Non-neutralizing antibodies induced by seasonal influenza vaccine prevent, not exacerbate A(H1N1)pdm09 disease

The association of seasonal trivalent influenza vaccine (TIV) with increased infection by 2009 pandemic H1N1 (A(H1N1)pdm09) virus, initially observed in Canada, has elicited numerous investigations on the possibility of vaccine-associated enhanced disease, but the potential mechanisms remain largely unresolved. Here, we investigated if prior immunization with TIV enhanced disease upon A(H1N1)pdm09 infection in mice. We found that A(H1N1)pdm09 infection in TIV-immunized mice did not enhance the disease, as measured by morbidity and mortality. Instead, TIV-immunized mice cleared A(H1N1)pdm09 virus and recovered at an accelerated rate compared to control mice. Prior TIV immunization was associated with potent inflammatory mediators and virus-specific CD8 T cell activation, but efficient immune regulation, partially mediated by IL-10R-signaling, prevented enhanced disease. Furthermore, in contrast to suggested pathological roles, pre-existing non-neutralizing antibodies (NNAbs) were not associated with enhanced virus replication, but rather with promoted antigen presentation through FcR-bearing cells that led to potent activation of virus-specific CD8 T cells. These findings provide new insights into interactions between pre-existing immunity and pandemic viruses.

Pro-inflammatory mediators were significantly elevated in TIV/Cal08 mice. Since virus titers of TIV/Cal08 mice were reduced, lung inflammatory mediators were expected to be reduced. Surprisingly however, pro-inflammatory mediators (IL-1β , MIP-1β , IL-6, MCP-1) were significantly elevated in TIV/Cal08 compared to PBS/Cal08 mice at 5dpi, while low in TIV/PBS and TIV/Bris59 mice ( Fig. 2A). Lung histology of TIV/Cal08 mice also revealed significant mononuclear cell infiltration compared to controls (Fig. 2B) and scored highest in lymphocyte infiltration as well as small airway and bronchiolar inflammation (Supplemental Fig. 1A). Flow cytometric analysis revealed significant infiltration of phagocytic cells including subsets of CD11c + cells and CD11b + cells in TIV/Cal08 mice's lungs compared to controls both in absolute cell numbers and percent (Fig. 2C, Supplemental Fig. 1B,C). Cytokines associated with T cell effector function (IL-12p40, TNFα , IFNγ ) (Fig. 2D) and chemokine associated with lymphocyte recruitment (IP-10) (Supplemental Fig. 1D) were also significantly elevated in TIV/Cal08 mice compared to controls. Accordingly, significantly more CD8 T cells (Fig. 2D), B cells and their Ig class-switched B cell subsets, follicular helper T cells (T FH ) (Supplemental Fig. 2D) were found in lungs of TIV/Cal08 mice compared to controls.

Virus-specific CD8 T cells were significantly induced in TIV/Cal08 mice. Reduction of virus titers
upon infection primarily depends on pre-existing, neutralizing Abs. However, NNAbs raised against nuclear protein (NP) have also been shown to protect mice from lethal infection [29][30][31][32][33] . In particular, LaMere and colleagues have shown that protection through NP-reactive NNAbs involves CD8 T cells and FcR 34 . Furthermore, León and colleagues have demonstrated that NP-immune complexes prolong cross-presentation by DCs to CD8 T cells 35 .
