Employing XIAP to enhance the duration of antigen expression and immunity against an avian influenza H5 DNA vaccine.

Immunological Investigations, Early Online: 1–17, 2015 ! Informa Healthcare USA, Inc. ISSN: 0882-0139 print / 1532-4311 online DOI: 10.3109/08820139.2014.988718

Employing XIAP to Enhance the Duration of Antigen Expression and Immunity Against an Avian Influenza H5 DNA Vaccine

Immunol Invest Downloaded from informahealthcare.com by Kainan University on 04/10/15 For personal use only.

Seyed-Elias Tabatabaeizadeh,1 Mohammad Reza Bassami,1,2 Alireza Haghparast,1,2 and Hesam Dehghani1,3 1

Division of Biotechnology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran, 2 Veterinary Biotechnology Research Group, Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran, and 3 Embryonic and Stem Cell Biotechnology Research Group, Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran DNA vaccine represents a powerful approach for prevention of avian H5N1 influenza infection. Yet, DNA vaccine-induced immune responses might be limited by the short duration of antigen expression. As a strategy to enhance adaptive immune responses elicited by a hemagglutinin 5 (H5) DNA vaccine, we explored the effect of co-administration of a DNA encoding X-linked inhibitor of apoptosis protein (XIAP) as a modulator of apoptosis and a stimulator of inflammatory signaling. In cultured cells as early as 24 hours (h), we found that the DNA vaccine encoded H5 antigen was a potent stimulator of apoptosis, and the H5 pro-apoptotic activity was significantly suppressed by the co-expression of full-length XIAP or mutant XIAP (DRING). However, full-length XIAP showed a higher potency than mutant XIAP (DRING) in the inhibition of H5-induced apoptosis. We also compared the immunizing ability of transmembrane and secretory forms of H5. Mice vaccinated (twice with 3-week intervals) with the secretory form of H5 showed higher hemagglutination inhibition (HI) antibody titers than mice vaccinated with the transmembrane form of H5. Furthermore, co-administration of XIAP with the secretory form of H5 resulted into a stronger antibody response than the transmembrane form of H5. Our findings suggest that in the design of DNA vaccines for a given pro-apoptotic antigen, using an antiapoptotic molecular adjuvant and the secretory form of antigen may be a greater stimulus to induce immune responses. Keywords Apoptosis, avian H5N1 influenza, DNA vaccine, XIAP

INTRODUCTION The H5N1 subtype of the avian influenza virus can cause a severe and fatal disease in humans and poultry. Susceptibility of the viral genome to mutation and reassortment might produce a virus with human to human transmission ability, and result into a pandemic and global crisis (Herfst et al., 2012; Imai et al., 2012). Among avian influenza viruses, highly pathogenic avian Correspondence: Hesam Dehghani, Ferdowsi University of Mashhad, Faculty of Veterinary Medicine, Division of Biotechnology, Azadi Square, Mashhad 9187195786, Islamic Republic of Iran. E-mail: [email protected]


