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1 This information is current as of December 26, References Subscription Permissions Alerts A Novel Vaccination Strategy Mediating the Induction of Lung-Resident Memory CD8 T Cells Confers Heterosubtypic Immunity against Future Pandemic Influenza Virus Yu-Na Lee, Young-Tae Lee, Min-Chul Kim, Andrew T. Gewirtz and Sang-Moo Kang J Immunol 2016; 196: ; Prepublished online 10 February 2016; doi: /jimmunol This article cites 51 articles, 13 of which you can access for free at: Why The JI? Submit online. Rapid Reviews! 30 days* from submission to initial decision No Triage! Every submission reviewed by practicing scientists Fast Publication! 4 weeks from acceptance to publication *average Information about subscribing to The Journal of Immunology is online at: Submit copyright permission requests at: Receive free -alerts when new articles cite this article. Sign up at: Downloaded from by guest on December 26, 2018 The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD Copyright 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: Online ISSN:

2 The Journal of Immunology A Novel Vaccination Strategy Mediating the Induction of Lung-Resident Memory CD8 T Cells Confers Heterosubtypic Immunity against Future Pandemic Influenza Virus Yu-Na Lee,* Young-Tae Lee,* Min-Chul Kim,*, Andrew T. Gewirtz,* and Sang-Moo Kang* The currently used vaccine strategy to combat influenza A virus (IAV) aims to provide highly specific immunity to circulating seasonal IAV strains. However, the outbreak of 2009 influenza pandemic highlights the danger in this strategy. In this study, we tested the hypothesis that universal vaccination that offers broader but weaker protection would result in cross protective T cell responses after primary IAV infection, which would subsequently provide protective immunity against future pandemic strains. Specifically, we used tandem repeat extracellular domain of M2 (M2e) epitopes on virus-like particles (M2e5x VLP) that induced heterosubtypic immunity by eliciting Abs to a conserved M2e epitope. M2e5x VLP was found to be superior to strainspecific current split vaccine in conferring heterosubtypic cross protection and in equipping the host with cross-protective lung-resident nucleoprotein-specific memory CD8 + T cell responses to a subsequent secondary infection with a new pandemic potential strain. Immune correlates for subsequent heterosubtypic immunity by M2e5x VLP vaccination were found to be virusspecific CD8 + T cells secreting IFN-g and expressing lung-resident memory phenotypic markers CD69 + and CD103 + as well as M2e Abs. Hence, vaccination with M2e5x VLP may be developable as a new strategy to combat future pandemic outbreaks. The Journal of Immunology, 2016, 196: I nfluenza A viruses (IAVs) are divided into subtypes based on hemagglutinin (HA) and neuraminidase (NA) proteins on the surface of the virus (1). At present, 18 different HA and 11 different NA molecules are known to exist. Wild birds are the primary natural reservoir for most subtypes of IAVs (2). The interspecies transmission often causes a devastating consequence. For example, three pandemics in the 20th century, in 1918 (H1N1), 1957 (H2N2), and 1968 (H3N2), resulted in many millions of deaths worldwide (3). Furthermore, the 2009 H1N1 influenza pandemic has claimed 18,500 laboratory-confirmed deaths in.200 countries (4). The genomes of pandemic viruses originated either wholly or partly from nonhuman reservoirs, and the HA genes ultimately derived from avian influenza viruses (5). It can be a panic if novel subtypes such as H7N9 or highly pathogenic H5N1 IAV acquire effective transmissibility among humans. Development of a universal influenza vaccine has been a challenge since the first vaccination a half century ago. Heterosubtypic immunity has mainly been demonstrated in animal models with virus infections (6 9), and there is also evidence for the *Center for Inflammation, Immunity & Infection, Institute for Biomedical Sciences, Georgia State University, Atlanta, GA 30303; and Animal and Plant Quarantine Agency, Anyang, Gyeonggi-do 14089, Republic of Korea Received for publication July 22, Accepted for publication January 8, This work was supported by National Institutes of Health/National Institute of Allergy and Infectious Diseases Grants AI105170, AI119366, and AI (to S.