Scientific RepoRts | 6:37341 | DOI: 10.1038/srep37341 Therefore, reduced virus titers in TIV/Cal08 mice compared to PBS/Cal08 mice, despite the non-neutralizing property of TIV-induced Abs (Fig. 1), indicate a possible involvement of immune complexes, FcR and memory CD8 T cells during the secondary responses. Indeed, Fcγ R (CD32/16) of TIV/Cal08 mice was significantly upregulated in all CD11b + cells, except neutrophils (Supplemental Fig. 2A). Further, immunohistochemistry of lung sections revealed visually higher NP staining in TIV/Cal08 mice compared to controls (Fig. 3A), suggesting that NNAbs were associated with higher intracellular NP levels. Accordingly, flow cytometric analysis showed that subsets of phagocytic cells (CD11c hi and CD11b + Ly6C lo cells) of TIV/Cal08 mice contained significantly more intracellular NP signals than PBS/Cal08 mice (Fig. 3B, Supplemental Fig. 2B,C). Higher viral protein signals in APCs were associated with significant recruitment of NP-or HA-pentamer-specific CD8 T cells (Fig. 3C). In addition, secretion of IFNγ , granzyme-B (granB) or IL-4 by T cells was readily detected ex vivo as well as upon in vitro stimulation in TIV/Cal08 mice (Fig. 3D,E, Supplemental Fig. 2D,E). Comparable granB, TNFα or IFNγ -secretion upon stimulation indicates significant conservation of T cell epitopes between Cal08 and Bris59 viruses. These data suggest a significant role for FcR-bearing cells in virus-uptake and subsequent T cell activation in TIV/Cal08 mice. IL-10R signaling prevented disease exacerbation in TIV/Cal08 mice. Upregulation of pro-inflammatory mediators and potent CD8 T cell activation could potentially lead to immunopathology, yet comparable morbidity between TIV/Cal08 and PBS/Cal08 mice (Fig. 1G) indicates a counteracting mechanism at play. Previous studies have shown that lung inflammation is controlled by IL-10 produced by CD4 and CD8 T cells during acute primary influenza infection 36 . Therefore, IL-10 may contribute to balanced immune responses in TIV/Cal08 mice. Accordingly, lung IL-10 was significantly elevated in TIV/Cal08 compared to control groups (Fig. 4A). Further, significantly higher IL-10, TGFβ -secretion from Treg (CD4 + CD25 + Foxp3 + ) cells ex vivo as well as IL-10 secretion from in vitro-stimulated CD4 T cells were detected in TIV/Cal08 mice compared to controls (Fig. 4B). To further investigate the role of IL-10R signaling in TIV/Cal08 mice, TIV-mice were treated with 0.5 mg IL-10R-blocking or isotype control Abs on − 1, + 1 and 3 dpi. Upon Cal08 infection, α IL-10R-TIV/Cal08 Figure 1. TIV-immunization induced non-neutralizing yet cross-reactive, weak-binding Abs that attenuated Cal08 infection. (A-E) Balb/c mice (5 mice/group) were i.m. immunized with 9 μ g TIV/100 μ l or PBS. At d30 post-immunization, the sera, BAL and nasal washes were collected for characterization of TIVinduced Abs. (A) Functional neutralizing activity of sera was measured by HI and MN titers against wild-type Bris59 or Cal08 virus. (B,C) Virus-binding ability of sera was measured by ELISA against WIV Bris59 or Cal08 virus or His-tagged rHA Bris59 or Cal07 proteins. (D) Sera Ab binding strength for Bris59 or Cal08 virus were measured by biolayer interferometry. Student t-test was used for comparison of titers between the 2 groups (P < 0.05) (E) Mucosal surface (BAL and nasal washes) Ab responses were measured by ELISA against WIV Bris59 or Cal08 virus. (F,G) TIV or mock (PBS)-immunized mice (5 mice/group) were infected with 5 × 10 6 pfu Cal08 virus (TIV/Cal08, PBS/Cal08 group) or Bris59 virus (TIV/Bris59 group) at d > 30 post-immunization. Control mice were TIV-immunized, mock-infected (TIV/PBS group). All mice were sacrificed at d5 post-Cal08 infection. (F) Lung virus titers at 5 dpi were assessed via plaque assay on MDCK cells. Unpaired t test with Welch's correction was used to compare TIV/Cal08 vs. PBS/Cal08 groups (P < 0.05), as data points in TIV/ PBS and TIV/Bris59 could not be transformed for one-way ANOVA. (G) BW was monitored for 5 days postinfection. Two-way ANOVA with Bonferroni's multiple comparison test was used for P values. *Comparison between TIV/Cal08 vs. PBS/Cal08 groups (P < 0.001), ! comparison between TIV/Cal08 vs. TIV/PBS groups (P < 0.001), † Comparison between TIV/Cal08 vs. TIV/Bris59 groups (P < 0.001). The error bars represent standard error of the mean (SEM). The data are a representative of 5 experiments. mice lost more BW than control Ab-TIV/Cal08 mice or PBS/Cal08 mice at all time-points and was significant at 5 dpi (Fig. 4C). However, this was not due to a direct suppression of virus replication, as virus titers were comparable regardless of IL-10R-blocking in TIV/Cal08 mice (Fig. 4C). Despite similar virus titers, α IL-10R-treated mice showed significantly higher NP-specific CD8 T cells and Fcγ R expression in CD11c hi cells compared to control Ab-treated mice (Fig. 4D), suggesting that regulation by IL-10R signaling may operate at the level of cross-presentation. Together, these data suggest that regulatory mechanism partially mediated by IL-10R signaling prevented severe outcomes in TIV/Cal08 mice.