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influenza (HPAI) H5N1 has created the greatest threat to humans in recent years. Since 2003, 694 laboratory confirmed cases of H5N1 infection (resulting in 402 deaths) have been reported from 16 countries (WHO, 2015). In addition, the 2009 pandemic H1N1 virus has a high genetic compatibility with the avian H5N1 virus. As identified by experimental molecular changes in hemagglutinin, a reassortant H5/H1N1 virus (derived from the hemagglutinin of an H5N1 virus and the seven segments from a 2009 pH1N1 virus) was able to recognize human-type receptors (Imai et al., 2012; Octaviani et al., 2010). It has recently been shown that without a need for reassortment in an intermediate host, HPAI A/H5N1 viruses have the potential to evolve directly to gain airborne transmission capability between mammals (Herfst et al., 2012). These findings emphasize the importance of intensive research on the development of safe and efficacious vaccines against the potential pandemics caused by H5 viruses. DNA vaccine has attracted widespread attention due to its simple manufacturing and its efficacy to induce both humoral and cell-mediated immune responses (Kutzler & Weiner, 2008). The ease of mass production and stability of DNA vaccines make them a proper vaccine candidate for the pandemic preparedness (Kim & Jacob, 2009). In preclinical trials, influenza DNA vaccine encoding hemagglutinin antigen has caused specific and protective immune responses against influenza virus (Dai et al., 2013; Ogunremi et al., 2013; Zhou et al., 2012). During the initial stages of infection, hemagglutinin is responsible for the attachment and penetration of virus into the cells, and antibodies produced against the hemagglutinin have the major role in protection against influenza virus (Andrew et al., 1987). Thus, among hemagglutinin, nucleoprotein, and matrix 2 antigens, hemagglutinin genebased immunization has induced the most efficacious protective immunity against high challenge doses of H5N1 (Rao et al., 2010). However, still there is a need the immune responses elicited by hemagglutinin based DNA immunization to be further improved. Immunogenicity of DNA vaccines is limited due to the short duration of antigen production (Greenland et al., 2007). The adaptive immune responses might be able to destroy antigen-producing myocytes as early as 10 days after DNA injection, leading to the clearance of antigen-producing cells (Davis et al., 1997; Geiben-Lynn et al., 2008). Immune-mediated myocyte clearance is dependent on either the emergence of antigen-specific adaptive immune responses, or antigen-induced apoptosis. While the immune-mediated clearance of host cells have been extensively studied (Davis et al., 1997; GeibenLynn et al., 2008), there are no previous studies on the apoptosis of host cells induced by the vaccine antigen. The vaccine DNA encoded antigen with proapoptotic activity may act faster than adaptive immune responses in the destruction of antigen-producing cells, which might lead to the short duration of antigen production and the low levels of immune responses. Thus, using an anti-apoptotic protein may inhibit the programmed cell death process which is initiated by pro-apoptotic antigens and/or cytotoxic T lymphocyte (CTL) activities, leading to the enhancement of antigen production and immunity. X-linked inhibitor of apoptosis protein (XIAP) is the most efficient inhibitor of apoptosis among inhibitor of apoptosis (IAP) family of proteins that potently inhibits effector caspase activation, and apoptosis stimulated by both the


Employing XIAP to Enhance Vaccine-Induced Immunity

intrinsic and extrinsic apoptotic pathways (Conte et al., 2001; Deveraux et al., 1997; Deveraux et al., 1998; Rehm et al., 2006; Takahashi et al., 1998; Wilkinson et al., 2004). XIAP contains three tandem copies of an 70 amino acid domain termed the baculoviral IAP repeat (BIR) and a RING zinc finger domain near the C terminus (Takahashi et al., 1998). XIAP modulates multiple signaling pathways, including nuclear factor-kappa b (NF-iB), transforming growth factor-beta (TGFb), and mitogen-activated protein kinase (MAPK) signaling (Lu et al., 2007; Sanna et al., 2002; Yamaguchi et al., 1999). Participation of XIAP in activation of NF-iB and MAPK signaling pathways results into the production of inflammatory cytokines and co-stimulatory molecules, and leads to an enhanced stimulation of innate and adaptive immunity (Bauler et al., 2008; Lu et al., 2007; Rigaud et al., 2006). XIAP has been previously used as a molecular adjuvant aimed to enhance CTL immune responses against human papillomavirus type-16 (HPV-16) and severe acute respiratory syndrome (SARS) infections (Azizi et al., 2005; Kim et al., 2003). These studies employed intradermal and subcutaneous administration routes to directly target the DNA encoding XIAP to dendritic cells (DCs) leading to increased survival of transduced DCs and enhanced antigen-specific CTL responses (Azizi et al., 2005; Kim et al., 2003). In addition to the immune-mediated clearance of the host cell, it has been previously observed that the intra- or extra-cellular location of DNA encoded antigens (transmembrane, intracellular, or secretory forms of the antigen) might also be effective in modulation of immune responses. The secretory and transmembrane forms of glycoprotein D from bovine herpesvirus 1 antigen were able to induce faster and stronger humoral immune responses than those elicited by the intracellular form (Lewis et al., 1999). In this study, we have hypothesized that the H5 antigen produced by a DNA vaccine is able to induce apoptosis. To increase the duration of antigen production, we have co-expressed XIAP to enhance the survival of antigenproducing myocytes and to induce an inflammatory condition required for myocytes to express several co-stimulatory molecules (Wiendl et al., 2005), leading to stronger stimulation of B cells and humoral responses. In addition, we reasoned that employing the secretory form of H5 would result in higher availability of antigen in the draining lymph nodes in order to prime immune cells. Our results indicate that a DNA vaccine expressing H5 protein of influenza virus in cultured cells enhanced antigen-induced cell death during 24 h, and that XIAP was able to inhibit this cell death. In addition, mice immunization using co-delivery of XIAP and the secretory form of H5 resulted into a significant increase in the influenza HI antibody titers.