-M.K.). The NP H-2K d tetramers were provided by the National Institutes of Health Tetramer Core Facility (Contract HHSN C). Address correspondence and reprint requests to Dr. Sang-Moo Kang, Center for Inflammation, Immunity & Infection, Institute for Biomedical Sciences, Georgia State University, Atlanta, GA address: skang24@gsu.edu Abbreviations used in this article: BMDC, bone marrow derived dendritic cell; EID 50, 50% egg infectious dose; HA, hemagglutinin; HI, hemagglutination inhibition; IAV, influenza A virus; M2e, extracellular domain of M2; NA, neuraminidase; p.i., postinfection; T RM, lung-resident memory T; VLP, virus-like particle. Copyright Ó 2016 by The American Association of Immunologists, Inc /16/$30.00 presence of cross-protective immunity in humans (10 12). It is indicated that cell-mediated immunity in particular CD8 + CTLs contributes to heterosubtypic immunity (6, 13). Current influenza vaccines are not effective in inducting virusspecific CD8 + T cells (14 16). Therefore, there is a concern that seasonal vaccination is not effective in preventing future pandemic strains. The extracellular domain of M2 (M2e) is well conserved across human influenza A subtypes (17, 18). Therefore, M2ebased vaccines have been investigated as a promising candidate for a universal influenza vaccine with broad-spectrum protection (19 21). Virus-like particles (VLP) presenting highly conserved M2e tandem repeat epitopes (M2e5x VLP) were demonstrated to be effective in conferring cross protection against H1, H3, and H5 subtype influenza viruses by reducing lung viral loads and morbidity (22). In this study, we hypothesized that M2e5x VLP immunization would induce cross protective M2e Abs as well as prevent severe disease while enabling the induction of virusspecific memory T cells after primary infection. This study demonstrates that M2e5x VLPs could be more effective in inducing M2e Abs and conferring heterosubtypic cross protection than current split vaccines. More significantly, after primary infection, mice that were immunized with M2e5x VLP but not split vaccines were found to acquire strong heterosubtypic immunity and CD8 + lung-resident memory T (T RM ) cells specific for highly conserved influenza nucleoprotein, conferring long-lived cross protection. Materials and Methods Viruses, vaccine, and M2e5x VLP IAV A/California/04/2009 (H1N1; a gift from Dr. Richard Webby), reassortant A/Vietnam/1203/2004 (rgh5n1 containing H5N1-derived NA and HA with polybasic residues removed and six internal genes from A/PR/8/ 1934), A/Philippines/2/1982 (H3N2; a gift from Dr. Huan Nguyen), and reassortant A/Mandarin Duck/Korea/PSC24-24/2010 (avian rgh5n1 con-

3 2638 VACCINATION FOR CROSS-PROTECTIVE T RM CELLS taining HA with polybasic residues removed, NA and M genes from A/PSC24-24, and the remaining backbone genes from A/PR/8/1934 virus) were propagated in 10-d-old embryonated hen s eggs as previously described (23). Purified viruses were produced by treating the virus with formalin at a final concentration of 1:4000 (v/v) as described previously (24). Commercial influenza monovalent split vaccine (Green Flu-S; Green Cross) derived from A/California/7/2009 NYMC X-179A (H1N1) virus was used in this study. M2e5x VLP that contain tandem repeat of heterologous M2e derived from human (23), swine (13), and avian (23) influenza viruses was prepared using the insect cell expression system as described previously (22). Immunization and challenge Female BALB/c mice (6 8 wk old) were i.m. immunized with 0.6 mg human split vaccine (total protein) or 10 mg M2e5x VLP or PBS at weeks 0 and 4. Immunized mice were then intranasally challenged with a sublethal dose (0.8 3 LD 50 ) of A/California/04/2009 (H1N1) virus at 4 wk after boost immunization. At 4 mo after primary infection, groups of mice were challenged with a 10 LD 50 of rgh5n1 A/Vietnam/1203/2004. After challenge with IAVs, survival rate and weight loss were monitored daily for 14 d postinfection (p.i.). All animal experiments presented in this study were approved by the Georgia State University Institutional Animal Care and Use Committee review boards. Determination of Ab responses and lung viral titers The Ab levels specific to M2e or influenza virus (2 mg/ml) were evaluated by ELISA as previously described (25). Receptor destroying enzyme treated (Denka Seiken) serum samples were used for hemagglutination inhibition (HI) assay as previously described (26, 27). Lung viral titers were determined as described in detail previously (28). Briefly, the 50% egg infectious dose (EID 50 ) in 10-d-old embryonated hen s eggs was determined with 10-fold serial dilutions of the supernatant, incubated for 48 h at 37 C, and calculated by the Reed-Muench method (29). Flow cytometric analysis For cell phenotype analysis, the cells were stained with fluorophore-labeled surface markers. Anti-mouse CD16/32 was used as an Fc receptor blocker, and then, an Ab mixture, which contained anti-mouse CD4-PE Cy7, CD8a-V500, CD44-allophycocyanin Cy7, CD62L-PerCP, CD69-FITC, CD103 Pacific Blue, and