Development of a Mouse Model to Explore CD4 T Cell Specificity, Phenotype, and Recruitment to the Lung after Influenza B Infection

The adaptive T cell response to influenza B virus is understudied, relative to influenza A virus, for which there has been considerable attention and progress for many decades. Here, we have developed and utilized the C57BL/6 mouse model of intranasal infection with influenza B (B/Brisbane/60/2008) virus and, using an iterative peptide discovery strategy, have identified a series of robustly elicited individual CD4 T cell peptide specificities. The CD4 T cell repertoire encompassed at least eleven major epitopes distributed across hemagglutinin, nucleoprotein, neuraminidase, and non-structural protein 1 and are readily detected in the draining lymph node, spleen, and lung. Within the lung, the CD4 T cells are localized to both lung vasculature and tissue but are highly enriched in the lung tissue after infection. When studied by flow cytometry and MHC class II: peptide tetramers, CD4 T cells express prototypical markers of tissue residency including CD69, CD103, and high surface levels of CD11a. Collectively, our studies will enable more sophisticated analyses of influenza B virus infection, where the fate and function of the influenza B-specific CD4 T cells elicited by infection and vaccination can be studied as well as the impact of anti-viral reagents and candidate vaccines on the abundance, functionality, and localization of the elicited CD4 T cells

Synergy between the classical and alternative pathways of complement is essential for conferring effective protection against the pandemic influenza A(H1N1) 2009 virus infection.

The pandemic influenza A(H1N1) 2009 virus caused significant morbidity and mortality worldwide thus necessitating the need to understand the host factors that influence its control. Previously, the complement system has been shown to provide protection during the seasonal influenza virus infection, however, the role of individual complement pathways is not yet clear. Here, we have dissected the role of intact complement as well as of its individual activation pathways during the pandemic influenza virus infection using mouse strains deficient in various complement components. We show that the virus infection in C3-/- mice results in increased viral load and 100% mortality, which can be reversed by adoptive transfer of naïve wild-type (WT) splenocytes, purified splenic B cells, or passive transfer of immune sera from WT, but not C3-/- mice. Blocking of C3a and/or C5a receptor signaling in WT mice using receptor antagonists and use of C3aR-/- and C5aR-/- mice showed significant mortality after blocking/ablation of C3aR, with little or no effect after blocking/ablation of C5aR. Intriguingly, deficiency of C4 and FB in mice resulted in only partial mortality (24%-32%) suggesting a necessary cross-talk between the classical/lectin and alternative pathways for providing effective protection. In vitro virus neutralization experiments performed to probe the cross-talk between the various pathways indicated that activation of the classical and alternative pathways in concert, owing to coating of viral surface by antibodies, is needed for its efficient neutralization. Examination of the virus-specific complement-binding antibodies in virus positive subjects showed that their levels vary among individuals. Together these results indicate that cooperation between the classical and alternative pathways not only result in efficient direct neutralization of the pandemic influenza virus, but also lead to the optimum generation of C3a, which when sensed by the immune cells along with the antigen culminates in generation of effective protective immune responses

Diverse Epitope Specificity, Immunodominance Hierarchy, and Functional Avidity of Effector CD4 T Cells Established During Priming Is Maintained in Lung After Influenza A Virus Infection

One of the major contributions to protective immunity to influenza viruses that is provided by virus-specific CD4 T cells is delivery of effector function to the infected lung. However, there is little known about the selection and breadth of viral epitope-specific CD4 T cells that home to the lung after their initial priming. In this study, using a mouse model of influenza A infection and an unbiased method of epitope identification, the viral epitope-specific CD4 T cells elicited after infection were identified and quantified. We found that a very diverse specificity of CD4 T cells is primed by infection, including epitopes from hemagglutinin, neuraminidase, matrix protein, nucleoprotein, and non-structural protein-1. Using peptide-specific cytokine EliSpots, the diversity and immunodominance hierarchies established in the lung-draining lymph node were compared with specificities of CD4 T cells that home to the lung. Our studies revealed that CD4 T cells of all epitope specificities identified in peripheral lymphoid tissue home back to the lung and that most of these lung-homing cells are localized within the tissue rather than the pulmonary vasculature. There is a striking shift of CD4 T cell functionality that enriches for IFN-γ production as cells are primed in the lymph node, enter the lung vasculature, and finally establish residency in the tissue, but with no apparent shifts in their functional avidity. We conclude that CD4 T cells of broad viral epitope specificity are recruited into the lung after influenza infection, where they then have the opportunity to encounter infected or antigen-bearing antigen-presenting cells