MATERIALS AND METHODS Plasmid DNA Four plasmids were constructed and employed: (1) a plasmid encoding transmembrane form of H5 fused to four copies of complement receptor 2 binding peptide (P28) (pCAGGS-H5-P284), (2) a plasmid encoding secretory form of H5 fused to four copies of P28 (pCAGGS-sH5-P284), (3) a plasmid encoding full-length XIAP fused to 6Myc tag (pcDNA3-6Myc-XIAP), and

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Figure 1. Map and expression analysis of H5 and XIAP constructs: (a) Schematic representation of (I) the wild-type transmembrane form of the H5 protein fused to 4 copies of P28 (H5-P284); (II) the secretory form of the H5 protein lacking the transmembrane and cytoplasmic domains fused to 4 copies of P28 (sH5-P284); (III) the wild-type full length XIAP fused to 6Myc tag; and (IV) the mutant XIAP (XIAP (DRING)) lacking the RING domain. Rectangles indicate protein domains. Linkers are composed of (G4S2)2. TM indicates transmembrane. (b) CHO cells were transfected with 2 mg of each expression plasmid. Cell lysates and supernatants of cells transfected with H5 constructs and cell lysates of cells transfected with XIAP constructs were electrophoresed on a 10% polyacrylimide gel. Lane 1, protein marker (Fermentas, Lithuania); Lane 2, H5-P284 in cell lysate; Lane 3, H5-P284 in the supernatant; Lane 4, sH5-P284 in cell lysate; Lane 5, sH5-P284 in the supernatant; Lane 6, 6-Myc-XIAP; Lane 7, XIAP(DRING); and Lane 8, empty vector control (H5 antibody).

(4) a plasmid encoding XIAP with deleted RING zinc domain (pcDNA3XIAP(DRING)) (Figure 1a). pCAGGS (BCCMÔ, Belgium) and pcDNA3.1 (Invitrogen Corp., Carlsbad, CA) eukaryotic expression vectors were used as backbone plasmids for the expression of hemagglutinin and XIAP genes, respectively. It was previously reported that fusion of multimers of the P28 peptide to an antigen may enhance the level of immunogenicity (Bergmann-Leitner et al., 2007; Bower & Ross, 2006). Thus, hemagglutinin sequence (GenBank ABQ45850) from the influenza A virus (A/chicken/India/NIV33487/2006,



