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Mosaic H5 Hemagglutinin Provides Broad Humoral and Cellular Immune Responses against Influenza Viruses Attapon Kamlangdee, Brock Kingstad-Bakke, Jorge E. Osorio Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin—Madison, Madison, Wisconsin, USA

ABSTRACT

The most effective way to prevent influenza virus infection is via vaccination. However, the constant mutation of influenza viruses due to antigenic drift and shift compromises vaccine efficacy. This represents a major challenge to the development of a cross-protective vaccine that can protect against circulating viral antigenic diversity. Using the modified vaccinia Ankara (MVA) virus, we had previously generated a recombinant vaccine against highly pathogenic avian influenza virus (H5N1) based on an in silico mosaic approach. This MVA-H5M construct protected mice against multiple clades of H5N1 and H1N1 viruses. We have now further characterized the immune responses using immunodepletion of T cells and passive serum transfer, and these studies indicate that antibodies are the main contributors in homosubtypic protection (H5N1 clades). Compared to a MVA construct expressing hemagglutinin (HA) from influenza virus A/VN/1203/04 (MVA-HA), the MVA-H5M vaccine markedly increased and broadened B cell and T cell responses against H5N1 virus. The MVA-H5M also provided effective protection with no morbidity against H5N1 challenge, whereas MVA-HA-vaccinated mice showed clinical signs and experienced significant weight loss. In addition, MVA-H5M induced CD8ⴙ T cell responses that play a major role in heterosubtypic protection (H1N1). Finally, expression of the H5M gene as either a DNA vaccine or a subunit protein protected mice against H5N1 challenge, indicating the effectiveness of the mosaic sequence without viral vectors for the development of a universal influenza vaccine. IMPORTANCE

Influenza viruses infect up to one billion people around the globe each year and are responsible for 300,000 to 500,000 deaths annually. Vaccines are still the main intervention to prevent infection, but they fail to provide effective protection against heterologous strains of viruses. We developed broadly reactive H5N1 vaccine based on an in silico mosaic approach and previously demonstrated that modified vaccinia Ankara expressing an H5 mosaic hemagglutinin prevented infection with multiple clades of H5N1 and limited severe disease after H1N1 infection. Further characterization revealed that antibody responses and T cells are main contributors to protection against H5N1 and H1N1 viruses, respectively. The vaccine also broadens both T cell and B cell responses compared to native H5 vaccine from influenza virus A/Vietnam/1203/04. Finally, delivering the H5 mosaic as a DNA vaccine or as a purified protein demonstrated effective protection similar to the viral vector approach.

I

nfluenza viruses still pose a serious threat for both humans and animals. Every year, seasonal influenza infects an estimated one billion people around the globe, resulting in 3 to 5 million severe cases (1). Avian influenza viruses, especially highly pathogenic avian influenza (HPAI) H5N1 viruses, have also caused public health concerns and have the potential to cause the next influenza pandemic. The main intervention strategy against both seasonal and pandemic influenza viruses is vaccination. Conventional vaccination methods against influenza viruses include inactivated (whole virus, split, or subunit) and live-attenuated influenza vaccine (LAIV). These vaccines provide protection by inducing a hemagglutinin (HA)-specific neutralizing antibody. The efficacy of inactivated vaccines and LAIVs are strongly affected by antigenic mismatch between circulating and vaccine strains. Inactivated vaccine effectiveness is about 50 to 70% based on age and health condition but can be as low as 30% (2). LAIVs are, in general, more broadly protective than inactivated vaccines and can have up to 93% overall efficacy against matched strains (3). However, LAIVs are still significantly less effective against mismatched strains. In addition, seasonal vaccine strains need to be selected each year to match the circulating strain. Inactivated vaccines from specific strains of H5N1 virus have been generated and stockpiled, in case of a pandemic, but this vaccine is also less effective against heterologous H5N1 strains (4).

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Several vaccine approaches have attempted to address the high genetic variation of influenza viruses that can occur via antigenic drift and shift. One such strategy is based on choosing immunogens from conserved regions of the HA stalk domain (5, 6) or highly conserved proteins such as the M2 gene (7, 8). Alternative approaches have attempted to centralize the virus antigenic sequences by using a consensus algorithm (9). Recently, a novel “mosaic” approach has been introduced in the field of human immunodeficiency virus (HIV) vaccinology and has been shown to provide broader protection against mismatched HIV strains than natural or consensus-derived antigens (10). The algorithm selects and recombines potential 9mer to 12mer “chunks” into a full-length protein in an attempt to preserve naturally occurring T