TIV-immunized mice cleared Cal08 virus and recovered faster than controls following infection.
Heightened, yet tightly-regulated immune responses of TIV/Cal08 mice not only protected the mice from disease exacerbation, but also promoted viral clearance, which correlated with accelerated kinetics of virus-specific CD8 T cell responses (Fig. 5A,B, Supplemental Fig. 3A). While NP-specific CD8 T cells of PBS/Cal08 mice gradually reached peak levels at 10dpi, TIV/Cal08 mice demonstrated significantly higher NP-specific CD8 T cells at 3 dpi, peaked at 7 dpi, then contracted at 10 dpi. Accelerated CD8 T cell activation, in turn, correlated with accelerated induction of CD11c hi cells and their accumulation of intracellular NP signals as well as upregulation of MHC class I (H-2 d ) and MHC class II (I-A d ) (Fig. 5C, Supplemental Fig. 3B). Consequently, TIV/Cal08 mice also recovered faster than PBS/Cal08 mice (Fig. 5D). While TIV/Cal08 mice fully reached the BW level of TIV or TIV/Bris59 mice by 13 dpi, PBS/Cal08 mice's BW remained lower at all time-points and failed to reach the original BW even at 21 dpi. The immune-homeostasis was also restored by 21 dpi, as IL-10 + CD4 T cells (Fig. 5E) or granB + CD8 T cells (Supplemental Fig. 3C) that were acutely elevated at 5 dpi, became comparable between TIV/Cal08 and PBS/Cal08 mice at 21 dpi. Interestingly, virus-specific antibody-secreting cells (ASCs) were developed in all infected mice at NNAbs were not associated with enhanced disease. Recent studies on VAERD in the pig model showed a pathological role of HA2-binding NNAbs in promoting viral membrane fusion of A(H1N1)pdm09 virus 22 . In addition, severe human A(H1N1)pdm09 cases were correlated with high levels of NNAbs with low avidity and complement-activating activity 37,38 . In the current study, however, the mere presence of NNAbs was not sufficient to lead to severe A(H1N1)pdm09 disease and the complement depletion did not change the disease course (Discussion). To test if TIV-associated A(H1N1)pdm09 disease could be manifested by differential Ag/Ab ratios, TIV-mice were infected with sub-lethal doses (5 × 10 3 -5 × 10 6 pfu) of Cal08 virus as an attempt to increase the NNAbs/virus ratio. All TIV-immune mice lost BW proportionally to infection dose at a range of 4-16% of the original BW, yet disease was not precipitated at any dose (Supplemental Fig. 4A). Further, lung virus titers at 5dpi were either lower (at 5 × 10 5 -5 × 10 6 pfu/mouse) than or equal (at 5 × 10 3 -5 × 10 4 pfu/mouse) to those of PBS/ Cal08 mice (Fig. 6A). Despite reduced virus titers, several inflammatory mediators including MCP-1, IL-1β , IL-6 and NP-specific CD8 T cells, granB + CD8 T cells and CD11c hi cells were elevated in TIV/Cal08 compared to PBS/ Cal08 mice at the two higher infection doses ( Fig. 6B-D, Supplemental Fig. 4B,C), while Mip-1β was consistently higher in TIV/Cal08 mice regardless of infection doses (Supplemental Fig. 4D). As an alternative approach to increase NNAbs, TIV-mice were boost-immunized prior to Cal08-infection. Despite a significant increase in Bris59-specific HI titers and Cal08-binding NNAbs measured by ELISA (Supplemental Fig. 4E), A(H1N1)pdm09 morbidity based on BW changes was also not exacerbated (data not shown). Therefore, higher NNAb/virus ratios were not associated with morbidity of TIV/Cal08 mice during sub-lethal