Vaccine 32 (2014) 2703–2711

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Heat shock protein gp96 adjuvant induces T cell responses and cross-protection to a split influenza vaccine Ying Ju a,1 , Hongxia Fan a,1 , Jun Liu a , Jun Hu a , Xinghui Li a , Changfei Li a , Lizhao Chen a , Qiang Gao b , George F. Gao a , Songdong Meng a,∗ a CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences (CAS), No.1 West Beichen Road, Chaoyang District, Beijing 100101, China b Sinovac Biotech Co., Ltd, Beijing, China

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Article history: Received 6 November 2013 Received in revised form 6 February 2014 Accepted 13 March 2014 Available online 1 April 2014 Keywords: gp96 Influenza vaccine Cross protection T cell response Conserved epitope

a b s t r a c t The commonly used inactivated or split influenza vaccines induce only induce minimal T cell responses and are less effective in preventing heterologous virus infection. Thus, developing cross-protective influenza vaccines against the spread of a new influenza virus is an important strategy against pandemic emergence. Here we demonstrated that immunization with heat shock protein gp96 as adjuvant led to a dramatic increased antigen-specific T cell response to a pandemic H1N1 split vaccine. Notably, gp96 elicited a cross-protective CD8+ T cell response to the internal conserved viral protein NP. Although the split pH1N1vaccine alone has low cross-protective efficiency, adding gp96 as an adjuvant effectively improved the cross-protection against challenge with a heterologous virus in mice. Our study reveals the novel property of gp96 in boosting the T cell response against conserved epitopes of influenza virus and its potential use as an adjuvant for human pre-pandemic inactivated influenza vaccines against different viral subtypes. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction The outbreak of swine-origin, 2009 influenza A (H1N1) virus (pH1N1) in Mexico and its rapid spread throughout the world posed a great threat to public health. This novel strain is antigenically and genetically distinct from seasonal H1N1 influenza strains [1]. The vaccine-induced protective neutralizing antibodies (Abs) that target the outer HA and NA proteins of influenza viruses are highly strain specific, and thus, the current vaccines against seasonal influenza strains are ineffective for pH1N1 due to the variability of influenza A virus. Current split-virion vaccines with or without aluminum adjuvant induce a highly humoral immune response but fail to induce cytotoxic T-lymphocyte (CTL) immunity, which plays a utilitarian role in inducing cross-protection against various subtypes of influenza virus as T cell epitopes are generally derived from conserved internal components of the virus [2–4]. In addition,

Abbreviations: DCs, dendritic cells; HI, hemagglutination inhibition; MN, microneutralization; APCs, antigen presentation cells. ∗ Corresponding author. Tel.: +86 10 64807350; fax: +86 10 64807351/ +86 10 64807381. E-mail address: [email protected] (S. Meng). 1 Denotes equal contribution. http://dx.doi.org/10.1016/j.vaccine.2014.03.045 0264-410X/© 2014 Elsevier Ltd. All rights reserved.

current influenza vaccine production capacity is limited for meeting large-scale vaccination against the emergence of pandemic influenza strains that have acquired interspecies transmission to human (e.g., the 2009 H1N1 swine virus or the more recent H7N9 avian virus in China) [5]. The application of adjuvant could be used either to promote types of immunity (e.g., T cell response) or to increase the response to a vaccine to reduce the antigen doses such that more people can be vaccinated in the case of a new influenza outbreak. Currently, two major types of adjuvants are used in human vaccines, i.e., aluminum salts-based adjuvants and oil-in-water emulsions. Aluminum salts are mainly used to improve humoral immune responses and the polarized Th2 cell response in a vaccine, likely via NLRP3 inflammasome activation [6] or the release of the endogenous danger signal, uric acid [7,8]. However, their capability to stimulate a cellular immune response is rather limited [8]. The oil-in-water emulsion-based adjuvants (such as AS03 (GlaxoSmithKline (GSK)) [9,10], MF59 (Novartis) [11–14], and AF03 (Sanofi Pasteur) [15,16]) are currently the most widely used component for influenza vaccines and promote a mixed Th1 and Th2 cell response, likely by activating dendritic cells (DCs) or increasing antigen uptake [17]. However, M59 adjuvant seasonal influenza vaccine does not effectively induce cross-protective immunity against pH1N1 infection [14], although a broader antibody response