allophycocyanin-labeled tetramer (National Institutes of Health Tetramer Core Facility) specific for influenza NP H-2K d (TYQRTRALV) (30) was used to treat the cells. To evaluate intracellular cytokine production, lung cells were stimulated with NP peptide-pulsed bone marrow derived dendritic cells (BMDCs) for 5 d as previously described (31 33), surface-stained for anti CD11c-PE Cy7, anti CD4-allophycocyanin, and anti CD8a-PE Abs, and then were permeable using the Cytofix/Cytoperm kit (BD Biosciences). Intracellular cytokines were revealed by staining the cells with anti IFN-g allophycocyanin-cy7 Abs. All Abs except tetramers were purchased from ebioscience or BD Biosciences. Stained cells were analyzed using LSR Fortessa (BD Biosciences) and FlowJo software (Tree Star). Preparation and in vitro stimulation of BMDCs BMDCs were prepared from bone marrow cells of C57BL/6 treated with 10 ng/ml mouse GM-CSF for 6 d. BMDCs were stimulated with 5 mg/ml H-2K d restricted NP peptide (TYQRTRALV) at cells/ml in six-well plates for 2 h. After wash, BMDCs were cocultured with allogeneic BALB/c lung cells with the ratio of 1:10 for BMDCs to lung cells. After 5 d, the cells were washed, and the activation of the T cells was assessed by flow cytometry. In vivo protection assay of immune sera It was reported that M2e-specific Abs contributed to cross protection, although these M2e Abs lack in vitro virus-neutralizing activity (22, 34 36). To further determine whether M2e5x immune sera would contribute to cross-protection against different subtypes of IAVs, we carried out an in vivo protection assay as previously described (22, 37). In brief, heatinactivatedimmunizedornaiveseraweremixedwithalethaldose(103 LD 50 ) of A/Vietnam/1203/2004 (rgh5n1) or a lethal dose (6 3 LD 50 )of A/Philippines/2/1982 (H3N2) or A/Mandarin Duck/Korea/PSC24-24/ 2010 (avian rgh5n1 with avian M2) and incubated at room temperature for 30 min. Naive BALB/c mice were infected with a mixture of virus and sera and monitored for their survival rates and weight loss for 14dp.i. In vivo depletion of immune cells Lung-resident CD8 + T cells were depleted by intranasal injection of rat mab clone 2.43 (10 mg/mouse; BioXCell, West Lebanon, NH) 4 d before challenge. The population of CD8 + T cells in the spleen, lungs, and mediastinal lymph nodes was confirmed by flow cytometry at day 4 after inoculation. Statistical analysis Statistical analyses were done using GraphPad Prism software (GraphPad). Data are presented as means 6 SEM. Differences between groups were analyzed by one-way ANOVA or two-way ANOVA where appropriate. The p values,0.05 were regarded as significant. Results M2e5x VLP is superior to split vaccine in conferring cross protection As seen in the 2009 pandemic and outbreaks of avian influenza viruses, current vaccination is not prepared for preventing a future new strain with different antigenicity. As a vaccination strategy toward a pandemic preparedness effort, we evaluated the immunogenicity of M2e5x VLP and split vaccines. At 21 d after boost vaccination of mice with M2e5x VLP or split vaccine, mice developed M2e-specific (Fig. 1A) or virus-specific (Fig. 1B) Abs, respectively. As an indicator of virus-neutralizing activity, the mice immunized with split vaccine showed homologous HI titers up to of log 2 (Fig. 1C). However, sera from M2e5x VLP-immunized mice showed no HI activity against 2009 H1N1 virus. To compare heterosubtypic cross protective efficacy, immune mice were challenged with a reassortant A/H5N1 virus (Fig. 1D). The 2009 H1N1 split vaccine group showed severe weight loss and did not survive lethal infection with A/H5N1 virus. In contrast, M2e5x VLP immune mice were 100% protected despite moderate weight loss. These results suggest that M2e5x VLP can confer superior protection compared with split vaccine when a new pandemic strain emerges. Split vaccine is effective in conferring homologous protection The efficacy of split vaccine immunization was tested by challenging immune mice with a sublethal dose of 2009 H1N1 homologous virus (Fig. 2). Mice immunized with homologous split vaccine did not develop any clinical signs following infection and did not display a loss in body weight (Fig. 2A). Mice vaccinated with M2e5x VLP showed a slight loss ( 10%) in body weight and then rapidly recovered to normal weight. In contrast, PBS mock control mice developed severe body weight loss from days 5 to 7 p.i. and showed a significant delay in recovery. To better assess the protective efficacy, lung viral titers were determined (Fig. 2B). The development of clinical signs correlated with virus titers in the lungs at day 4 p.i. The lung viral titers of the split-vaccinated group ( Log 10 EID 50 /ml) were significantly lower than those in the PBS control group ( Log 10 EID 50 /ml; p, 0.001) and the M2e5x VLP-immunized group ( Log 10 EID 50 /ml; p, 0.01). Moreover, the lung viral titers of the M2e5x VLP-immunized group were 31.6-fold lower than those in the PBS control group, which is statistically significant (p, 0.05). These results indicate that split vaccine is effective in inducing immunity to homologous IAV vaccine strain. M2e5x VLP does not hamper the induction of virus-specific CD8 T cell responses in lungs after primary infection We hypothesized that M2e5x VLP immunization would be more effective in inducing T cell responses to conserved NP epitopes than split vaccination during homologous primary infection. At 4 d and 4 mo p.i. with 2009 H1N1 virus, the frequency of CD8 + T cells

4 The Journal of Immunology 2639 FIGURE 1. M2e5x VLP is superior to split vaccine in conferring heterosubtypic protection. BALB/c mice (n = 10 per group) were i.m. immunized with M2e5x VLP or split vaccine. Blood samples were collected at 3 wk after immunization, respectively. IgG Abs specific for M2e peptide (A) or inactivated 2009 H1N1 virus (B) were measured in prime (p) and boost (b) immune sera. (C) HI titers. HI titers were determined by standard methods using 4 HA units of inactivated A/California/04/2009 (H1N1) virus and 1% chicken erythrocyte suspension. (D) Superior cross protection by M2e5x VLP. Groups of mice (n = 4/group) were challenged with a 10 LD 50 of A/Vietnam/1203/2004 (rgh5n1) virus at 4 wk after boost immunization. Body weight changes were monitored for 14 d. Data are representative of three independent experiments that are highly reproducible. Error bars indicates mean 6 SEM. specific for the NP epitope was assessed by staining lung cells with a tetramer specific for this epitope, respectively. At 4 d p.i., the frequency of NP specific CD8 + T cells in the lungs of split, M2e5x VLP, and PBS-immunized mice were , , and , respectively (Fig. 3A). At an early time post primary infection, M2e5x VLP immune mice showed 2-fold higher levels of percentages in NP specific CD8 + T cell responses in lungs than split vaccine immune mice. The cellularity of NP specific CD8 + T cells in lungs was detected at high levels in the PBS group and M2e5x VLP-immunized group than the split vaccine immune group (Fig. 3C). At 4 mo p.i., the frequency (Fig. 3B) and cellularity (Fig. 3D) of NP specific CD8 + T cells in the lungs of split-vaccinated mice were reduced to a background level. In contrast, the mean percentages of NP specific CD8 + T cells in the lungs from M2e5x VLP-vaccinated mice were higher than the split vaccine group and maintained for.4 mo (Fig. 3A, 3B) prior to the secondary infection. The PBS control mice showed an increase in the percentages of NP specific CD8 + T cells at a later time point compared with that at day 4 p.i. Importantly, the cellularity of NP specific CD8 + T cells in the M2e5x VLP group was maintained at high levels comparable to those of the PBS control group at early and late time points (Fig. 3C, 3D). Therefore, these results suggest that M2e immunity allowing limited replication without severe disease during primary infection is effective in inducing virus-specific CD8 + T cell responses. HI assay is the standard measurement for the presence of neutralizing Abs. At 4 mo p.i. with 2009 H1N1, all vaccinated or PBS control mice showed high HI titers against 2009 H1N1 virus (Fig. 3E) but no HI activity against heterosubtypic A/H5N1 virus (Fig. 3F). The M2e5x VLP group showed high levels of M2especific Abs at 5 mo after boost vaccination, but the PBS control or split vaccine group did not (Fig. 3E), even if they were infected with 2009 H1N1 virus, indicating that split vaccine immunization even after subsequent viral infection is not still effective in inducing M2e-specific Abs. M2e5x VLP does not prevent establishing secondary heterosubtypic immunity after primary infection M2e5x VLP immune mice maintained M2e Abs and NP specific CD8 + T cell responses even at 4 mo after primary infection. We tested whether non-ha immunity would confer protection against an unexpected future virus. Groups of mice were challenged with a 10 LD 50 of A/H5N1 virus as a second follow-up FIGURE 2. Split vaccine is effective in conferring homologous protection. Groups of mice (n = 10/group) were challenged with a 0.8 LD 50 of A/California/04/2009 (H1N1) virus at 4 wk after boost vaccination. Body weight (A) was monitored for 14 d. Lung viral titers (B) were determined at 4 d p.i. by an egg infection assay (n =4/ group). Data are representative of two independent experiments. Data represent mean 6 SEM. Statistical significance was determined by one-way ANOVA.