Received 15 April 2016 Accepted 9 May 2016 Accepted manuscript posted online 18 May 2016 Citation Kamlangdee A, Kingstad-Bakke B, Osorio JE. 2016. Mosaic H5 hemagglutinin provides broad humoral and cellular immune responses against influenza viruses. J Virol 90:6771–6783. doi:10.1128/JVI.00730-16. Editor: D. S. Lyles, Wake Forest School of Medicine Address correspondence to Jorge E. Osorio, [email protected]. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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cell epitopes (11). This approach increased the breadth and depth of cellular immune responses against multiple strains of HIV-1 (10). It has been speculated that this mosaic approach conserves structurally conserved sequences better than consensus sequences, which may result in a more naturally folded, functional protein (11). We previously reported the use of the mosaic approach to generate a modified vaccinia Ankara-vectored vaccine expressing a mosaic H5 HA (MVA-H5M) (12). This vaccine protected mice against H5N1 virus clades 0, 1, 2.2, and 2.2.1 and also provided heterosubtypic protection against H1N1 virus (A/Puerto Rico/8/ 1934). However, homosubtypic and heterosubtypic protection induced by MVA-H5M has not been well characterized. Here, we characterized the immunological responses elicited by MVAH5M and also compared MVA-H5M to MVA expressing H5 HA from influenza virus A/VN/1203/04. In addition, the efficacy of the H5M gene expressed as either a DNA vaccine or subunit protein against H5N1 challenge was evaluated to further assess the effectiveness of the mosaic approach for the development of a broadly cross-protective influenza vaccine. MATERIALS AND METHODS Cells and viruses. Chicken embryo fibroblasts (CEFs), human embryonic kidney 293 (HEK293) cells, and Madin-Darby canine kidney (MDCK) cells were obtained from Charles River Laboratories, Inc. (Wilmington, WA), and the American Type Culture Collection (Manassas, VA), respectively. Cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics. CEFs were used for propagating MVA. The HPAI H5N1 virus A/Vietnam/ 1203/04 (A/VN/1203/04, clade 1) was kindly provided by Yoshihiro Kawaoka (University of Wisconsin—Madison, Madison, WI). HPAI H5N1 viruses A/Hong Kong/483/97 (A/HK/483/97, clade 0), A/Whooper swan/Mongolia/244/05 (A/Ws/MG/244/05, clade 2.2), and A/Chicken/ Egypt/01/08 (A/Ck/Egypt/1/08, clade 2.2.1) and seasonal influenza viruses, including A/Puerto Rico/8/34 (PR8;H1N1) and A/Aichi/2/1968 (H3N2), were kindly provided by Stacey Schultz-Cherry and Ghazi Kayali (St. Jude Children’s Research Hospital, Memphis, TN). The amino acid similarities between these viruses and the mosaic H5 HA include A/Vietnam/1203/04 (97.7%), A/Hong Kong/483/97 (98.4%), A/Whooper swan/ Mongolia/244/05 (98.9%), and A/Chicken/Egypt/01/08 (96.8%). In addition, mosaic H5 has 76.4 and 56.1% amino acid similarities to the seasonal influenza viruses A/Puerto Rico/8/34 and A/Aichi/2/1968, respectively. All viruses were propagated and titrated in MDCK cells with DMEM containing 1% bovine serum albumin and 20 mM HEPES, except for seasonal influenza viruses that were added with TPCK (tolylsulfonyl phenylalanyl chloromethyl ketone)-treated trypsin. Viruses were stored at ⫺80°C until use. Viral titers were determined and expressed as 50% tissue culture infective dose(s) (TCID50). All experimental studies with HPAI H5N1 viruses were conducted in a biosafety level 3⫹ (BSL3⫹) facilities in compliance with the University of Wisconsin—Madison Office of Biological Safety. Animal studies. Female 5-week-old BALB/c mice (The Jackson Laboratory, Bar Harbor, ME) were used in all animal studies. All mouse studies were conducted at University of Wisconsin—Madison animal facilities and were approved by the Interinstitutional Animal Care and Use Committee (IACUC). Challenge experiments involving H5N1 viruses were conducted at animal BSL3⫹ (ABSL3⫹) facilities. Challenge studies with seasonal influenza viruses were conducted under BSL2 conditions to facilitate animal monitoring. Deep isoflurane euthanasia (with a ⬎5% concentration), followed by cervical dislocation, was performed according to IACUC guidelines. Production of recombinant mosaic H5 protein (H5M) and DNAexpressing mosaic H5 gene (pCMV-H5M). The mosaic H5 gene from the previous study (12) was synthesized and cloned into pTriEx-4 Neo vector

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(Novagen, Germany), followed by transformation into Escherichia coli for clonal selection. Bacterial strains containing pTriEx expressing the mosaic H5 gene (pTriEx-H5M) plasmid were then selected and cultured, and plasmids were extracted using a Maxiprep kit (Zymo Research, Irvine, CA). Purified pTriEx-H5M plasmids were electrotransformed into HEK293 cells using Bio-Rad gene pulser at 300 V and 960 ␮F. Transfected cells were then plated into T-175 plates and collected at 48 h after incubation with CelLytic M buffer (Sigma-Aldrich, St. Louis, MO). The cell pellet solution was then incubated for 1 h and centrifuged at 15,000 ⫻ g for 15 min to separate the supernatant from the pellet and genetic materials. Recombinant H5M protein was then purified using a Ni-NTA column (Zymo Research). The purified H5M protein was analyzed by Coomassie blue gel staining and Western blot analysis. A DNA vaccine plasmid expressing mosaic H5 HA (pCMV-H5M) was constructed. Briefly, the mosaic H5 DNA sequence was cloned into pCMV vector under a cytomegalovirus (CMV) promoter. A positive plasmid was selected and amplified in E. coli and confirmed by Sanger DNA sequencing. Then, plasmid DNA containing mosaic H5 was precipitated onto 1.6-␮m gold particles at a rate of 4 ␮g of DNA per mg of gold to make gene gun bullets (13). Each bullet contained 1 ␮g of plasmid DNA. Vaccination and viral challenge. Groups of 5-week-old BALB/c female mice were used as an animal model for vaccine studies. For the MVA-based vaccine, mice were vaccinated with 1.25 ⫻ 107 PFU of recombinant MVA expressing mosaic H5 (MVA-H5M) (12), MVA expressing native HA from influenza virus A/Vietnam/1203/04 (MVA-HA) (14), or MVA expressing green fluorescent protein (MVA-GFP) (12) via the intradermal (i.d.) route. For two-dose vaccination studies, mice were given 1.25 ⫻ 107 PFU of MVA-H5M, MVA-HA, or MVA-GFP at 4 weeks postpriming. For the purified H5M vaccine, 2 ␮g of protein was mixed with 0.2% alum adjuvant before given into mice intraperitoneally (i.p.). Boosts were given 4 weeks postpriming. For DNA vaccine (pCMV-H5M), 4 ␮g of DNA plasmids was coated onto gold particles and delivered i.d. on the abdominal area using a Helio gene-gun system (Bio-Rad, Berkeley, CA) with 300 lb/in2 helium pressure. A boosting dose was given at 4 weeks postpriming for the two-dose study. At 6 weeks postvaccination, mice were challenged by intranasal (i.n.) instillation, while they were under isoflurane anesthesia, with 100 50% lethal dose(s) (LD50) of influenza virus A/Hong Kong/483/97 (4 ⫻ 103 TCID50) or A/Ck/Egypt/1/08 (3.56 ⫻ 104 TCID50) contained in 20 ␮l of phosphate-buffered saline (PBS). Three mice from each group were euthanized at days 2 and 5 postchallenge, and lung tissues were collected for viral titrations and histopathology. For virus isolation, lung tissues were minced in PBS using a mechanical homogenizer (MP Biochemicals, Solon, OH), and the viral titers in the homogenates were quantified by plaque assay on MDCK cells. The remaining lung tissue was fixed in 10% buffered formalin. The remaining animals in each group were observed daily for 14 days, and survival and clinical parameters, including clinical scores and body weights, were recorded. Mice showing at least a 20% body weight loss were humanely euthanized. In vivo depletion of T cells. In order to study the role of T cells elicited by the MVA-H5M vaccine, CD4⫹ and CD8⫹ T cells were immunodepleted. Groups of 5-week-old BALB/c mice (n ⫽ 8) were primed i.d. with 1.25 ⫻ 107 PFU of either MVA-H5M or MVA-GFP constructs. On days 38 and 41 postpriming, immunized mice were treated i.p. with a combination of 50 ␮g of anti-CD4 monoclonal antibody (MAb; clone GK1.5, Rat IgG2b) and 125 ␮g of anti-CD8a MAb (clone 2.43, rat IgG2b) (Bio X Cell, West Lebanon, NH). Control groups included untreated immune and nonimmune animals. Mice were then challenged i.n. with 4 ⫻ 103 TCID50 units of influenza virus A/Hong Kong/483/97 (H5N1) at days 42 postpriming. Mice were treated again with depleting antibodies at 3, 7, and 10 days postchallenge. The depletion efficiency in peripheral blood mononuclear cells on the day of infection was ⬎99% for both T cell subsets, as analyzed by flow cytometry by staining with anti-mouse CD4 FITC (RM4-5) and anti-mouse CD8a PerCP (53-6.7) MAbs (BD Biosci-