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against different virus strains is observed with M59 adjuvant [18]. Given the critical role of T cell-mediated immune responses in cross-protection against a broad range of variant strains, it is urgently necessary to explore new adjuvants for influenza A vaccine that can augment T cell responses [19–21]. The heat shock protein (HSP) gp96 (glucose-regulated protein, GRP94) is one of the most abundant proteins in the endoplasmic reticulum. The distinctive characteristics of gp96 include its capability to bind to antigenic peptides derived from tumors, viruses, and intracellular bacteria, as well as cross-presentation of associated peptides to MHC class I molecules and the subsequent activation of peptide-specific cytotoxic T lymphocyte (CTL)-mediated immune responses [22–27]. In addition, gp96 interacts with toll-like receptors (TLR)-2, TLR-4, and TLR-9, induces secretion of TNF-␣, IL-1␤, and IL-12 from APCs, and activates innate immunity [28–30]. Further, autologous gp96 tumor vaccines have been tested in clinical trials for treatment of various cancers [31]. Previous studies in our lab show that, similar to naturally purified native gp96, the methylotrophic yeast Hansenula polymorpha-expressed recombinant gp96 as adjuvant elicits a strong antiviral T cell mediated immune response [32]. Here, we used a pH1N1 vaccine with recombinant gp96 protein vaccination regimen to stimulate virus-specific antibody and cellular immune responses in mice and evaluated the efficiency of gp96adjuvanted vaccine for protection against heterologous influenza infection. 2. Materials and methods 2.1. Vaccines, viruses, and peptides The H1N1 split-virus vaccine was produced by Sinovac Biotech (Beijing China). The seed virus was prepared from the reassortant vaccine virus NYMC X-179A (A/California/7/2009), which is recommended by the World Health Organization (WHO) for the development of 2009 pandemic H1N1 vaccines and supplied by the US Centers for Disease Control and Prevention. Recombinant mouse gp96 protein was prepared and purified as previously described [32]. Influenza virus mouse-adapted A/PR/8/34 was prepared by inoculation of 9-day-old embryonated chicken eggs at 37 ◦ C for 48–72 h. The infectivity of the A/PR/8/34 virus was determined by titration in MDCK cell monolayers, and the virus titer is expressed as plaque forming units per mL. The kd -restricted epitope NP147-155 (TYQRTRALV) peptide and a control peptide, HBcAg87–95 Kd -restricted epitope SYVNTNMGL, were synthesized by GL Biochem Ltd. (Shanghai, China).

CO2 inhalation followed by cervical dislocation. Spleen samples were collected at day10 for mice given single immunization on day 1, or at day 24 for mice given twice immunizations on days 1 and 14, and splenocytes were dispersed with a syringe plunger. Splenocytes were cultured in RPMI 1640 medium (GIBCO, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (FCS), 25 ␮g/mL streptomycin, and 100 IU/mL penicillin in a humidified atmosphere containing 5% CO2 at 37 ◦ C. 2.3. Hemagglutination inhibition (HI) and neutralization antibody assay The HA-specific antibody titers were measured by HI and microneutralization (MN) assays. The HI assays were performed according to established procedures using 1% Turkey erythrocytes and 4 hemagglutinating units of virus [33]. The levels of neutralizing antibody were measured by MN assays as previously described [34]. Antisera to A/California/7/2009 or A/PR/8/34 virus from immunized or infected mice were used as positive controls, and normal mice sera were used as negative control. 2.4. Vaccine-specific antibody titer detected by ELISA The split H1N1 vaccine was diluted in coating buffer (0.1 M carbonate/bicarbonate, pH 9.6) to a concentration of 10 ␮g/mL HA for plate coating. Plates were incubated with 100 ␮L of serum dilutions from vaccinated mice for 2 h and then with a 1:5000 dilution of the appropriate isotype-specific antibody (IgG, IgG1, or IgG2a) conjugated to horseradish peroxidase for 1 h at 37 ◦ C. Plates were visualized by the addition of 100 ␮L tetramethylbenzidine (TMB) substrate. 2.5. Intracellular cytokine staining and flow cytometry The indicated populations of T cells (5 × 105 cells per sample) were stained with various fluorochrome-conjugated Abs against surface markers of interest. PerCP-Cy5.5-conjugated anti-mouse CD3, PE-conjugated anti-mouse CD4, FITC-conjugated anti-mouse CD8, and APC-conjugated anti-mouse IFN-␥ were purchased from eBioscinence. Intracellular IFN-␥ staining was performed using a Cytofix/Cytoperm kit (BD Pharmingen). Cells were permeabilized with permeabilization buffer. Four-color flow cytometric analyses were performed using FACSCalibur and CellQuest software (BD Biosciences) after staining. 2.6. Cytokine ELISA