5 2640 VACCINATION FOR CROSS-PROTECTIVE T RM CELLS FIGURE 3. M2e5x VLP is effective in mediating the induction of NP-specific CD8 + T cells after primary infection. The percentages (A and B) or cellularity (C and D) of CD8 + T cells are presented after staining of lung cells with NP H-2K d tetramer. The cell numbers are expressed per lung tissue. The lung cells are obtained at 4 d (A and C) and 4 mo (B and D) p.i. with A/California/04/2009 (H1N1) virus (n = 4/group). (E and F) HI titers were determined in serum samples collected from the mice at 4 mo p.i. with 2009 H1N1 virus (n = 10/group). HI titers were determined by standard methods using 4 HA units of inactivated 2009 H1N1 virus (E) or A/Vietnam/1203/2004 (rgh5n1) virus (F). (G) Anti-M2e IgG Abs in sera at 4 mo p.i. with 2009 H1N1 virus. Data are representative of two independent experiments. Data represent mean 6 SEM. Statistical significance was determined by one-way ANOVA. **p, 0.01, ***p, infection after 4 mo. The split-vaccinated group showed 22% body weight loss and 40% survival rate and a significant delay in recovery of body weight in surviving mice (Fig. 4A, 4B). By contrast, all mice immunized with M2e5x VLP did not show weight loss, resulting in 100% protection. The M2e5x VLP-immunized group lowered lung viral loads by 10,000-fold close to a detection limit ( Log 10 EID 50 /ml, p, 0.001) (Fig. 4C), similar to the PBS mock control group that showed severe weight loss during primary infection. The splitvaccinated group could not control lung viral replication displayinghightiters( Log 10 EID 50 /ml). Thus, these results provide evidence that M2e5x VLP immunization followed by primary infection can be superior to split vaccine in equipping the host with heterosubtypic immunity against a novel influenza strain in future. Lung-resident memory CD8 + T cells secreting IFN-g have a correlation with subsequent heterosubtypic immunity Equivalent protection between the M2e5x VLP and PBS mock control groups suggested a possible role of T cells in the heterosubtypic immunity. At 4 d after secondary infection of H1N1- exposed mice with A/H5N1, CD8 + T cells specific for the NP epitope in the lung were determined by tetramer and intracellular IFN-g staining after stimulation with peptide-pulsed dendritic cells. The mean percentages (Fig. 5A, 5B) and cellularity (Fig. 5D) of NP specific CD8 + T cells were observed at significantly higher levels in the lungs from the PBS control group than those from the split-vaccinated group. Moreover, the frequency of NP specific CD8 + T cells in lungs from M2e5x VLP-vaccinated mice were increased and higher than those from FIGURE 4. Mice with M2e5x VLP immunization are completely protected against secondary heterosubtypic influenza virus. Groups of mice (n = 10/ group) were challenged with a 10 LD 50 of A/Vietnam/1203/2004 (rgh5n1) virus at 4 mo after first infection. Survival rate (A) and body weight changes (B) were monitored for 14 d. (C) Lung viral titers were determined at 4 d p.i. by an egg infection assay (n = 4/group). Data are representative of two independent experiments. Data represent mean 6 SEM. Statistical significance was determined by one-way ANOVA. ***p,

6 The Journal of Immunology 2641 FIGURE 5. Mice with M2e5x VLP vaccination further increase NP-specific CD8 + T RM cells during secondary heterosubtypic IAV infection. Groups of mice (n = 3/group) were challenged with a 10 LD 50 of A/Vietnam/1203/2004 (rgh5n1) virus at 4 mo after first infection. Lung cells were isolated from mice at day 4 p.i. (A) NP H-2K d tetramer + -specific CD8 + T cells in lungs. Flow cytometric analysis showing lung CD8 + T cells that are stained with NP H-2K d tetramers after gating total lung CD8 + T cells. (B) Percentages of NP H-2K d tetramer + CD8 + T cells in the total CD8 + T cells. (C) Representative histograms showing CD69 expression on gated NP H-2K d tetramer + CD8 + T cells in the lung. NP H-2K d tetramer + lung CD8 + T cells were gated first to determine CD69 expression. (D) The cellularity of NP H-2K d tetramer + CD8 + T cells in the total CD8 + T cells is expressed as the number per lung. (E) Representative histograms showing CD103 expression on gated NP H-2K d tetramer + CD8 + T cells in the lung. NP H-2K d tetramer + lung CD8 + T cells were gated first to determine CD103 expression. (F) Percentages of IFN-g expression in total lung CD8 + T cells after in vitro stimulation with NP peptide-pulsed BMDCs. Lung cells were cocultured with peptide-pursed BMDCs with the ratio of 1:10 for BMDC to lung cells. After 5 d, the cells were washed, and IFN-g expressing T cells were assessed by flow cytometry. After gating CD8 + T cells, IFN-g positive events were evaluated by flow cytometry of intracellularly stained cells. Data are representative of two independent experiments. Data represent mean 6 SEM. Statistically significance was determined by one-way ANOVA. *p, 0.05, ***p, Ctrl DC, control dendritic cells; Pulsed DC, peptide-pulsed dendritic cells. split-vaccinated mice, implicating the presence of pre-existing lung memory