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ence, Franklin Lakes, NJ) (data not shown). The mice were then monitored for morbidity and mortality for 2 weeks. The role of CD8⫹ T cells against H1N1 (PR8) virus was also evaluated. Groups of 5-week-old BALB/c mice (n ⫽ 8) were primed i.d. with 1.25 ⫻ 107 PFU of either MVA-H5M or MVA-GFP, and then vaccinated mice were treated i.p. with 125 ␮g of anti-CD8a MAb (clone 2.43, rat IgG2b; Bio X Cell) at days 38 and 41 postpriming. Control groups included untreated immune and nonimmune animals. Mice were challenged i.n. with 6.15 ⫻ 103 TCID50 units of influenza virus PR8 (H1N1) at days 42 postpriming. Mice were treated again with depleting antibodies at days 3, 7, and 10 postchallenge and monitored for morbidity and mortality for 2 weeks. Passive serum transfer. Passive serum transfer with sera from MVAH5M-vaccinated mice and challenged with H5N1 virus was used to confirm the role of antibody against homosubtypic virus. At 6 weeks postvaccination, sera were collected and pooled from mice that received one dose of MVA-H5M vaccine (1.25 ⫻ 107 PFU). The neutralizing antibody titer against influenza virus A/HK/483/97 in pooled sera was 256. For passive transfer, 10 naive mice received 500 ␮l of either pooled serum or PBS (controls) i.p. Sera were collected at 12 h from serum-transferred mice to detect the level of circulating neutralizing antibodies. At 24 h posttransfer, mice were challenged i.n. with 4 ⫻ 102 TCID50 of influenza virus A/HK/ 483/97 (H5N1). We used a lower dose of A/HK/483/97 as 4 ⫻ 103 TCID50 equal to more than 100 LD50 (data not shown). Survival and weight loss were monitored for 14 days. Three mice were sacrificed at day 5 postchallenge for nasal and lung viral titers. The in vivo study of nonneutralizing antibodies against H1N1 virus was also conducted. Groups of mice (5 weeks old, 8 mice per group) received 500 ␮l of either MVA-H5M serum or PBS i.p. Mice were then challenged with 6.15 ⫻ 103 TCID50 of influenza PR8 (H1N1) virus at 24 h posttransfer. Survival and weight loss were monitored for 14 days. Three mice were sacrificed at day 3 postchallenge for the determination of nasal and lung viral titers. ADCC activity against H1N1. The antibody-dependent cell cytotoxicity (ADCC) activity was analyzed by using a Promega ADCC reporter bioassay core kit (Promega, Madison, WI). Briefly, a 96-well enzymelinked immunosorbent assay (ELISA) plate (catalog no. 9018; Corning, Corning, NY) was coated overnight with 600 ng of purified HA from influenza virus A/Puerto Rico/8/1934 (H1N1) (BEI Resource, catalog no. NR-19240)/well in PBS. The wells were then washed multiple times with PBS to remove the unbound proteins. Positive ADCC target cells or Raji cells (Promega) were then added to the control wells. We used an antiCD20 MAb (clone H299/B1, mouse IgG2a; Promega) as a positive control. Heat-inactivated (56°C for 1 h) serial dilutions of pooled serum from vaccinated mice and control anti-CD20 antibodies were added to respective wells containing 25 ␮l of ADCC assay buffer (25 ␮l of serum per well). Each plate was incubated for 45 min at 37°C. After the incubation, 25 ␮l of ADCC bioassay effector cells was then added to each well, followed by incubation at 37°C for 6 h in a humidified CO2 incubator. Each plate was then removed from the incubator, and 75 ␮l of Bio-Glo reagent (Promega) was added to each well. The luminescence activities were read in a glow-type luminescence read capability machine (Veritas Microplate Luminometer; Promega). Graphs of data were prepared based on the relative light units versus the log10 antibody dilution. ELISA for detecting HA-binding antibodies. An ELISA was used to detect binding antibodies against the HA protein. Briefly, purified HA from influenza virus A/Vietnam/1203/04 (BEI, catalog no. no. NR-10510) or virus A/Puerto Rico/8/1934 (H1N1) (BEI, catalog no. NR-19240) was coated in Costar 96-well EIA/RIA plates (Corning) at 200 ng/ml in carbonate coating buffer, followed by incubation at 4°C overnight. The plates were then washed three times in PBS– 0.1% Tween 20 (PBS-T), and 100 ␮l of 5% skim milk plus 2% FBS in PBS-T were added to each well to block nonspecific bindings. The plates were incubated for 1 h at ambient temperature, and diluted mouse serum samples were then added to each well, followed by incubation for 1 h at an ambient temperature. After an incu-