2.2. Immunization of mice Female BABL/c mice (6- to 7-week-old) were purchased from the Peking University Experimental Animal Center. All animal procedures were conducted in accordance with “the regulation of the Institute of Microbiology, Chinese Academy of Sciences of Research Ethics Committee,” The protocol was approved by the Research Ethics Committee of the Institute of Microbiology, Chinese Academy of Sciences. Mice were housed under specific pathogenfree (SPF) conditions. H1N1 split-virus vaccine was mixed with gp96 protein, and the mixture was incubated for 30 min at 4 ◦ C. The mice were then intraperitoneally immunized with PBS, splitvirus vaccine alone, or H1N1 split-virus vaccine adjuvanted with 20 ␮g gp96 at days 1 and 14, respectively. Blood samples were collected by orbital sinus puncture at day 14 for mice receiving single immunization on day 1, or at day 28 for mice receiving twice immunizations on days 1 and 14. Mice were euthanized by

Splenocytes (106 mL−1 ) were incubated with split virus vaccine containing 10 ␮g/mL HA in RPMI-1640 medium with 10% FCS. Supernatants were collected after 3 days of stimulation, and the levels of IFN-␥, TNF-␣, IL-4, and IL-6 were measured by ELISA (eBiosciences, San Jose, CA) following the manufacturer’s instructions. 2.7. IFN- enzyme-linked immunospot (ELISPOT) assay ELISPOT assays were performed according to the manufacturer’s instructions. Briefly, isolated splenocytes (5 × 105 cells/well) and split vaccine containing 20 ␮g/mL HA, 20 ␮g/mL NP147–155 peptides, or 20 ␮g/mL inactivate whole virus antigen of A/Puerto Rico/8/34(H1N1) were added to the well and incubated at 37 ◦ C for 48 h. Each test condition was performed in triplicate. The spots were counted and analyzed with an ImmunoSpot S5 Versa Analyzer (Cellular Technology Limited, US).

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Fig. 1. Immune responses induced by different doses of split pH1N1 vaccine with or without gp96 adjuvant. BALB/c mice were immunized with the indicated doses of pH1N1 vaccine once at day 1 or twice at days 1 and 14 with or without gp96 as adjuvant. Serum HI (A) and neutralizing Ab titers (B) were determined at day 14 for mice given single immunization on day 1, or at day 28 for mice given twice immunizations on days 1 and 14. Splenocytes from each group were harvested and stimulated with vaccine antigens or BSA as a negative control for background evaluation. IFN-␥ secretion was measured by ELISPOT assays (C). Data show means ± SD of five mice. * P < 0.05; ** P < 0.01 by t-test compared with no gp96 immunization. Data are representative of two independent experiments.

2.8. Challenge infections

3. Results

With intranasal ether anesthesia, mice were challenged with 104 PFU of PR8 virus in a total volume of 50 ␮L 10 days after the final immunization. The weight loss and mortality rate of infected mice were recorded every other day, ending at 14 days following infection.