infection. NNAbs potentiated recruitment and activation of memory CD8 T cells in TIV/Cal08 mice. In contrast to the initial hypothesis, our findings so far suggest that TIV-immune mice are protected through mechanisms involving NNAbs, virus-specific CD8 T cells and FcR-bearing phagocytic cells, similar to heterosubtypic immunity 34,[39][40][41][42] . To delineate the key contributors for potent CD8 T cell response in TIV/Cal08, but not in PBS/ Cal08 mice in the current context, sera or splenic CD8 T cells from TIV-mice were adoptively transferred into naïve mice (Fig. 7). For each naïve mouse that received 1 × 10 7 isolated CD8 T cells, NP-specific and HA-specific CD8 T cells were 0.52% (5 × 10 4 ) and 0.12% (1 × 10 4 ) CD8 T cells, respectively, based on tetramer staining. All mice were then infected the next day with Cal08 virus. Efficient reduction of lung virus titers at 5 dpi required both sera and CD8 T cells, which was also correlated with highest activation of NP-specific CD8 T cells and their maximum granB-secretion (Fig. 7A,B, Supplemental Fig. 5A). These data indicate that both NNAbs and memory CD8 T cells are required for efficient protective immunity, with NNAbs potentiating CD8 T cell responses. Interestingly, CD8 T cells alone were sufficient to induce the majority of pro-inflammatory mediators as well as IL-10, while both sera and CD8 T cells were required for full IL-1β induction (Fig. 7C, Supplemental Fig. 5B). This indicates that CD8 T cell-mediated killing, rather than infection-induced cell death, is a trigger/amplifier of these proteins. The presence of sera was associated with intracellular NP signal within CD11c hi cells (Fig. 7D) and CD11b + monocytes (Supplemental Fig. 5C), indicating that accumulation of intracellular viral Ag is facilitated by NNAbs. While intracellular viral Ag could represent intracellular infection or receptor-mediated uptake, transfer of sera alone led to superior CD4 T cell recall responses (Fig. 7D) upon in vitro stimulation, supporting the latter scenario. It also confirms that NNAbs themselves do not exhibit pathological activity. Collectively, these data suggest that NNAbs cooperate with FcR-bearing cells to activate CD8 T cells.

Discussion
An important observation from the Canadian sentinel reports is that despite the association of seasonal TIV with increased risk of A(H1N1)pdm09 infection, disease severity measured by hospitalization and death was not exacerbated 12,13 . Although our study is not set to test the increased risk of A(H1N1)pdm09 infection among TIV-immune mice, our findings show that when TIV-immune mice were infected with Cal08 virus, the disease measured by morbidity and mortality was not worsened. While animal studies utilizing pigs and ferrets 22,28 have suggested that prior vaccination was associated with enhanced respiratory disease, our findings contrast with them in that TIV did not enhance disease and that viral titers were reduced by TIV-immunization even though TIV-sera exhibited no detectable neutralizing activity against Cal08 (Figs 1F, 4C, 5A, 6A and 7A). The reason for discrepancy is not entirely clear, but it is noteworthy that WIV was used as immunogen and lung virus replication was not directly measured in the pig model 22 and that TIV did not elicit detectable HI titers, but only ELISA-measurable Abs in the ferret model 28 .
