Influenza virus.

This unit contains several methods for infecting mice with influenza virus. It also includes protocols needed to propagate influenza virus in hen eggs, quantitate virus titers (in tissue culture medium and in influenza-infected mouse serum), and adopt human isolates of influenza for growth in mice. Methods for measuring the 50% mouse lethal dose are also included. Finally, protocols for generating anti-influenza cytotoxic T lymphocytes (CTL) from splenocyte precursors and harvesting pulmonary CTL following respiratory tract challenge of mice with influenza virus are provided.

Immunogenicity and efficacy of DNA vaccines encoding influenza A proteins in aged mice.

Influenza is a leading cause of morbidity and mortality in older persons. The current influenza vaccine is only modestly successful, in part because of an age-related decline in immunogenicity and also because it induces only type-specified immunity. To overcome this, we evaluated DNA vaccines encoding A/PR8/34 haemagglutinin (HA) and nucleoprotein (NP) in young and aged BALB/c mice. Control mice were given formalin-inactivated A/PR8/34, control DNA, or a non-lethal dose of PR8. Aged mice given HA DNA developed slightly lower anti-HA serum antibodies than young mice; however, both young and aged mice were protected from a homotypic PR8 challenge. Following vaccination with NP DNA, both young and aged mice developed anti-NP bulk cytotoxic T-lymphocyte (CTL) activity and pCTL frequency similar to control animals. When challenged with a low dose of A/HK/68 (H3N2) influenza virus, both young mice and aged mice showed significant protection as measured by inhibition of weight loss. When challenged with a relatively high dose of A/HR/68 (H3N2) influenza virus, however, the anti-NP vaccine only partially protected young mice and failed to protect aged mice. These data demonstrate that DNA-based vaccines are immunogenic in aged animals, but suggest that factors other than the age-related decline in CTL activity also contribute to the increased morbidity and mortality of influenza in the elderly.

Interleukin 12 administration enhances Th1 activity but delays recovery from influenza A virus infection in mice.

Interleukin 12 (IL-12) directs the differentiation of undifferentiated T helper (Th0) cells to T helper type 1 (Th1) cells and induces a cell-mediated immune response. To evaluate the effect of IL-12 on the course of influenza A virus infection, BALB/c mice were administered a daily intraperitoneal dose of 1000 ng of IL-12 or saline on days -1 to +4 for a total of six treatments. The treatment generally enhanced Th1-mediated responses. IFNgamma lung concentrations were 1193 +/- 275 pg/100 microl in controls and 3693 +/- 745 pg/100 microl in IL-12-treated mice at day 5. IFNgamma levels were undetectable at day 13 in controls and 1335 +/- 220 pg/100 microl in IL-12-treated mice. Cytokine production was also assessed at the single-cell level for mediastinal lymph nodes. IL-12 treatment increased the number of IL-2- and IFNgamma-producing cells and decreased the number of IL-4- and IL-10-producing cells. IL-12 treatment decreased the anti-influenza antibody response, especially anti-influenza IgG1 antibody resulting in an increased IgG2a/IgG1 ratio. Primary pulmonary CTL activity on day 5 was low for both groups (10% specific lysis). Secondary CTL activity at day 11 was higher for control mice than for IL-12-treated mice on day 11 (44 versus 34%), but not on day 13. Despite this overall enhancement of Th1-mediated immune functions, the IL-12 treatment increased severity of the disease. Following infection, control and IL-12-treated mice decreased their body weight to approximately 75% of their initial weight. After day 5, the control mice started to recover, while IL-12-treated mice did not begin recovering until day 9. Pulmonary viral titers were 1.6 +/- 0.3 TCID50 in controls at day 5 compared to 2.4 +/- 0.3 for IL-12-treated mice (P < 0.01). In addition, control mice had significantly less severe inflammation and damage on histologic examination. Serum TNFalpha concentrations, undetectable in control mice, were elevated by IL-12 treatment up to 80 pg/ml at day 5 and decreased to zero at day 13. It is concluded that IL-12 administration to influenza-infected mice induces a switch from a Th2- to a Th1-mediated response, but inhibits recovery probably through induction of TNFalpha.

Influenza infection of beta 2-microglobulin-deficient (beta 2m-/-) mice reveals a loss of CD4+ T cell functions with aging.