3.1. Optimization of the formulation of the split pH1N1 vaccine using gp96 as adjuvant

2.9. Lung viral titers assay For viral titer analyses, mice were sacrificed at day 4 after challenge with PR8 virus, and viral titers in the lung were determined by plaque assays on MDCK cells [35]. 2.10. Statistical analysis The statistical significance of the difference between two groups were determined by the two-tailed Student’s t test and was set to P < 0.05.

To determine the optimal dose of pH1N1 split virus vaccine, mice were immunized once at day 1 or twice at days 1 and 14 with serially diluted doses (0.03, 0.15, 1.5, and 15 ␮g HA), respectively, with or without gp96 as adjuvant. HI and neutralizing Ab titers were determined in mice receiving one immunization at day 14, or in mice receiving two immunizations at day 28. A geometric mean titer (GMT) was concluded for each group. A significant adjuvant effect of gp96 was observed in the group receiving a single dose of 0.15 ␮g HA vaccine. Mice that received one gp96-adjuvanted split virus vaccine containing 0.15 ␮g HA exhibited an approximately 2-fold increase in HI titer compared to the non-adjuvanted group 14 days after vaccination (P < 0.01). There was also an increasing trend in HI titers in mice receiving two doses of gp96-adjuvanted vaccine compared to no gp96 immunization, but the difference was not significant (Fig. 1A).

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Fig. 2. Humoral immune responses induced by gp96 adjuvant. BALB/c mice were immunized twice with split pH1N1 vaccine containing 0.15 ␮g HA with or without gp96 adjuvant or PBS as a negative control. (A) HI and MN assays were performed at day 28 for mice given twice immunizations on days 1 and 14. (B) Serum anti-HA IgG, IgG1, and IgG2a titers were determined by ELISA assays. (C) The ratios of IgG2a:IgG1 titers are shown. Data show means ± SD of five mice. * P < 0.05; ** P < 0.01 by t-test compared to non-adjuvanted immunization. Data are representative of two independent experiments.

For the neutralizing Ab titers, gp96 adjuvant led to significantly higher Ab levels under both one dose and two doses of 0.15 ␮g HA vaccination (Fig. 1B). These results suggest that gp96 adjuvant can elicit a greater Ab response for the pH1N1 split vaccine. Meanwhile, the T cell response generated by the pH1N1vaccine with or without gp96 adjuvant was evaluated as demonstrated by IFN-␥ ELISPOT assays. As shown in Fig. 1C, immunization of mice with gp96 adjuvant resulted in significantly greater numbers of spot forming cells (SFCs) in groups receiving 0.15, 1.5, or 15 ␮g HA immunization doses compared to no gp96 treatment (P < 0.01). Notably, a dramatic increase (3-fold) of the specific T cell response in the group receiving two doses of gp96-adjuvanted 0.15 ␮g HA vaccine was observed relative to the group receiving one dose of vaccine (P < 0.01). We therefore chose two doses of 0.15 ␮g HA in the vaccine for further study of the gp96 adjuvant. 3.2. gp96 Enhances antigen-specific humoral and T cell immune responses in BALB/c mice Female BALB/c mice were immunized twice with the pH1N1 split vaccine containing 0.15 ␮g HA with or without gp96 adjuvant. HI and neutralizing Ab titers were measured on day 28. As can be seen in Fig. 2A and B, co-immunization with gp96 led to an increase in HI and neutralizing Ab titers. To further substantiate the type of Ab subtypes elicited by gp96, anti-HA Ab isotypes IgG2a and IgG1 were analyzed by ELISA using sera collected on day 28. Immunization with gp96 adjuvant significantly increased total IgG, IgG1, and IgG2a levels (Fig. 2C). The IgG2a subclass Ab response is characteristic of the Th1 type, while IgG1 is Th2 type. The ratio