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bation period, the plates were washed with PBS-T three times. Goat anti-mouse IgG polyclonal antibody conjugated to horseradish peroxidase (HRP; Thermo Scientific, Rockford, IL) at a 1:5,000 dilution or goat anti-mouse IgA conjugated to HRP (Thermo Scientific) at 1:2,000 was then added to each well, followed by incubation for 1 h at ambient temperature, followed by three washes with PBS-T. 3,3=,5,5=-Tetramethylbenzidine (TMB) substrate solution was added, followed by incubation for 20 min until a blue color was observed. Then, 1 N phosphoric acid was added to stop the reaction. The absorbance reactivity in each well was measured at 450 and 630 nm. PRNT. We performed a plaque reduction neutralization test (PRNT) as described previously. Briefly, we used protein G-HP SpinTrap columns (GE Healthcare Life Sciences, Pittsburgh, PA) to purify total IgG from serum obtained from vaccinated mice. We followed the manufacturer’s protocol, except that plasma was incubated with the protein G-Sepharose for 1 h with mild shaking instead of the standard 5 min. Eluted IgG was buffer exchanged in PBS by using an Amicon Ultracel-30K centrifugal filter unit (Millipore, Billerica, MA) with a 30-kDa molecular mass cutoff in a swinging-bucket rotor. Protein concentrations were measured using the Quick-Start Bradford protein assay (Bio-Rad). Influenza A/Puerto Rico/8/1934 stock virus was diluted to approximately 50 PFU/well, followed by incubation with 3-fold serial dilutions of total IgG for 1 h at room temperature. Twelve-well plates were seeded with MDCK cells and washed twice with PBS. Then, 300 ␮l of antibody-virus mixture was placed over the MDCK monolayer for 45 min at 37°C. The antibody-virus mixture was then aspirated off and washed once with PBS. Next, 1 ml of agar overlay containing the appropriate antibody concentration and TPCK-treated trypsin was added to each well. Plates were incubated for 2 days at 37°C and then fixed with 10% formalin plus crystal violet at room temperature for 1 h. Plaques were visualized and counted under a light microscope. An anti-human influenza virus A (H1N1, H2N2) MAb (clone C179, mouse IgG2a) (TaKaRa Bio, Inc., Shiga, Japan) was used as a positive control. This MAb binds to the stalk-HA region and neutralizes influenza A virus subtypes H1, H2, H5, and H6 (15, 16). ICS of T cells. T cell responses were analyzed by using an intracellular cytokine staining (ICS) assay, as previously described (12, 14). Briefly, vaccinated BALB/c mice were euthanized 6 weeks postpriming (n ⫽ 6), and the spleens were aseptically removed. Splenocytes from individual animals were suspended in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 100 IU of penicillin/ml, 100 ␮g of streptomycin/ml, and 0.14 mM ␤-mercaptoethanol. Red blood cells were lysed with 1⫻ BD Pharm Lyse buffer (BD Biosciences, San Jose, CA). After washing with RPMI 1640 medium and centrifugation, the cells were resuspended in the same medium, and 106 splenocytes were plated on Ubottom 96-well plate. Splenocytes were stimulated with H5N1 HA or H1N1 HA peptide pools in a 200-␮l total volume for 16 h in the presence of brefeldin A (BD GolgiPlug; BD Biosciences). The cells were stained intracellularly for gamma interferon (IFN-␥) conjugated to allophycocyanin (XMG1.2) and for interleukin-2 (IL-2) conjugated to phycoerythrin (JES6-5H4) after surface staining of CD4-fluorescein isothiocyanate (RM4-5) or anti-mouse CD8a PerCP (53-6.7). All antibodies were from BD Biosciences, except where noted. The samples were acquired on a BD FACSCalibur flow cytometer and analyzed using Flowjo (v10.0.6) software (TreeStar, Inc., Ashland, OR). The background cytokine level from medium-treated group was subtracted from the cytokine level for each treated sample. The frequency of cytokine-positive T cells was presented as the percentage of gated CD4⫹ or CD8⫹ T cells. Statistical analysis. Student t tests and one-way analysis of variance were used to evaluate viral lung titers, antibody titers, and T cell responses between groups. Survival analyses were performed to assess vaccine effectiveness against challenge viruses. Probability values of 0.05 were considered significant. Prism (v6) software (GraphPad, La Jolla, CA) was used for all statistical analyses.