A novel vaccine strategy to overcome poor immunogenicity of avian influenza vaccines through mobilization of memory CD4 T cells established by seasonal influenza

Avian influenza vaccines exhibit poor immunogenicity in humans. We hypothesized that one factor underlying weak B cell responses was sequence divergence between avian and seasonal influenza hemagglutinin proteins, thus limiting the availability of adequate CD4 T cell help. To test this, a novel chimeric hemagglutinin protein (cH7/3) was derived, comprised of the stem domain from seasonal H3 hemagglutinin and the head domain from avian H7. Immunological memory to seasonal influenza was established in mice, through strategies that included seasonal inactivated vaccines, Flumist, and synthetic peptides derived from the H3 stalk domain. After establishment of memory, mice were vaccinated with H7 or cH7/3 protein. The cH7/3 Ag was able to recall H3-specific CD4 T cells, and this potentiated CD4 T cell response was associated with enhanced early germinal center response and rapid elicitation of Abs to H7, including Abs specific for the H7 head domain. These results suggest that in pandemic situations, inclusion of CD4 T cell epitopes from seasonal viruses have the potential to overcome the poor immunogenicity of avian vaccines by helping B cells and conferring greater subtype-specific Ab response to viral HA.This work was supported by National Institutes of Health Grant HHSN272201400005C (to A.J.S.), Leading Advanced Projects for medical innovation from the Japan Agency for Medical Research and Development (JP18am001007), and the National Institute of Allergy and Infectious Diseases–funded Center for Research on Influenza Pathogenesis under Grant HHSN272201400008C (to Y.K.). A.T.D. was supported in part by National Institutes of Health Grant 5T32AI007285

Complement deficient mice infected with A(H1N1)pdm09 virus show prolonged infection.

<p>WT (C57BL/6) and complement deficient mice (C3^-/-, C4^-/- and FB^-/- mice on C57BL/6 background) were infected intranasally with the pandemic influenza virus (450 TCID₅₀), euthanized at day 4 or 7 post-infection, and lungs were harvested for estimation of the virus load. Virus titer was determined in the lung homogenates by TCID₅₀ analysis. C3^-/- and C4^-/- mice retained significantly higher virus load at day 7 post-infection compared to the WT mice. Bars represent mean ± SD. *p < 0.05 (Mann–Whitney Rank Sum test, IBM PASW). <i>ns</i> indicates not significant.</p

A(H1N1)pdm09 virus neutralization by human complement.

<p>A) CP-mediated neutralization of A(H1N1)pdm09 virus by NHS in the presence or absence of antibody. B) AP-mediated neutralization of A(H1N1)pdm09 virus by NHS in the presence of Mg-EGTA. C) Alternative pathway-mediated neutralization of A(H1N1)pdm09 virus in the presence of antibody and Mg-EGTA. D) AP-mediated neutralization of H3N2-A/Perth virus by NHS in the presence of Mg-EGTA. E) AP-mediated C3b deposition on H3N2-A/Perth09, and A(H1N1)pdm09 viruses in the presence or absence of antibody. F) A(H1N1)pdm09 virus neutralization by C1q depleted sera in the presence or absence of antibody. All the neutralization experiments were performed in an essentially similar manner to that performed with mouse complement. Results represent mean ± SD for three independent experiments except for panel E, which shows mean of two independent experiments. *p ≤ 0.05.</p

Complement deficient mice infected with A(H1N1)pdm09 virus show increased lung inflammation.