The balance between immune protection vs. immunopathology of virus-specific CD8 T cells during influenza virus infection determines the disease outcome 43 . There is ample evidence showing protection 44-48 mediated by CD8 T cells as well as immunopathology, especially when regulatory mechanisms including costimulatory/inhibitory signals, are altered [49][50][51][52][53][54][55] . In our study, TIV/Cal08 mice showed overall better disease outcomes as measured by body weight, mortality, virus titers and viral clearance than PBS/Cal08 mice, instead of worsened disease outcomes. Importantly, these outcomes were associated with elevated inflammatory mediators and cytotoxic T cell activity in the lung. Thus, local lung injury and immunopathology may possibly have occurred and temporarily compromised the lung functions of TIV-immune mice. However, they did not lead to poor outcome, characteristic of immunopathology described in other animal studies following respiratory infections. Immune-regulatory mechanisms including IL-10R signaling may have prevented the weight loss. Multiple regulatory mechanisms appeared to operate in TIV/Cal08 mice, as α IL-10R-treated TIV/Cal08 mice, despite modest aggravation in morbidity during the 5 dpi, recovered at a faster rate than PBS/Cal08 mice during 14-days monitoring (data not shown). Thus, during the 2009 pandemic, immune-competent individuals such as middle-aged adults, even though mostly affected by A(H1N1)pdm09 infection, may have been able to prevent unchecked immunopathology, while individuals with immune-dysregulation may have been prone to severe outcomes. Interestingly, obesity (46%) was the most common underlying medical condition associated with A(H1N1)pdm09 fatal cases 56 , and NNAbs are generally categorized as non-neutralizing by in vitro functional assays that do not encompass potential in vivo functions such as Ab-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) 58 . These pathways, by engaging cellular components, can cause lysis of infected cells and induction of inflammation as mechanisms to limit virus replication. We have previously reported that non-hemagglutinating Abs can induce lysis of virus-infected MDCK cells by peritoneal inflammatory macrophages in an FcR-dependent manner 40 . In addition, recent studies by DiLillo and colleagues and by Tan and colleagues have demonstrated the significance of NNAbs against H1 and H7 for in vivo protection 59,60 . These NNAbs are raised against the HA head and are dependent on the Fc-Fcγ R interaction for in vivo protection, as in the case for broadly-neutralizing HA-stalk binding Abs 61 . Therefore, it is conceivable that NNAbs from TIV-immunized mice in our current study confer protection against Cal08 virus through ADCC by NK cells or macrophages. Notably, FcR levels were increased in the majority of CD11b + cells in TIV/Cal08 mice (Supplemental Fig. 2A). However, no evidence of increased NK cell frequency or activation (IFNγ secretion) was observed by flow cytometry in the current study setting (data not shown). We also investigated the potential role of NNAbs for CDC in TIV/Cal08 mice. Although TIV/Cal08 mice secreted Abs (IgM and IgG3) that could potentially activate complement, complement-depletion did not directly affect BW, virus titers, NP-specific CD8 T cells or NP + CD11c hi cells (Supplemental Fig. 6). Nonetheless, it remains possible that these in vivo functions of TIV-sera may surface at a different stage of protective immunity or exhibit long-term impact. Indeed, recent studies have shown that ADCC-inducing Abs are readily detectable in normal human sera 62 , or increased following seasonal influenza vaccine as well as avian influenza vaccine 63,64 . Therefore, further investigation utilizing specific gene-knockout mice will help clarify crucial in vivo CDC or ADCC functions of TIV-sera.