of IgG2a:IgG1 Abs in mice immunized with gp96 adjuvant was much higher (around 166-fold) than in mice without gp96 treatment (P < 0.01), indicating that gp96 treatment shifts the immune response toward a Th1 phenotype. Next, we investigated if gp96 could induce pH1N1-specific T cell responses. Splenocytes from mice immunized with the pH1N1 split vaccine with or without gp96 adjuvant were collected and analyzed at day 10 after the second immunization. A dramatic difference in antigen-specific T cell activation was observed between mice treated with or without gp96 by ELISPOT assays (P < 0.01; Fig. 3A). Mice immunized with gp96 adjuvant exhibited a 8.8-fold increase in SFCs. Similar results were obtained for IFN-␥-secreting CD8+ T cells assays, with a 7.3-fold increase under gp96 immunization (P < 0.01; Fig. 3B). In addition, we measured the production of IFN-␥, TNF-␣, IL-6, and IL-4 secreted by splenocytes stimulated in vitro with the pH1N1 vaccine. As shown in Fig. 3C, the levels of Th1-type cytokines IFN-␥ and TNF-␣ secreted by splenocytes from mice co-immunized with gp96 were 3.5- and 3-fold greater, respectively, than mice without gp96 immunization (both P < 0.01). In contrast, gp96 immunization did not increase the levels of Th2-type cytokines IL-4 and IL-6 by splenotyctes. These data demonstrate that gp96 adjuvant shifts the immune response to pH1N1 vaccine toward a Th1 phenotype. 3.3. gp96-Adjuvanted pH1N1 vaccination protects against heterologous H1N1 virus challenge To evaluate whether the T cell immune response induced by gp96 could lead to protection from a heterologous influenza virus

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Fig. 3. Cellular immune responses induced by gp96 adjuvant. Splenocytes from mice immunized with pH1N1 split vaccine with or without gp96 adjuvant or PBS as a negative control were harvested at day 24 for mice given twice immunizations on days 1 and 14. (A) Vaccine antigen-specific T cell responses were determined by IFN-␥ ELISPOT assays. (B) Flow cytometric analysis was performed to identify IFN-␥-producing CD8+ T cells. (C) Secretion of IFN-␥, TNF-␣, IL-6, and IL-4 by splenocytes stimulated with vaccine antigens for 3 days were determined by an ELISA assays. Data show means ± SD of five mice. * P < 0.05; ** P < 0.01 by t-test, compared to pH1N1 vaccine immunization alone. Data are representative of two independent experiments.

Fig. 4. gp96 Adjuvant promotes cross-protection against a heterologous H1N1 virus challenge for the pH1N1 split vaccine. BALB/c mice were immunized twice with split pH1N1 vaccine with or without gp96 adjuvant or PBS as negative control. Immunized mice were then intranasally challenged with 104 PFU PR8 virus 10 days after the second immunization. The weight loss (A) and mortality rate (B) of infected mice were recorded every other day, ending at 14 days following infection. Lung virus titers were measured on day 4 post-challenge (C). * P < 0.05 compared to pH1N1 vaccine immunization alone. Data show means ± SD of five mice from a representative experiment that was repeated two times with similar results.

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Fig. 5. gp96 Activates cross-protective T cell responses against heterologous H1N1 virus. BALB/c mice were immunized twice with split pH1N1 vaccine containing 0.15 ␮g HA with or without gp96 adjuvant or PBS as a negative control. (A) Levels of neutralizing Abs against pH1N1 and PR8 virus were determined by MN assays at day 28 after the first immunization. (B) Spleen lymphocytes from mice were stimulated with pH1N1 vaccine, inactivated PR8 antigens, or BSA as the negative control for background evaluation. pH1N1 vaccine and inactivated PR8 antigen-specific T cells were detected by IFN-␥ ELISPOT assays. (C) Splenocytes were either incubated with the Kd -restricted NP epitope NP147–155 peptide or control peptide HBcAg87–95 for background evaluation and assayed by IFN-␥ ELISPOT. (D) Splenocytes were stimulated with NP147–155 peptide in vitro for 3 days. Intracellular IFN-␥ staining and FACS analysis were performed to quantify the IFN-␥+ CD8+ T cell populations. * P < 0.05; ** P < 0.01 compared to pH1N1 vaccine immunization alone. Data show means ± SD of five mice from a representative experiment that was repeated two times with similar results.