<p>WT (C57BL/6) and complement deficient mice (C3^-/-, C4^-/- and FB^-/- mice on C57BL/6 background) were infected intranasally with the pandemic influenza virus (450 TCID₅₀), euthanized at day 4 (<i>panel A</i>) or 7 (<i>panel B</i>) post-infection, and lungs were harvested for histopathology. Sections were stained with H & E and are representative of each group (n = 6, in each group). Magnified view (400X) of the marked area for each panels are displayed in the bottom rows. C3 and FB deficiency was associated with increased inflammatory changes compared to C4 deficiency. Typical histopathological changes include: Emphysema (asterisk), Edema (star), Peri-bronchial (plus) and peri-vascular (filled arrowhead) lymphoid cell infiltrate, parenchymal MNC infiltration (unfilled arrowhead), Hyperplasia (filled arrow), and Sloughing (unfilled arrow). B = bronchi, H = hemorrhage, V = blood vessel, VC = vascular congestion. Original magnification = 100X.</p

Adoptive transfer of WT splenocytes or purified B cells to C3^-/- mice restores protection against the pandemic influenza virus infection.

<p>A) Schematic diagram showing the experimental setup. C3^-/- mice on C57BL/6 background were injected with 1 X 10⁶ splenocytes or purified T cells or B cells (WT or C3^-/-) via tail vein at day minus 7 pre-infection. Thereafter, they were infected intranasally with 450 TCID₅₀ of the virus and monitored for body weight loss and survival for 14 days post-infection. Control mice did not receive splenocytes. B) Adoptive transfer of splenocytes. <i>Left panel</i>, the percentage of body weight loss during the infection in mice receiving WT or C3^-/- splenocytes. Body weight was normalized to their initial weight. The number of animals utilized in each group is shown at the end of the line for that group. WT splenocytes to C3^-/- mice versus C3^-/- splenocytes to C3^-/- mice: *p < 0.05 and **p < 0.02. <i>Right panel</i>, the percentage of survival during the infection in C3^-/- mice receiving WT or C3^-/- splenocytes. Results represent mean ± SEM of two independent experiments. WT splenocytes to C3^-/- mice versus C3^-/- splenocytes to C3^-/- mice: ***p < 0.001. C) Adoptive transfer of purified splenic T or B cells. <i>Left panel</i>, percentage of body weight loss during the infection in C3^-/- mice receiving either T or B cells (WT or C3^-/-). Body weight was normalized to their initial weight. The number of animals utilized in each group is shown at the end of the line for that group. WT B cells to C3^-/- mice versus C3^-/- B cells to C3^-/- mice: **p < 0.02. <i>Right panel</i>, percentage of survival during the infection in C3^-/- mice receiving either T cells or B cells (WT or C3^-/-). WT B cells to C3^-/- mice versus C3^-/- B cells to C3^-/- mice: **p < 0.02.</p

Antibodies generated during early phase of the infection are critical in containing the virus infection.

<p>C3^-/- mice on C57BL/6 background, untreated or treated with immune sera (250μl each at day -1 and 6 post-infection), were infected intranasally with 450 TCID₅₀ of the virus in PBS and monitored for body weight loss and survival for 14 days post-infection. A) Percentage of body weight loss during the infection in mice untreated or treated with the indicated sera. Body weight was normalized to their initial body weight. The number of animals utilized in each group is shown at the end of the line for that group. Statistical comparisons were performed between C3^-/- immune sera treated and WT immune sera treated mice. Significant weight gain was observed in WT immune sera treated mice. *p < 0.05 and **p < 0.02. B) Percentage of survival during the infection in mice untreated or treated with the indicated sera. About 67% of the mice treated with WT immune sera recovered. C3^-/- immune sera treated versus WT immune sera treated mice: ***p < 0.001.</p