T cells after M2e5x VLP vaccination and primary infection. The early T cell activation marker CD69 was upregulated exclusively in the NP specific CD8 + T cell subset in the lungs from M2e5x VLP immune or PBS control mice, whereas the NP specific CD8 + T cell subset observed at a low level from split-vaccine mice was predominantly CD69 negative (Fig. 5C). Moreover, the integrin CD103, which binds E-cadherin on epithelial cells, was also upregulated on NP specific CD8 + T cells in the lungs from M2e5x VLP immune or PBS control mice, but not from split-vaccinated mice (Fig. 5E). Interestingly, NP specific IFN-g secretion was detected at a significantly higher level by CD8 + T cells in the lungs from M2e5x VLP immune mice (p, 0.05; Fig. 5F) or PBS control mice (p, 0.001) than that from split vaccine immune mice after in vitro stimulation with NP peptide-pulsed dendritic cells. These results suggest that generation of lung-resident memory CD8 + T cells secreting IFN-g in the vaccination and infection regimen plays a significant role in conferring subsequent heterosubtypic immunity. Virus-specific lung-resident memory CD8 + T cells play a critical role in heterosubtypic immunity We determined whether influenza virus-specific CD8 + T RM cells in lungs primed during first infection were required for protection against subsequent heterosubtypic IAV infection. It was reported

7 2642 VACCINATION FOR CROSS-PROTECTIVE T RM CELLS that resident and circulatory T cells in the lung following influenza infection could be differentiated using an i.v. Ab labeling approach (38). First, the condition of selectively depleting lungresident memory CD8 + T cells but not systemic CD8 + T cells was optimized with CD8 + T cell depleting Abs at a low dose via intranasal administration. Mice were challenged with a sublethal dose of 2009 H1N1 and then intranasally treated with a predetermined low amount of anti-cd8 Abs. Four days following anti-cd8 Ab treatment, total CD8 + T cells in the lungs, mediastinal lymph node, and spleens were observed to be maintained at similar levels between the anti-cd8 Ab treated and untreated mock control mice (Fig. 6D). In contrast, the frequency and cellularity of NP specific CD8 + T cells were significantly reduced by 3- to 4-fold in the lungs of anti-cd8 treated mice compared with PBS-treated control mice (Fig. 6A C). Therefore, intranasal administration of a low amount of anti-cd8 mab could be used as a tool to deplete preferentially lung-resident memory CD8 + T cells without significantly affecting the circulating systemic CD8 + T cells. Next, M2e5x immune mice and PBS control mice, both of which experienced primary infection with 2009 pandemic virus, were challenged with a 10 LD 50 of A/H5N1 virus after selective depletion of CD8 + T RM cells. Mice that were depleted of CD8 + T RM cells in lungs suffered from significant body weight loss, whereas mock-treated mice did not show body weight loss (Fig. 6E). M2e5x immune mice that were treated with CD8 + T cell depleting Abs showed a moderate loss of body weight ( 7%) and then fully recovered. Meanwhile, PBS control mice with CD8 + T cell depleting Abs showed a more substantial body weight loss.10% as well as a delay in recovery of weight loss, probably due to the lack of M2e Abs. Moreover, the lung viral titers of the M2e5x immune ( Log 10 EID 50 /ml; p, 0.01) or PBS control ( Log 10 EID 50 /ml; p, 0.001) groups that were treated anti-cd8 Abs were significantly higher than those in the mock-treated groups (Fig. 6F). These results provide strong evidence that virus-specific T RM cells in the lung play an essential role in conferring heterosubtypic immunity. M2e-specific Abs also contribute to heterosubtypic immunity M2e5x VLP immune mice were found to be better protected against secondary heterosubtypic virus challenge in a condition with depletion of lung-resident memory CD8 + T cells than the PBS control group (Fig. 6). Thus, we further evaluated whether M2e5x immune sera would contribute to cross-protection against different subtypes of IAVs. Naive mice were infected with a lethal dose of different strains of IAVs mixed with immune sera. Sera from naive or 2009 H1N1-exposed split-vaccinated mice did not provide any protection to naive mice (Fig. 7). In contrast, immune sera from mice vaccinated with M2e5x VLP conferred 67% protection to naive mice that were infected with A/H5N1 (Fig. 7A). Moreover, M2e5x VLP-immune sera granted 100% protection to naive mice that were infected with A/H3N2 (Fig. 7C) or A/PSC24-24 (avian FIGURE 6. CD8 + T RM cells play a critical role in conferring heterosubtypic immunity. Groups of mice were intranasally treated with PBS or anti-cd8 Ab at 4 mo after primary infection. (A) Selective depletion of CD8 + T RM cells after intranasal treatment with CD8 depleting Abs at a low dose. A representative flow cytometry profile shows lung CD8 + T RM cells that are stained with NP H-2K d tetramers. Lung CD8 + T RM cells were analyzed by flow cytometry at 4 d after mock (left panel) or anti-cd8 Ab (right panel) treatment (n = 