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FIG 1 Vaccinated mice were injected with a combination of anti-CD4⫹ and CD8⫹ MAbs and then challenged with HPAI A/HK/483/97 virus. (A) MVA-H5M-

vaccinated mice with CD4⫹ and CD8⫹ treatments were protected against H5N1 challenge. (B) No morbidities were observed after lethal challenge. (C) Lung viral titers were not detected at day 5 postchallenge after treatment. (D) In contrast, nasal viral replications were detected in the treatment group. Three mice from each group were sacrificed at day 5 postchallenge to determine the viral titers.

RESULTS

Immunodepletion of both CD4ⴙ and CD8ⴙ T cells. Immunodepletion studies were conducted to determine the role of T cells elicited by MVA-H5M vaccine on protection against H5N1 challenge. Mice were vaccinated with MVA-H5M vaccine, and both CD4⫹ and CD8⫹ subsets were then depleted with the combination of anti-CD4⫹ and CD8⫹ antibodies at 4 days and 1 day before challenge and again at 3, 7, and 10 days after challenge. Mice were challenged with 4 ⫻ 103 TCID50 units of HPAI influenza virus A/HK/483/97 at 6 weeks postpriming. No changes in morbidity and mortality were observed following antibody depletion (Fig. 1A and B). Lung and nasal wash samples were collected at days 5 postchallenge and analyzed for virus titers. While no virus was detected in the lungs of T cell-depleted mice, significant viral titers (an average of 1.58 ⫻ 105 TCID50/ml) were detected in the nasal wash (Fig. 1C and D). No virus was detected in either lung or nasal washes in the nontreatment group.

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Serum antibody from the MVA-H5M vaccine protected naive mice against H5N1. In order to verify the role of antibodies elicited by the MVA-H5M vaccine, passive serum transfer of pooled serum from MVA-H5M-vaccinated mice to naive mice was conducted. Ten mice received 500 ␮l of pooled sera 24 h before challenge with H5N1 influenza virus A/HK/483/97 (4 ⫻ 102 TCID50). At 12 h before challenge, mice were bled, and the mean neutralizing antibody titer was 1:213 (Fig. 2A). Mice that received pooled sera from MVA-H5M showed no signs of morbidity, and no mice succumbed to infection (Fig. 2B and C). In contrast, mice that received PBS succumbed to the infection and were humanely euthanized by 8 days postchallenge. Viral titers were detected in nasal washes and lung samples collected at day 5 postchallenge from PBS-transferred mice but not from MVAH5M serum-transferred mice (Fig. 2D). Cellular immune responses correlate with heterosubtypic protection against H1N1 virus. Since the MVA-H5M vaccine

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FIG 2 Mice received 500 ␮l of pooled sera or PBS i.p. and were challenged with influenza virus A/HK/483/97 24 h later. (A) At 12 h posttransfer, the mice were bled, and the mean neutralizing titer was determined to be 1:213. (B) MVA-H5M pooled sera protected naive mice against lethal H5N1 challenge, with no mortality. (C) MVA-H5M pooled sera prevent weight loss in naive mice, whereas all PBS-received mice succumbed to infection and died. (D) No viral titers were detected in either lung or nasal wash samples at day 5 postchallenge in mice that received pooled sera. Three mice from each group were sacrificed at day 5 postchallenge in order to determine the viral titers.

protected against H1N1 (influenza A/Puerto Rico/8/1934) virus (12) without detectable neutralization antibody titers (Fig. 3A), it was important to examine the role of ADCC and T cell responses. First, serum samples were analyzed by ELISA to characterize the IgG subtypes elicited against H1N1 HA protein. The MVA-H5M vaccine elicited antibody subtypes IgG2a, IgG1, IgG2b, and IgG3 against H1N1 HA protein (Fig. 3B). Antibodies capable of inducing ADCC were analyzed by a Promega ADCC reporter bioassay. Sera incubated with H1N1 HA protein, followed by incubation with effector Jurkat cells (for 12 h at 37°C in a CO2 incubator), did not induce a measurable ADCC reaction (Fig. 3C). In addition, the anti-HA stalk antibody has been shown to contribute to heterosubtypic protection against influenza virus (17). Plaque reduction neutralization tests (PRNTs) have been used to identify the existence of anti-stalk HA (17). Therefore, a PRNT was conducted to determine anti-HA stalk antibodies against H1N1 virus. The results showed no PRNT titer against H1N1 virus similar to the negative-control serum (MVA-GFP serum), whereas a positive-

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control MAb (C179 MAb) showed neutralizing activity at an antibody concentration of 5.5 ␮g/ml (Fig. 3D). Furthermore, T cell responses were then characterized against H1N1 HA peptide using ICS to detect IFN-␥-releasing CD4⫹ and CD8⫹ T cells. The results showed that MVA-H5M elicited high IFN-␥-CD8⫹ levels but not high IFN-␥-CD4⫹ levels against H1N1 HA peptides (Fig. 3E). Depletion of CD8⫹ T cells with anti-CD8⫹ MAbs (clone 2.43) was also conducted to confirm the role of CD8⫹ T cells against H1N1 virus challenge. The results demonstrated that mice succumbed to the infection after depleting CD8⫹ T cells (Fig. 3F). These results strongly suggest that CD8⫹ T cells are responsible for providing heterosubtypic protection against H1N1 virus. Finally, the role of nonneutralizing antibody against H1N1 virus had been confirmed in vivo with passive serum transfer against H1N1 challenge. The results showed that sera from MVA-H5M vaccine did not confer any protection against H1N1 virus, and all infected mice succumbed to the infection (Fig. 3F).

