccines and identification of new correlates of protection.

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