3/group). Results are shown for one of three independent and reproducible experiments. (B) Percentage of NP H-2K d tetramer + CD8 + T cells in the total lung cells. (C) The cellularity of NP H-2K d tetramer + CD8 + T cells in the total lung cells is expressed as the number per lung. (D) The cellularity of total CD8 + T cells in lungs, mediastinal lymph nodes (MLN), and spleens. The cell numbers are expressed as of per tissue. (E) Effects of lung-resident CD8 + T cell depletion on the efficacy of heterosubtypic immunity. Groups of mice (n = 4/group) were challenged with a 10 LD 50 of A/Vietnam/1203/2004 (rgh5n1) virus at 4 d after CD8-depleting Ab treatment intranasally, and body weight changes were monitored for 14 d. (F) Mice with CD8 Ab intranasal treatment fail to clear lung viral loads. Lung viral titers were determined at 4 d p.i. by an egg infection assay (n = 3/group). Data are representative of two independent and reproducible experiments. Data represent mean 6 SEM. Statistical significance was determined by one-way or two-way ANOVA where appropriate. Asterisks indicate significant differences (*p, 0.05, **p, 0.01, ***p, 0.001) compared with the results in the PBS control group. Pound symbols indicate significant differences ( # p, 0.05, ## p, 0.01, ### p, 0.001) compared with the results in the M2e5x group.

8 The Journal of Immunology 2643 FIGURE 7. Sera of M2e5x VLP-immune mice contribute to conferring cross protection. Mice (n = 3/group) were intranasally infected with a lethal dose of reassortant A/Vietnam/ 1203/2004 (A/H5N1) (A and B), A/Philippines/ 2/82 (A/H3N2) (C and D), and reassortant A/ Mandarin Duck/Korea/PSC24-24/2010 (avian rgh5n1) (E and F) virus mixed with 2-fold diluted immune sera or naive sera. Immune sera collected from vaccinated mice at 4 mo after challenge with 2009 H1N1 virus. Survival rates (A, C, and E) and body weight changes (B, D, and F) were monitored for 14 d. Data are representative of at least two independent experiments. Data represent mean 6 SEM. rgh5n1) with an avian type M2e (Fig. 7E). Moderate levels of morbidity (14 17% weight loss) depending on the virus strain used for infection were observed in protected mice. These results support that M2e-specific Abs generated following vaccination with M2e5x VLP but not immune sera from split vaccination contribute to cross protection during subsequent heterosubtypic lethal infection. Discussion The current strategy of seasonal influenza vaccination is based on immunity to HA, mainly aiming to induce vaccine strain-specific neutralizing Abs. When the predictionofacirculatingstrainin the next season is well matched with a chosen vaccine strain, the efficacy is sufficiently high. However, this strategy has a major drawback of being unable to protect against a new and/or unanticipated strain. Wild birds serve as a natural reservoir that presumably contains numerous possible combinations of HA and NA subtypes, consistently providing a source of introducing new strains into poultry. In addition, pigs are known to play a role as mixing vessels in generating diverse new reassortants from avian and human influenza viruses (39). These unavoidable natural reservoirs make it extremely difficult to predict a matching vaccine strain and determine which strain might be a next pandemic. The emergence of swine-origin 2009 H1N1 pandemic virus provides a good example of this major vulnerability of the current vaccination strategy. Hence, this study has focused on exploring a new paradigm of vaccination and providing mechanistic insight into surmounting these problems by inducing humoral and cellular immunity to highly conserved antigenic targets. We investigated two different outcomes of protection after first exposure to homologous or heterosubtypic virus in the context of split vaccine. In the scenario of homologous challenge infection, HA-based split vaccine was superior to M2e5x VLP, probably due to its induction of virus-neutralizing Abs, thus demonstrating that, indeed, the current strategy of influenza vaccination is highly effective if a circulating virus is matched with the vaccine strain. Also, this suggests that a strategy of inducing neutralizing Abs is more effective than nonneutralizing immunity such as M2e5x VLP vaccination. These results are consistent with previous studies reporting that M2e conjugate protein vaccines with adjuvants were shown to confer survival protection but not effective in preventing weight loss (40 42). Encouragingly, M2e5x VLP vaccination in the absence of adjuvants was effective in preventing weight loss as well as broadening cross protection against H1, H3, and H5 subtype IAVs (22, 43). Thus, as shown in this study, it is highly significant that a universal vaccination strategy can provide superior cross protection to current vaccines in the scenario of when a first infecting virus is antigenically different from a vaccine strain. Protective mechanisms by M2e immunity include anti-m2e specific IgG Abs, FcgRs,NKcells,CD4 + and CD8 + T cells, alveolar macrophages, and dendritic cells (34, 37, 44, 45).