challenge, pH1N1-vaccinated mice with or without gp96 adjuvant were intranasally challenged with 104 PFU of PR8 influenza virus 10 days after receiving the second vaccination. By days 6-9 post-challenge, all BALB/c mice immunized with pH1N1 vaccine alone or PBS suffered typical influenza symptoms and displayed a significant body weight loss and shiver (Fig. 4A). All died at day 8 (Fig. 4B). In contrast, although mice co-immunized with gp96 exhibited moderate weight loss after PR8 challenge, these mice quickly restored weight from day 7 post-challenge. All mice coimmunized with gp96 survived following the challenge with lethal infection of PR8 virus (Fig. 4B).Viral titers in mice lungs were measured at day 4 after challenge. As shown in Fig. 4C, the lung viral titers of mice co-immunized with gp96 were approximately 1000fold lower than mice immunized with H1N1 vaccine alone or PBS (P < 0.05). These data indicate that the split pH1N1vaccine alone has low cross-protective efficiency, and gp96 as adjuvant effectively induced cross-protection against a heterologous virus. 3.4. An internally conserved epitope-specific CTL response elicited by gp96 contributes to cross-protection against the heterologous virus Viral neutralization assays were first performed to measure vaccine-induced humoral cross-immunity. Sera were collected to detect neutralization Abs against the homologous pH1N1 and

heterologous virus PR8 at day 14 after the second immunization of pH1N1 vaccine. Both mice vaccinated with and without gp96 adjuvant developed a neutralization Ab response against the homologous pH1N1 virus, but there were no neutralizing Ab responses to the heterologous PR8 virus in either group (Fig. 5A), indicating that gp96-induced cross-protection cannot be attributed to an Ab response. The T cell response was then assayed to measure crossprotective immunity. Spleen lymphocytes from immunized mice were stimulated with pH1N1 vaccine or inactivated PR8. As shown in Fig. 5B, gp96-adjuvanted vaccine effectively induced a vaccine-specific T cell response (as shown by ELISPOT assays), and also induced a significantly greater (∼3.7-fold) heterologous PR8specific T cell response. As cross-protective immunity to different influenza subtypes is mediated by CTLs that recognize conserved internal components of the virus [3,4], we further tested if gp96ajuvanted vaccine could elicit viral nucleoprotein (NP)-specific T cell immunity. Spleen lymphocytes from immunized mice were stimulated with a dominant Kd -restricted NP epitope NP147–155 peptide or control peptide HBcAg87–95 . The gp96-adjuvanted vaccine dramatically increased the number of NP epitope-specific IFN-␥+ lymphocytes by ∼7.6-fold by ELISPOT assays compared to non-adjuvanted vaccine (Fig. 5C). Similar results were obtained for analysis of the number of IFN-␥-secreting CD8+ T cells, with a 14-fold increase under gp96 immunization (P < 0.01; Fig. 5D).

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Table 1 Conservation of Kd -restricted CTL epitope NP147–155 and its flanking sequences in different influenza A virus strains. Strain A/California/04/2009(H1N1) A/Brevig Mission/1/1918(H1N1) A/WSN/1933(H1N1) A/Brisbane/59/2007(H1N1) A/Puerto Rico/8/34(H1N1) A/Hong Kong/1073/99(H9N2) A/bar-headed goose/Qinghai/3/2005(H5N1) A/Japan/305/1957(H2N2) A/Hong Kong/16/1968(H3N2) A/Viet Nam/1203/2004(H5N1) A/Taiwan/S02076/2013(H7N9) A/Taiwan/T02081/2013(H7N9)

NP(137-165) amino acid sequences MIWHSNLNDA –––––––––– –––––––––– –––––––––T –––––––––– –––––––––– –––––––––– –––––––––T –––––––––T –––––––––– –––––––––– ––––––––––

Of note, the NP147–155 epitope is highly conserved among different clades of influenza virus by multiple sequence alignment (Table 1). Together, these results indicate that gp96 adjuvant induces cross-protection against heterologous influenza strains by activation of cross-protective T cell responses.