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FIG 3 CD8⫹ T cells elicited by MVA-H5M vaccine play a role in controlling and protection against H1N1 virus. (A) No neutralizing antibodies were detected against PR8 (H1N1) virus. (B) IgG antibodies were detected against H1N1 HA protein. (C) No elevated ADCC activity was detected in the sera of mice given MVA-H5M vaccine against PR8 HA. (D) No plaque-neutralizing antibodies against H1N1 virus from MVA-H5M vaccine were detected. (E) CD8⫹ T cell responses against H1N1 HA peptides were detected in MVA-H5M-vaccinated mice, whereas there were no detectable CD4⫹ T cells. (F) CD8⫹ T cell depletion and passive serum transfer studies were performed in response to H1N1 challenge to confirm that CD8⫹ T cells, but not antibodies, were providing heterosubtypic protection against H1N1 virus. The starting dilution of anti-CD20 MAbs in the ADCC bioassay was 9 ␮g/ml. Anti-HA MAb clone C179 was used as a positive control to demonstrate the anti-stalk HA neutralizing antibody in a PRNT assay.

Mosaic sequence vaccine elicits broader immune responses compared to native sequence vaccine. To test the hypothesis that mosaic HA can elicit broader protection than native or wild-type HA, the immune responses between the mosaic H5 (MVA-H5M)

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and native sequence (VN/1203/04) vaccines (MVA-HA) were compared. First, ICS was conducted to compare MVA-H5M and MVA-HA vaccines. Splenocytes from both MVA-H5M and MVA-HA vaccinated mice were collected at 6 weeks postpriming

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FIG 4 Comparison of cellular immune responses between mosaic HA and native HA vaccines. (A and B) The MVA-H5M vaccine elicited broader IFN-CD4⫹

T cell epitope coverage than did the MVA-HA vaccine. (C and D) The MVA-H5M vaccine elicited broader IL-2-CD8⫹ T cell epitope coverage than did the MVA-HA vaccine.

and incubated with a panel of H5N1 HA peptides, followed by surface staining with anti-CD4⫹ and anti-CD8⫹ MAbs and intracellular staining with anti-IFN-␥ and anti-IL-2 MAbs. The MVAH5M elicited broader T cell epitope coverage, specifically for IFN␥-CD4⫹ and IL-2-CD8⫹ cells, compared to the MVA-HA vaccine (Fig. 4). Also, the MVA-H5M elicited broader serum neutralizing antibodies compared to MVA-HA, especially against influenza virus A/Ck/Egypt/01/08 (Fig. 5A and B). Furthermore, antibodysecreting-cell enzyme-linked immunospot (ELISPOT) or B-cell ELISPOT assays showed that MVA-H5M elicited higher and broader B cell responses than did the MVA-HA vaccine (Fig. 5C and D). Vaccine efficacy between mosaic H5 vaccine and native H5 vaccine. Since MVA-H5M vaccine elicited broader immune responses compared to MVA-HA vaccine, an animal challenge study was conducted to compare their efficacy. BALB/c mice were primed with either MVA-H5M (mosaic) or MVA-HA (native; VN/1203/04)) vaccine and then challenged with 3.56 ⫻ 104 TCID50 of influenza A/Ck/Egypt/01/08 (H5N1) virus at 6 weeks postpriming. The MVA-H5M vaccine elicited neutralizing antibody titers (1:64 endpoint titer) against influenza virus A/Ck/ Egypt/01/08, whereas no measurable neutralizing antibody response was elicited by the MVA-HA vaccine (Fig. 6A). Mice

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vaccinated with MVA-H5M were completely protected against H5N1 virus challenge, with no observed morbidity and mortality (Fig. 6B and C). Although the mice were protected from H5N1 virus lethality, MVA-HA-vaccinated mice showed clinical signs and significant weight loss (Fig. 6C) after challenge. No virus was detected in the lungs and upper airway of MVA-H5M after H5N1 challenge, indicating that the MVA-H5M vaccine provided sterile immunity (Fig. 6D). In contrast, virus was detected in the lungs of mice vaccinated with MVA-HA after challenge (Fig. 6D). Recombinant mosaic H5 protein protects against avian influenza virus challenge. MVA has been widely used as a vaccine vector due to its ability to induce strong Th1, Th2, and innate immune responses (17, 18). To examine the role of the MVA vector on the broad protection conferred by the mosaic H5 vaccine, the H5M gene was cloned and expressed in a DNA vaccine vector (pCMV-H5M). The pCMV-H5M DNA vaccine effectively protected mice, with no clinical signs against H5N1 (3.56 ⫻ 104 TCID50 of A/Ck/Egypt/01/08 virus) challenge, when the vaccine was given as a single dose or as two doses (Fig. 7). However, low viral titers were found in both nasal cavity and lungs at 5 days postinfection. Furthermore, the H5M gene was also cloned into the pTriEx vector, and the expressed protein was purified, mixed with alum adjuvant, and evaluated as a vaccine. Groups of BALB/c

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FIG 5 Comparison of humoral immune responses between mosaic HA and native HA vaccines. (A) The MVA-H5M vaccine elicited serum neutralizing antibodies against influenza virus A/Ck/Egypt/01/08, whereas the MVA-HA vaccine did not. (B) No BAL fluid-neutralizing antibodies were detected in response to either vaccine. (C and D) The MVA-H5M vaccine elicited a greater B cell response than did the MVA-HA vaccine.

mice were primed or primed and boosted with purified mosaic H5 (pH5M)-alum mix, followed by influenza A/Ck/Egypt/01/08 (H5N1) virus challenge at 6 weeks postpriming or at 2 weeks postboosting. The pH5M with alum adjuvant elicited a high neutralizing antibody titer against the virus (Fig. 7A) and provided protection with no morbidity and mortality similar to a single dose of the MVA-H5M vaccine (Fig. 7B and C). When two doses of purified H5M were given to mice, no viral titers were detected in either nasal samples or the lungs. However, low viral titers were detected when animals were given only one dose of the vaccine (Fig. 7D). Increasing of IgG antibody in the lung after challenge. Since serum antibody responses are strongly correlated with homosubtypic protection, and high levels of serum IgG but not IgA were found in MVA-H5M-vaccinated mice (data not shown), we analyzed BAL samples for IgG and IgA both prior to and after challenge with H5N1 to determine their role in protection. BAL sam-

