Cytomegalovirus infection enhances the immune response to influenza

Abstract

Cytomegalovirus (CMV) is a β-herpesvirus present in a latent form in most people worldwide. In immunosuppressed individuals, CMV can reactivate and cause serious clinical complications, but the effect of the latent state on healthy people remains elusive. We undertook a systems approach to understand the differences between seropositive and negative subjects and measured hundreds of immune system components from blood samples including cytokines and chemokines, immune cell phenotyping, gene expression, ex vivo cell responses to cytokine stimuli, and the antibody response to seasonal influenza vaccination. As expected, we found decreased responses to vaccination and an overall down-regulation of immune components in aged individuals regardless of CMV status. In contrast, CMV-seropositive young adults exhibited enhanced antibody responses to influenza vaccination, increased CD8(+) T cell sensitivity, and elevated levels of circulating interferon-γ compared to seronegative individuals. Experiments with young mice infected with murine CMV also showed significant protection from an influenza virus challenge compared with uninfected animals, although this effect declined with time. These data show that CMV and its murine equivalent can have a beneficial effect on the immune response of young, healthy individuals, which may explain the ubiquity of CMV infection in humans and many other species.

Figure 1. Different immunological profiles in aging versus CMV seropositivity

The contribution of age and CMV to immunological and gene expression profiles was estimated by a combination of nuclear norm and the Elastic net methods (see Methods). The magnitude of the regression coefficients used to separate the classes yCMV− and oCMV− (CMV-independent age effect) or yCMV− and yCMV+ (age-independent CMV effect) are shown in light and dark grey bars, respectively. Only two parameters, the frequency of CD8⁺ CD28⁻ and CD8⁺ TEM cells, overlapped between these classification tasks. 14/16 (87.5%) of the parameters used to separate the yCMV− from yCMV+ classes were up-regulated in CMV, in contrast, the majority of parameters used to separate the yCMV− from oCMV− classes (16/23, 69.5%) were down-regulated in aging.

Figure 2. Young but not old CMV+ individuals have a better response to influenza vaccination

The geometric mean titer (GMT) for all three strains in the vaccine was calculated for each individual in the study and a standardized score (delta (Δ) post-pre GMT) for response was computed as described in Methods (y-axis). A higher response is observed in yCMV+ compared to yCMV− in the first (a) and second (b) year, as well as in an independent validation study conducted during the 2010-2011 influenza season (c). No significant differences were observed between oCMV− and oCMV+. Green bars = CMV-, yellow bars = CMV+. The age ranges for young and older individuals were 20-30 and 60->89 years, respectively (a); 22-32 (young) and 62->89 (older) years (b) and 19-44 years old (c).

Figure 3. Reduced viral titer and enhanced IAV-specific CD8⁺ T cell responses in early and established but not long-standing MCMV latency

Groups of C57BL/6 mice were mock infected or infected with 4×10⁴ PFU MCMV Smith strain i.p. and challenged with 10⁶ EID₅₀ of IAV ×31 i.n. 5 weeks (early latency) (a and d), 12 weeks (established latency) (b and e) or 9 months (long-standing latency) later (c and f). Seven days after IAV infection, influenza viral titer was determined (upper panel) and IAV-specific T cells were enumerated from the BAL of IAV+ MCMV− (MCMV−) or IAV+ MCMV+ (MCMV+) mice by tetramer staining for NP, PA and PB1-specific responses. Data are representative of three independent experiments with 4-6 mice per group in each experiment. Significance was determined by t-test for viral titer and using the Fisher’s combined probability test for comparison of specific T cell responses.

Figure 4. The effect of MCMV on cross-protection against influenza is IFN-γ-mediated

Groups of C57BL/6 mice or IFN-γ-deficient mice (on the C57BL/6 background) were mock infected or infected with 4×10⁴ PFU MCMV Smith strain i.p. and challenged 5-6 weeks later with 10⁶ EID₅₀ of IAV x31 i.n. (a) IAV lung viral titers from control (wild-type, WT) or IFN-γ-deficient co-infected (IAV+ MCMV+) mice were determined at day 7 after IAV infection in early MCMV latency (>5 weeks). (b) IAV-specific CD8⁺ T cells were enumerated from the BAL of WT and IFN-γ-deficient MCMV and IAV co-infected mice by tetramer staining for NP, PA and PB1-specific responses, 7 days after IAV infection. Significance was determined by t-test. Data are representative of two independent experiments with 3-5 mice per group. Significance was determined by t-test for viral titer and using the Fisher’s combined probability test for comparison of specific T cell responses.