Following influenza infection, aged mice have prolonged viral shedding that is presumably due to lower anti-influenza class I-restricted CD8+ CTL activity. To examine alternative viral clearance mechanisms in immunosenescense, we infected young (1.5-2.5 month) and aged (15-18 month) class I and CD8+-deficient beta 2m-/- mice with influenza A/Port Chalmers/1/73 (H3N2). We found that 40% of young beta 2m-/- mice were shedding virus from the lung on day 9 (mean titer of 0.3 log10 TCID[50]), with a maximal anti-influenza class II CTL activity of 68+/-2% on day 7. In contrast, 100% of aged beta 2m-/- mice were still shedding virus (mean titer of 3.0 log10 TCID[50]) from the lung on day 9 with a peak CTL activity of 15+/-6%. Aged beta 2m-/- mice also had significantly lower pulmonary IFN-gamma levels, serum anti-influenza neutralizing Ab, and anti-influenza mucosal IgA titers, as well as a less intense pulmonary inflammatory response on early days of infection. Th2-mediated cytokines were dysregulated. We conclude that there are multiple mechanisms responsible for the age-related delay in recovery from influenza. These include decreased anti-influenza class II-restricted CTL activity, pulmonary IFN-gamma levels, and serum neutralizing Ab. Taken together, these findings show a loss of CD4+ T cell functions with aging.

Serum antibody response to influenza vaccine in pulmonary patients receiving corticosteroids.

OBJECTIVE: Despite the recommendation that patients with chronic lung diseases--many of whom receive corticosteroids--receive annual influenza vaccination, it is not known whether corticosteroids influence antibody response to influenza vaccine in this population. The purpose of this study was to assess whether patients with pulmonary conditions receiving long-term corticosteroid therapy develop an adequate antibody response. DESIGN: We prospectively studied 39 consecutive candidates for influenza vaccination, 25 of whom were receiving corticosteroids for underlying lung diseases. Patients with immunosuppression besides corticosteroids were excluded. Serum samples were obtained prior to and 1 month after vaccination with inactivated trivalent influenza vaccine and assayed for antibodies to the three strains using a hemagglutination inhibition assay. No patients had any intercurrent illness compatible with influenza during the study period and patients receiving corticosteroids continued treatment with them during this time. RESULTS: A fourfold rise in antibody titer at 1 month to at least one component was seen in 21 of 25 (84%) of corticosteroid-treated patients, which was similar to patients not receiving corticosteroids (11/14, 79%). There was no corticosteroid-antibody, dose-response relationship. CONCLUSIONS: Patients with pulmonary conditions receiving corticosteroids can generate an adequate antibody response to killed influenza virus vaccine. Long-term therapy with corticosteroids should not preclude influenza vaccination in patients with chronic pulmonary diseases who are deemed vaccine candidates.

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 (Tc) of mice. Following influenza challenge, 2-mo-old female BALB/c mice demonstrated a significant fall in Tc. Female BALB/c mice 24 mo of age had lower baseline Tc than young mice and a larger fall in Tc 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 Tc of young, but not aged mice. There was a further fall in Tc with influenza challenge in young mice. These data imply that nesting is an important mechanism for maintaining Tc 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.

Pulmonary immune response of young and aged mice after influenza challenge.

After influenza challenge, aged mice have prolonged viral shedding that correlates with lower splenic cytotoxic T lymphocyte (CTL) activity. To evaluate the age-related pulmonary cell-mediated immune response to influenza, pulmonary lymphocytes were obtained from young and aged mice at various days after respiratory tract infection with nonlethal influenza A/PC/1/73 (H3N2) virus. In young mice, pulmonary CTL activity peaked at 48% +/- 2% on day 7 after infection. Pulmonary CTL activity peaked 1 day later in aged mice and at about half the activity (24% +/- 5%). The majority of the cells recovered from the lungs in both age groups were CD3+, CD8+ T cells. Histologic examination of the lungs revealed that aged mice had significantly less inflammation than young mice. Therefore, after influenza challenge there was a large influx of lymphocytes into the lungs of both young and aged mice, but the cells from young mice were more active on a per-cell basis. In a further experiment, challenge with a more virulent strain of influenza produced higher mortality in young mice than in aged mice. Thus the higher CTL activity of young animals leads to more rapid virai clearance, but this may be at a price to the host--that is, more immunopathologic damage.