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ples were collected from MVA-H5M-vaccinated mice prior to challenge and at day 5 postchallenge. IgG and IgA ELISAs were conducted with HA H5N1-coated plates. Small amounts of IgG antibodies and no IgA were detected in BAL samples prior to challenge. Interestingly, high IgG titers were detected 5 days after challenge (Fig. 8A and B). In order to confirm these findings, BAL samples were collected from naive mice that had passively received pooled sera from MVA-H5M-vaccinated mice. The results indicated an undetectable level of both IgG and IgA antibodies in BAL samples prior to challenge, but a surge in IgG levels was again found in lungs after challenge (Fig. 8C and D). DISCUSSION

In our previous studies, we used an in silico approach to generate a “mosaic” H5N1 HA and expressed it using the MVA vector (MVA-H5M) (12). Here, we examined the role of humoral and

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FIG 6 Mouse challenge study against avian influenza virus A/Ck/Egypt/01/08 comparing the mosaic HA vaccine (MVA-H5M) and the native HA vaccine (MVA-HA). (A) Neutralizing antibodies were elicited by MVA-H5M but not by MVA-HA. (B) Both MVA-H5M and MVA-HA protected against influenza virus A/Ck/Egypt/01/08 challenge. (C and D) Mice vaccinated with MVA-H5M vaccine showed no weight loss and no nasal or lung viral titers compared to mice vaccinated with MVA-HA, which showed morbidity with significant weight loss and nasal and lung viral titers. Three mice from each group were sacrificed at day 5 postchallenge for determination of the nasal and lung titers.

cellular immune responses elicited by this MVA-H5M vaccine in more depth, and we also compared immune responses and protection to the MVA-vectored expression of native H5 HA (MVAHA). As demonstrated above, the MVA-H5M vaccine provided broader T cell, B cell, and antibody responses against H5N1 viruses than did the MVA-HA vaccine. Protection against homosubtypic strains (H5N1) mainly correlated with neutralizing antibodies in serum. Serum IgG antibodies were also detected in the lung after challenge in mice that received pooled sera from MVAH5M-vaccinated mice. In addition, CD8⫹ T cells appeared to play an important role in heterosubtypic protection against H1N1 virus. The expression of the H5M gene as either a DNA vaccine or a subunit protein protected mice against H5N1 challenge, indicating the effectiveness of the mosaic protein itself as a vaccine antigen for inducing humoral immune responses against influenza virus. Antibodies are known to play an important role in protection against influenza virus since they can directly prevent viral attach-

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ment to the target epithelial cells (19). Our results show that strong antibody responses were elicited by MVA-H5M, and its significance in protection against H5N1 virus challenge was confirmed with T cell depletion and passive serum transfer studies (Fig. 1 and 2). Although the protection elicited by the MVA-H5M vaccine against H5N1 did not depend on T cells, viral replication in nasal wash samples was detected when the T cells were depleted (Fig. 1D), suggesting the importance of T cells on viral clearance in the upper airway. However, we did not detect viral titers from nasal wash samples of serum-transferred animals. The differences between nasal viral titers in T-cell-depleted animals and serumtransferred animals can be explained as follows. First, primed and naive CD4⫹ and CD8⫹ cells were depleted in the depletion study, but naive cells were not depleted in the passive serum transfer study, therefore naive T cells, or other CD4⫹ CD8⫹ cells could have contributed to control viral infection in the upper airway. Second, the challenge dose that was used in the passive serum transfer study (4 ⫻ 102 TCID50) was lower than the one used in

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FIG 7 (A) Recombinant mosaic H5 expressed as a purified protein (pH5M) or DNA elicited high levels of neutralizing antibodies after boosting. (B and D) The mosaic H5 proteins (both DNA and purified protein) provided 100% protection with no morbidity, similar to the MVA-H5M control. (D) When vaccinees were given both a prime and a boost, purified H5M (pH5M) prevented viral replications at day 5 postchallenge in both the lungs and the nasal cavity, similar to the MVA-H5M vaccine. Three mice from each group were sacrificed at day 5 postchallenge for determination of the viral titers.

depletion study (4 ⫻ 103 TCID50), so a lower dose could have been cleared from the upper airway by day 5 postchallenge. Further studies are needed to elucidate the role of both CD4⫹ and CD8⫹ T cells in controlling infections in the upper respiratory tract. It is possible that T cells play a role in controlling viral replication in the upper airway by direct killing (20). In addition, CD4⫹ T cells can contribute to protection by orchestrating immune responses using both cytokines and chemokines to control viral replication (20). Heterosubtypic protection against influenza viruses by vaccination has been linked to CD8⫹ T cells, anti-stalk HA antibodies, and ADCC responses (17, 19, 21, 22). MVA-H5M provided heterosubtypic protection against H1N1 virus without detectable neutralizing antibodies (12). The strong CD8⫹ T cell responses but lack of both ADCC antibody and PRNT titer against H1N1 indicates that CD8⫹ T cells are the main contributor to the het-

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erosubtypic protection against H1N1 challenge (Fig. 3). The role of CD8⫹ T cells against H1N1 virus was also confirmed by a depletion study with anti-CD8⫹ MAbs. Vaccinated mice succumbed to the infection when treated with depleting antibody (Fig. 3F). Finally, passive transfer of nonneutralizing H5 HA specific antibodies did not protect mice against heterosubtypic H1N1 virus challenge (Fig. 3F). These findings help confirm the importance of CD8⫹ T cells in controlling heterosubtypic protection (21, 23–26). It has previously been shown that the mosaic approach can broaden CD8⫹ T cell responses in HIV-1 vaccines with a 9-mer CD8⫹ T cell epitope approach (10, 27). However, our mosaic H5 sequence has been designed in order to broaden humoral immune responses by targeting CD4⫹ T cell epitopes with a 12-mer epitope approach (28). Designing the vaccines to target T helper cells can help broaden humoral immune response via T helper cell-B cell