4. Discussion Vaccination is the most effective way to control influenza outbreaks. However, the potential for a global influenza pandemic remains high with the constant emergence of swine pH1N1, new pathogenic avian influenza subtypes of H5N1, and the more recent H7N9 stains. Because the commonly used inactivated or split influenza vaccines are less effective in preventing heterologous virus infection, developing cross-protective influenza vaccines against the spread of a new influenza virus is an important strategy against pandemic emergence. In the present study, we demonstrated that gp96 adjuvant enhances the immune response to the pH1N1 vaccine, especially the T cell response. Compared to pH1N1 split vaccine alone, immunization with gp96 adjuvant induced higher HI and neutralizing Ab titers, and more importantly, led to a dramatically increased number of antigen-specific T cells and IFN-␥-producing CTLs. Immunization with gp96 elicited a crossreactive CD8+ T cell response to the internal conserved viral protein NP. The adjuvant effects of gp96 were also reflected by enhanced secretion of the Th1-type cytokines IFN-␥ and TNF-␣ by splenocytes from gp96-immunized mice. The superior T cell immune responses induced with the aid of gp96 correlated with improved cross-protection against a heterologous influenza strain. Therefore, our study reveals the novel property of gp96 in boosting T cell responses against conserved epitopes of influenza virus and its potential use as an adjuvant for human pre-pandemic inactivated influenza vaccines. Consistent with the results of previous studies [14,39,40], we observed that the inactivated and split influenza virus vaccines alone mainly induced Th2 responses and only minimal levels of Th1 responses. However, when co-administered with gp96 as adjuvant, the Th1 immune responses were significantly enhanced, as evidenced by higher IgG2a Ab titers, higher numbers of IFN-␥+ CD8+ T cells, and a dramatic increase in viral-specific T cell responses by ELISPOT assays. T cells, especially virus-specific CTLs, are believed to play key roles in reducing influenza virus loads and viral transmission, limiting progression of disease, inducing cross-protective immunity, and preventing the potential threat of the re-emergence of pandemic influenza. However, induction of CD8+ T cell responses through vaccination requires antigen loading of antigen presentation cells (APCs) (e.g., DCs), antigen cross-presentation to MHC class I molecules, and then T cell antigen receptor (TCR) cross-linking, as well as co-stimulation signaling and secretion of certain cytokines [3,41].

TYQRTRALV ––––––––– ––––––––– ––––––––– ––––––––– ––––––––– ––––––––– ––––––––– ––––––––– ––––––––– ––––––––– –––––––––

RTGMDPRMCS –––––––––– –––––––––– –––––––––– –––––––––– –––––––––– –––––––––– –––––––––– –––––––––– –––––––––– –––––––––– – – – – – – – –P–