Apart from HA-reactive NNAbs, NNAbs raised against NP have also been shown to confer protection 29,30,32,33 . Because in vivo protection by NP immunization correlates with CD8 T cells in these studies, CD8 T cells are thought to be the sole effector mechanism. However, recent studies have shown that such protection following NP immunization is also dependent on B cells and FcR 34,65,66 . Therefore, unlike HA-reactive NNAbs, NP-reactive NNAbs may confer protection mainly by enhancing cross-presentation of FcR-bearing cells. This idea has been recently demonstrated by León and colleagues that NP-immune complexes facilitate a prolonged antigen presentation by DCs and promote efficient memory CD8 T cell formation during the primary and secondary infection 35 . Our current findings are in line with these works that pre-existing NNAbs facilitate cross-presentation of internalized Ags to CD8 T cells, that are otherwise induced at significantly lower levels upon Cal08 infection in naïve mice (Fig. 7B, Supplemental Fig. 5A). Virus-specific CD8 T cells were in turn, directly associated with reduced virus titers (Fig. 7A). Under these conditions, the overall Ag-presentation machinery of CD11c hi DCs was significantly enhanced as shown by upregulation of FcR, MHC class I (H-2 d ) and MHC class II (I-A d ) expression  Fig. 2A,D). NP-reactive NNAbs may be poorly induced by TIV, and are rarely boosted by seasonal influenza vaccination in human 31 . In our current study, not all NNAbs of TIV-immunized mice were directed against HA (Fig. 1B,C), yet these NNAbs may be sufficient enough to induce potent CD8 T cell responses. Collectively, these data suggest a clinical potential of NP-reactive NNAbs for cross-protection against not only seasonal influenza but also A(H1N1)pdm virus infection.
Apart from FcR dependency for in vivo protection, neutralizing vs. NNAbs may lead to qualitatively different outcomes in the cross-presentation process, as shown by the sharp difference in virus-specific CD8 T cell responses as well as CD11c + cells between TIV/Cal08 vs. TIV/Bris59 mice (Figs 2C and 3C,D). Regardless of neutralizing activities, ICs would be internalized by phagocytic cells through Fc-FcR interaction. Intracellular signals regulating the cross-presentation process remains largely unknown, but studies have suggested that internalization alone is not sufficient for cross-presentation and may require additional intracellular pathways for efficient cross-presentation 67 . In addition, Ag-binding induces a conformational change in the heavy chain constant region of Ab 68 . Therefore, NNAbs-FcR interaction may transduce differential intracellular signals from neutralizing Abs-FcR interaction, which allow efficient cross-presentation. Interestingly, recent studies have shown that DCs can provide qualitatively different activation signals to virus-specific CD8 T cells in an epitope-specific manner 69 . Considering the role of the NP-immune complex for efficient cross-presentation 35 , it is tempting to speculate a correlation between NNAbs raised at different levels during the primary response and levels of CD8 T cell activation. Among FcR-bearing cells, both CD11c hi DCs and alveolar macrophages appear to promote cross-presentation to CD8 T cells. DCs are the most efficient cells for cross-presentation in vivo, thanks to reduced proteolysis compared to macrophages 67,70 . However, other studies have identified the role of macrophages for CD8 T cell activation 42 , while our current findings (Figs 3B, 5C and 7D) and recent studies 71 support the role of CD11c hi DCs.

Figure 7. NNAbs potentiated recruitment and activation of memory CD8 T cells in TIV/Cal08 mice.