9 2644 VACCINATION FOR CROSS-PROTECTIVE T RM CELLS A major drawback of seasonal vaccine strategy is that current vaccination regimes are not prepared for preventing a future pandemic outbreak. After primary protection against homologous virus as designed in the current vaccination strategy, split vaccination was not effective at all when a subsequent heterosubtypic virus was exposed 4 mo later, as evidenced by severe weight loss and low survival rates. Therefore, strain-specific immunity might make the host greatly vulnerable to severe infection with a new variant virus in the future. As demonstrated in this study, a possible mechanism is that immunization with split vaccine is unable to induce cross-protective CD8 + T RM cells in lungs during primary protection against a homologous strain. Therefore, split vaccine immune mice were not protected against a subsequent heterosubtypic H5N1 viral infection. To overcome these problems, our strategy was that vaccination with M2e5x VLP would protect from severe disease of heterosubtypic primary infection as well as equip the host with cross-protective immunity to a subsequent pandemic potential strain. T cells can recognize conserved epitopes in the internal proteins of IAVs, which is considered to be a major mediator providing heterosubtypic immunity by viral infection (7, 46, 47). Phenotypes of T cells that are responsible for conferring heterosubtypic immunity are not well understood yet, in particular after vaccination and infections. In this study, we observed that M2e5x VLP immune mice maintained a substantial level of NP specific CD8 + T cells for.4 mo after primary infection but not split vaccine immune mice, indicating that a certain level of viral replication is required. To obtain mechanistic insights into heterosubtypic immunity, phenotypes of NP specific CD8 T + cells were further determined during secondary infection using CD103 (a E integrin) and CD69, molecules traditionally associated with adhesion within epithelial layers and recent activation (48 50). The expression of CD69 and CD103 as phenotypic markers of T RM cells in lungs was highly upregulated on NP-specific CD8 + T cells in the lung from M2e5x VLP-immune mice but not from split-vaccinated mice. Intranasal treatment with CD8 + -depleting Abs at a low dose effectively depleted CD8 + T RM cells but not the circulating systemic CD8 + T cells. Interestingly, we found that mice depleted of CD8 + T RM cells displayed more body weight loss and higher viral loads in lungs compared with mock-treated mice. Consistent with our results, it was reported that a circulating population of memory T cells was not sufficient for heterosubtypic immunity (51, 52). Recently, Steinert et al. (53) reported that the number of T RM cells is underestimated by standard immunologic assays due to the limitation in the complete isolation of resident memory T cells from nonlymphoid tissues and the fact that some T RM cells showed CD69 - or CD103 2 phenotypes. Thus, findings in this study provide convincing evidence that pulmonary NP specific resident memory CD8 + T RM cells play a critical role in conferring heterosubtypic immunity, even though they might not fully represent total CD8 + T RM cells. Interestingly, the PBS control group that was treated with CD8-depleting Ab showed more severe weight loss and higher viral loads during secondary heterosubtypic infection compared with the M2e5x VLP-immune mice, supporting the roles of M2e Abs. M2e5x VLP-immune sera were found to grant higher protection to naive mice against a lethal dose of different H3 and H5 subtypes of IAV. Surprisingly, we found that lung viral clearance in M2e5x VLP-immune mice during secondary heterosubtypic infection was highly effective, completely preventing weight loss. Thus, the results in this study support that M2e-specific humoral immune responses by M2e5x VLP immunization are contributing to cross protection during primary infection as well as during secondary infection in addition to NP-specific CD8 + T RM cell responses. M2e5x VLP was effective in preparing the host with establishing heterosubtypic immunity during primary infection without severe disease as well as in eliciting and maintaining lung-resident memory CD8 + T cell responses against a future pandemic virus. Despite broader cross protection by a nonneutralizing conserved target based vaccine strategy, weaker protection during primary infection diminishes clinical significance. Disease severity after this M2e-based vaccination strategy may not be attenuated or could be differentially attenuated in at-risk human populations. As an alternative option, supplementing M2e5x VLP to split vaccines was found to significantly improve the cross protective efficacy by preventing morbidity (weight loss) compared with either vaccine alone (45). M2e conserved epitope supplemented HA split vaccination is expected to confer equivalent protection against seasonal influenza virus strains because vaccine strain-specific immunity was elicited by supplemented split vaccination in addition to the M2e immunity (45). However, long-term secondary cross protection after supplemented vaccination and subsequent heterosubtypic infections remains to be determined. This approach of supplemented vaccination could be a clinically viable option, significantly relieving the public health burden of annually updating seasonal vaccines. 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