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FIG 8 Immunoglobulin profiles in BAL samples. (A and B) IgG and IgA in BAL samples from mice vaccinated with the MVA-H5M vaccine. (A) Small amounts of IgG antibodies were found in the BAL fluid before challenge. (B) High levels of IgG antibodies were found 5 days after challenge with H5N1 virus. (C and D) IgG and IgA in BAL samples from naive mice that received pooled serum from MVA-H5M-vaccinated mice. (C) No IgG and IgA antibodies were detected before challenge. (D) High IgG antibody levels were found 5 days after challenge.

interaction (19). Our results indicated that the MVA-H5M vaccine elicited a broader humoral and cellular immune response than did MVA-HA (Fig. 4 and 5). The MVA-H5M vaccine elicited strong neutralizing antibodies and fully protected against a highly virulent H5N1 strain (influenza A/Ck/Egypt/01/08), whereas MVA-HA (native sequence) failed to elicit neutralizing antibody response and prevent viral infection in mice (Fig. 6). In addition, MVA-H5M increased B cell and T cell epitope coverage, especially IFN-CD4⫹ and IL-2-CD8⫹ T cells over native H5 vaccine (MVAHA), suggesting strong Th1 responses. Future studies will further characterize Th1 versus Th2 type responses to elucidate the role of T helper cells toward the induction of Th2 type cytokines and cells. Our studies also demonstrate that the broad protection conferred by the MVA-H5M is mainly due to the nature of the immune responses induced by the mosaic approach and is not necessarily because of the intrinsic nature of the MVA vaccine vector. MVA virus is a highly immunogenic vector that elicits strong innate immune responses that link to adaptive immune responses (29, 30). In order to dissect the role of MVA-based expression on the observed protection, we tested the immunogenicity and efficacy of the H5M as a subunit protein (pH5M) or a DNA vaccine (pCMV-H5M). The results showed that the H5M subunit protein

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protected against H5N1 challenge with no observed morbidity and mortality, suggesting that a subunit vaccine could also be further pursued in preclinical trials. This approach would reduce concerns that preexisting immunity against MVA might reduce the effectiveness of the mosaic vaccine approach. Parenteral inactivated influenza vaccines have been reported to induce strong serum anti-HA IgG antibody response that is necessary for preventing infection, whereas i.n. vaccines have shown to induce high levels of mucosal IgA (31, 32). IgA and IgM can be secreted into respiratory mucosal tissues to prevent viral infection in respiratory tract (32). However, a small amount of serum IgG can also be diffused into the respiratory mucosa (31, 32). The MVA-H5M vaccine was delivered i.d. and elicited high serum IgG antibody titers, but no serum IgA in mice. In addition, a low level of IgG in BAL fluid was detected before challenge, whereas no IgA was observed. These findings suggest that a small fraction of serum IgG can be diffused from serum into the lung (Fig. 8A), a finding which is consistent with a previous study (32). High level of IgG in BAL fluid after challenge were observed, which indicated high serum diffusion into the respiratory tract after influenza virus infection, similar to previous reports (Fig. 8B) (32, 33). Similarly, we observed the same phenomenon after passive serum transfer of

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pooled sera into naive mice, whereas there was a significant increase in HA specific IgG in the lungs of mice after challenge (Fig. 8C and D). These observations confirmed that IgG antibodies detected in BAL fluid came from pooled sera that had been transferred but not from local plasma cells. By 5 days postchallenge, there has not been enough time for naive B cells to secret IgG against viral infection (34). The present study suggests that small amounts of IgG antibodies can be diffused from the blood into the lungs and covered respiratory tract before challenge. Once the infection has occurred, the virus will compromise the epithelial integrity and trigger inflammatory responses that cause high levels of leakage of serum IgG into the respiratory lumens, which helps to prevent viral infection in the lung (32, 33, 35). However, some of the antibody that is detected in the BAL samples of vaccinated mice may have been generated locally by plasma cells in response to infection. In summary, using the mosaic approach we successfully generated influenza vaccine candidates that generate broad immune responses. The MVA-H5M vaccine provides protection against H5N1 virus with strong neutralizing antibodies that can diffuse into the lung and prevent infection. The vaccine also elicited CD8⫹ T cells against heterosubtypic H1N1. Importantly, the mosaic HA approach generates a better vaccine than does the traditional native sequence, providing broader cellular and humoral immune responses. The mosaic H5 sequence provides broad protection regardless of the delivery method, as shown with the subunit and DNA vaccine. Finally, by incorporating other influenza virus HA subtypes into the existing vaccine, we could generate a “universal influenza vaccine” that would provide broad protection against circulating influenza viruses, and this vaccine could be stockpiled for the next pandemic.

6. 7.

8.

9.

10.

11.

12.

13.

14.

ACKNOWLEDGMENTS We thank Matt Aliota (University of Wisconsin—Madison [UW-Madison]) for critical review of the manuscript and Tony Goldberg (UWMadison), Thomas Friedrich (UW-Madison), Suresh Marulasiddappa (UW-Madison), and Stacy Shultz-Cherry (St. Jude Hospital) for suggestions and guidance in experiments. A.K., B.K.-B., and J.E.O. conceived and designed the experiments. A.K. performed the experiments. A.K. analyzed the data. J.E.O. contributed reagents, materials, and analysis tools. A.K., B.K.-B., and J.E.O. wrote the paper.

15. 16.

17.

FUNDING INFORMATION This work was partially funded by a University of Wisconsin Fall Competition award.

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