Splenocytes and sera were collected from TIV (9 μ g)-immunized Balb/c mice (10 mice/group) at d > 30 postimmunization and pooled. CD8 T cells were isolated via MACS and adoptively transferred to naïve Balb/c mice (1 × 10 7 cells/mouse) with or without 200 μ l sera (4-5 mice/recipient group). One day following adoptive transfer, recipients were infected with 5 × 10 6 pfu/mouse Cal08 virus. Control mice were PBS-transferred and then infected. All mice were sacrificed at d5 post-infection. A potential role of NNAb in TIV/Cal08 mice for ADE was also suspected, as intracellular NP signals in CD11c hi DCs (Figs 3B and 7D) and simultaneous activation of virus-specific CD8 T cells (Fig. 3C,D), could represent an enhanced intracellular infection following NNAbs-mediated internalization, as described for dengue or HIV infections 72 . However, recent studies have shown that it is the endocytosed exogenous virus particles rather than intracellular infection that leads to significant cross-presentation in myeloid DCs 73 . In addition, opsono-phagocytosis of ICs by macrophages significantly contributed to the viral clearance of influenza 74 . To address whether intracellular infection occurs following FcR-mediated Ag-uptake in DCs or macrophages, mixtures of serially diluted TIV-sera and Cal08 or Bris59 virus (2 × 10 3 TCID50/ml) were incubated with bone marrow-derived DCs or macrophages (BM-Mɸ ) overnight and intracellular virus replication was assessed. While both viruses were able to infect BM-Mɸ (Supplemental Fig. 7A,B) or DCs (data not shown), the presence of TIV-sera did not increase M-gene expression compared to the virus control, or sustained intracellular infection during longer incubation. Suppression of virus titers were associated with induction of anti-viral responses including tripartite motif-containing protein 21 (TRIM21) 75 , retinoic acid-induced gene-I (RIG-I) 76 genes and IFNβ secretion in a sera Ab dose-dependent manner (Supplemental Fig. 7C). Thus, FcR-bearing cells of TIV/ Cal08 mice may be resistant to intracellular infection upon internalization of ICs, but are poised to readily promote an anti-viral state. In agreement with this idea, recent studies have shown that influenza replication is abortive in DCs 77 . These features of FcR-bearing cells may have prevented ADE in TIV/Cal08 mice.
Our current findings provide significant insights into the interaction between pre-existing immunity and pandemic influenza virus. By definition, pandemic virus exploits the lack of neutralizing Abs in the general population, yet has to face pre-existing NNAbs and memory CD8 T cells that can play a significant role in determining disease outcome, apart from virulence of the pandemic virus itself. The current study highlighted the role of seasonal vaccine-induced NNAbs for protection against A(H1N1)pdm09 infection rather than enhancement of disease, through the mechanisms engaging FcR-bearing cells and pre-existing CD8 T cells.

Mice, cells and viruses.
Balb/c mice were purchased from Jackson Laboratory. Mouse handling, bleeding, infection and immunization were previously described 78 . Collection of tissues including spleen and lungs was performed after euthanizing mice with a lethal dose of Avertin (Sigma-Aldrich). Bronchoalveolar lavage (BAL) was collected by injecting 1 ml PBS + 0.5% bovine serum albumin (BSA) through the trachea with an 18G catheter. Nasal washes were collected by passing 0.5 ml PBS + 0.5% BSA through the nasal passage. Madin-Darby canine kidney (MDCK) cells were maintained in Dulbecco's modified Eagle's medium containing antibiotics, glutamine and 10% fetal bovine serum and used for plaque assays as previously described 78 . Influenza viruses were propagated in 11 day-old embryonic chicken eggs and clarified allantoic fluid was collected as previously described 78 . This study was approved by CDC Institutional Animal Care and Use Committee and was conducted in an Association for Assessment and Accreditation of Laboratory Animal Care International accredited animal facility. This study was carried out in strict accordance with the Animal Welfare Act regulations of the United States Department of Agriculture and Public Health Service Policy on Humane Care and Use of Laboratory Animals, administered by the National Institutes of Health. All efforts were made for animal welfare, including influenza virus infection under light anesthesia with Avertin and daily monitoring of body weight.
Adoptive transfer. TIV-immunized mice (at > d30 post immunization) were bled for collection of sera and then euthanized for collection of spleens. After single cell suspension, resting CD8 T cells were isolated by negative selection by magnetic-activated cell sorting (MACS; Miltenyi Biotec) and approximately 1 × 10 7 CD8 T cells/200 μ l PBS were injected through the tail-vein into naïve Balb/c mice. Pooled sera (200 μ l/mouse) were injected i.v. into naïve mice. Sera-transferred mice were subsequently bled to evaluate the HI titers. One day following adoptive transfer, mice were infected i.n. with 5 × 10 6 pfu/mouse Cal08 virus. Five days following infection, lungs were collected for assessment of immune responses.