Because conventional inactivated vaccines do not effectively induce CTLs, two current strategies are used to increase the magnitude of cellular immunity to vaccination and elicit robust cross-protection. One is the use of conserved viral antigens (e.g., NP and M2) or T cell epitopes to provide cross-protective immunity and develop universal influenza vaccines [2,18,42–46]. The other is to incorporate mucosal adjuvants (e.g., chitosan, TLR3-specific double-stranded RNA oligonucleotide, or polyI:polyC) into the vaccines for intranasal immunization [13,47,48]. It is also attractive to incorporate adjuvants into the current widely used split or inactivated vaccines to induce cross-clade protective immunity. To date, only a few studies have addressed this issue. The addition of the TLR4 agonist glucopyranosyl lipid adjuvant-stable emulsion, TLR9 agonist CpG oligodeoxynucleotides, or the oil-in-water emulsion MF59 (which may increase uptake of antigen by DCs) has been shown to enhance the breadth of the Ab response against drifted influenza variants [13,49,50]. Moreover, Hong et al. [39] show that cationic lipid/noncoding DNA complex (CLDC)-adjuvanted inactivated influenza virus vaccine induces higher levels of virus-specific CD4+ and CD8+ T cells, likely by engagement with TLR7/9 and increased antigen uptake by DCs, and provides significant crossprotection from a viral challenge with a different subtype. In this study, heat shock protein gp96, which has been proven to be safe in multiple clinical trials [31], was chosen as adjuvant due to its novel immune modulation ability in both innate and adaptive immunity. In our previous studies [36–38], titration of gp96 dose (0, 0.5, 5, 10, 20, 50, 100 and 200 ␮g) showed that immunization with 10–20 ␮g of gp96 induced the highest CTL response in mice. In this study, we therefore chose 20 ␮g of gp96/mice as an optimal dose for immunization. After immunization, gp96 complexed with antigens is taken up by macrophages and dendritic cells through the CD91 receptor. Antigens associated with gp96 can be cross-represented through MHC class I molecules and effectively activate an antigen-specific CD8+ T cell response [24,37,38,51,52]. In addition, gp96 itself can interact with the toll-like receptors (e.g., TLR-2, TLR-4, and TLR-9) of APCs and induce the secretion of Th1 type cytokines (TNF-␣, IL-1␤, and IL-12) [24,29,30]. We demonstrated that the cellular immunity elicited by the addition of gp96 as adjuvant to split pH1N1 vaccine could cross-protect BALB/c mice from the challenge of a heterologous virus stain. The effective rate of protection against infection with a lethal dose of a different subtype reached 100% after immunization with gp96-adjuvanted vaccine. gp96 adjuvant dramatically increased the NP protein-derived epitope NP147–155 -specific T cell response. NP147–155 , an immunodominant Kd -restricted epitope located on the conserved internal protein NP, remains invariant throughout the evolution of influenza viruses (Table 1). In addition, the flanking sequences of NP147–155 which may affect the epitope processing and presentation are in general also conserved among different influenza A virus strains, except for a few mutations in A/Brisbane/59/2007(H1N1), A/Japan/305/1957(H2N2), A/Hong

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Kong/16/1968(H3N2) and A/Taiwan/T02081/2013(H7N9). This suggests that the stimulation of the conserved peptide-specific CD8+ T cell response by gp96 adjuvant may contribute to the crossprotection against infection with heterologous viruses. We currently cannot totally exclude a possible contribution from Abs or another effector cellular mechanism for the gp96-mediated cross-protection against the heterologous virus, though no obvious cross-protective neutralizing Ab responses were observed in gp96-immunized mice (Fig. 5A). More studies are needed to assess the breadth of cross-reactive epitope-specific CTL responses toward the internal viral proteins, including NP, M1, and PB1, and the efficiency of gp96-adjuvant inactivated or split influenza vaccines for broad range cross-protection. Also, the different efficiency of split pH1N1 and inactive PR8 vaccine antigens for cell internalization and procession in APCs could possibly affects the results of ELISPOT assay (Fig. 5). In addition, it will be of interest to determine the capability of gp96-adjuvanted vaccines to induce memory T cells, as these specific cells can respond more rapidly to challenge and are important for cross-protection. In a recent study, we show that gp96 immunization could provide long-term protective anti-tumor immunity and T memory responses [51]. In conclusion, this study has significant implications for the application of gp96 as adjuvant for current split or inactivated influenza virus vaccines. We demonstrated that gp96 adjuvant significantly enhances the magnitude and quality of protective T cell immunity induced by pH1N1 split influenza vaccine and thereby increases the efficacy of pH1N1 vaccine against a heterologous influenza challenge. These results provide evidence that gp96 has particular potential for vaccine strategies to induce a cross-reactive CTL response toward the internal conserved viral proteins, and may provide a promising new approach for protection against newly emerging epidemic and pandemic influenza viruses by split or inactivated influenza virus vaccines.

Notes Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Financial Support. This work was supported by grants from the National Natural Science Foundation of China (Grant numbers 31230026, 91029724, 81021003, and 81102018); a grant from Major State Basic Research Development Program of China (grant number 2014CB542602); a grant from the Beijing Natural Science Foundation (Role of heat-shock protein gp96 in antigen presentation and development of new gp96-based vaccines, grant number 5112021); and the Merieux Research Grant program.

Acknowledgments We thank Yajing Li and Fang Cai for technical help.

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