Assessment of Ab response and Ab-secreting cells (ASCs). Microneutralization (MN)
, HI assay were previously described 78 . Sera and mucosal (BAL and nasal washes) Abs binding to influenza viruses were analyzed by ELISA. Briefly, Nunc 96 well plates (Maxi-sorb) were coated with 100 HAU WIV or 2 μ g/ml His-tagged rHA proteins, then blocked with 4% BSA in PBS-Tween for 1 hr. Two or ten-fold dilutions of samples are added to the plates for 2 hr at room temperature or overnight at 4 °C. Plates were then developed by biotin-anti-mouse IgG/IgA followed by streptavidin (SA)-HRP (Southern Biotech). The signals were developed using 1 x TMB (ebioscience) and measured at 450 nm using a plate reader (Biotek). Ab binding strength was measured by biolayer interferometry on an Octet Red instrument (Fortebio, Inc.) using H1N1 recombinant HAs as previously described 79 . The frequency of ASCs in spleen was measured by ELISpot assay as previously described 80 . Briefly, ELISpot plates (Millipore) were coated with 100 HAU WIV overnight and blocked with cRPMI-1640 media. Dilutions of cells were added to plates and incubated overnight at 37 °C. Plate-bound Abs were probed by biotin-anti-mouse IgG, SA-alkaline phosphatase (Southern Biotech), then Vector Blue Substrate Kit (Vector Lab). Spots were counted using an ImmunoSpot ® ELISPOT reader (Cellular Technology Ltd.).

Assessment of inflammatory mediators in lung lysates and lung histology sections.
Pro-inflammatory mediators in lung lysates were analyzed using Bio-Plex Pro TM mouse Cytokine Assay-7-plex on a Bio-Plex System (Bio-Rad) according to the manufacturer's instructions. IP-10 was measured using IP-10 ELISA kit (Abcam). For histology and immunochemistry of lung sections, mouse lungs were fixed by perfusion with 10% formalin (W/V) via the vena cava. After removal, lungs were placed in 70% ethanol before routine paraffiin embedding and processing into 0.5 mm sections. Lung histology and immunochemistry were performed on 10% formalin (W/V)-fixed sections by staining with hematoxylin and eosin (H&E) or mouse anti-NP (CDC scientific resources), biotinylated α − mouse IgG and hematoxylin (Biocare). Whole slides were scored based on lymphocyte infiltration as well as small airway and bronchiolar inflammation.
Assessment of antibody-dependent enhancement of infection using bone marrow-derived macrophages (BM-Mɸ) culture. Mouse BM cells were incubated with recombinant M-CSF (2 ng/ml) for 7 days with media replenishment on d4. For in vitro assessment of ADE, serial dilutions (1:40 through 1: 1280) of sera from TIV-immunized mice were incubated with 2 × 10 3 TCID/ml Bris59 or Cal08 virus for 2 hr, then placed onto BM-Mɸ overnight. Culture supernatants were analyzed for type I IFN by ELISA and cells were lysed for qPCR for the M gene. Alternatively, TIV-sera and virus mixtures were added to cells for 1 hr, then unbound or free viruses were washed away prior to incubation for 3 days. The viruses in the supernatants were assessed at d3 via plaque assay.

Statistics.
One-way ANOVA analysis with a Bonferroni post-test was used for comparison of multiple groups. For comparison between Cal08-vs. Bris59-responses within the group, a student t-test was used. For analysis of body weight (BW) changes, two-way ANOVA with Bonferroni's multiple comparison test was used. For statistical designations, *denotes P < 0.05; **denotes P < 0.01; ***denotes P < 0.001.