H5N1, clade 2.2) was fused to 4 copies of P28 sequence to make H5P284 (Figures 1a-I). Between hemagglutinin and P28 and between P28 repeats, linkers were placed which were composed of two repeats of four glycines and two serines (G4S2)2. Because the used hemagglutinin sequence belonged to a highly pathogenic influenza virus, basic cleavage site (PQGERRRKKRGL) was replaced with PQRESRGL to maximize the safety of the H5 DNA vaccine. The final protein sequence was back-translated and optimized for murine expression using Gene Designer 2.0 software (DNA 2.0; Menlo Park, CA). The coding gene was synthesized by GenScript (Piscataway, NJ). The synthesized gene was cloned into the pCAGGS to derive pCAGGSH5-P284 plasmid. The secretory version of H5-P284, sH5-P284, was generated by deleting the transmembrane and cytoplasmic domains of hemagglutinin. This was accomplished by using an XhoI restriction enzyme (Fermentas, Lithuania) digesting two restriction sites on either side of the region encoding the transmembrane and cytoplasmic domains. The resulting linear plasmid was self-ligated to make pCAGGS-sH5-P284 (Figure 1a-II). The plasmid encoding full length XIAP, pcDNA3-6Myc-XIAP (Figure1a-III), was a kind donation from Robert G. Korneluk (Apoptosis Research Centre, Children’s Hospital of Eastern Ontario Research Institute, Ottawa, Ontario, Canada). Since the RING domain of XIAP does not contribute to the in vitro inactivation of caspase 3, caspase 7 and caspase 9 (Takahashi et al., 1998), we also examined the inhibitory effect of the RING deleted XIAP (about 1/3 shorter than XIAP) on the H5 induced apoptosis.A plasmid encoding a mutant version of XIAP containing three tandem BIRs lacking the RING domain, pcDNA3-XIAP(DRING), was constructed using a one-step polymerase chain reaction method with Pfu DNA polymerase (Fermentas, Lithuania) employing the pcDNA3-6Myc-XIAP as the template and using 50 -GCTAAGCTTGATCCA TGACTTTTAACAGTT-30 as the forward primer and 50 -TCTCTCGAGCTATTC TAACAGATATTTGCACC-30 as the reverse primer. The PCR product was digested with HindIII and XhoI (Fermentas, Lithuania) and was subcloned into pcDNA3.1 (Fig. 1a-IV) to make pcDNA3XIAP(DRING). The plasmids were amplified in Escherichia coli strain, TOP10, were isolated in a miniprep scale using Plasmid DNA Isolation Kit (DENAzist Asia, Iran), and also were isolated in a large scale using standard alkaline lysis method. Verification of subclonings was performed by appropriate restriction enzyme digestions, gel electrophoresis, and sequencing. Purity and concentration of plasmid preparations were determined by measurements of optical density at 260 nm and 280 nm by a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE) and gel electrophoresis. Cell Transfections and Expression Analysis To evaluate the expression of H5-P284, sH5-P284, XIAP and XIAP (DRING), the Chinese hamster ovary (CHO) cell line was transfected with 2 mg of each plasmid DNA using TurboFect transfection reagent (Thermo Scientific) in 6well tissue culture plates according to the manufacturer’s protocol. After 24 h, supernatants were collected and stored at 20 C. Cell lysates were collected in 200 ml of RIPA lysis buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.1%

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SDS, 50 mM Tris, pH 8.0) and stored at 20 C. For western blot analysis, supernatants or cell lysate protein (20 mg) were mixed with SDS loading buffer (Fermentas, Lithuania) and loaded onto 10% SDS-polyacrylimide gel followed by electrophoretic blotting onto a nitrocellulose membrane (Problat MN 0.45 mm, Takara, Otsu, Japan). The membranes were incubated with polyclonal antibodies against: H5 (1:100; GD, Cat. No. VLDIA042) and XIAP (1:500; Santa Cruz Biotechnology, Cat. No. sc-11426). Detection of primary antibodies was performed by HRPconjugated secondary antibodies, goat anti-chicken (1:15,000; GenScript; Cat. No. A00165), and donkey anti-rabbit (1:15,000; Santa Cruz Biotechnology; Cat. No. sc-2313). Immunoreactive bands were detected by Amersham Biosciences ECL Western blotting detection reagents (GE Healthcare Biosciences, Buckinghamshire, UK) and visualized by G-Box gel documentation system (Syngene, Cambridge, UK). Apoptosis Assay and Flow Cytometry Analysis To determine the apoptotic and anti-apoptotic effects of H5 and XIAP, four test groups: (1) pCAGGS-H5-P284 + empty vector, (2) pCAGGS-H5-P284 + pcDNA3-6Myc-XIAP, (3) pCAGGS-H5-P284 + pcDNA3-XIAP (DRING), and (4) empty vector control were examined. CHO cells were transfected with 1 mg of each plasmid (a total of 2 mg for two plasmid groups) and 2 mg of the empty vector in the control group. Plasmids were transfected using TurboFect transfection reagent in triplicates according to the manufacturer’s protocol. After 24 h, the percentage of the apoptotic cells was measured by an annexin V fluorescein isothiocyanate (FITC)-propidium iodide (PI) apoptosis detection kit (Santa Cruz Biotechnology) according to the manufacturer’s instruction. Annexin V-FITC/PI and flow cytometry analysis made it possible to quantify the percentages of the H5 antigen induced apoptotic cells and to quantify and compare the percentages of cells with enhanced survival coexpressing XIAP. Transfected cells were harvested and washed with PBS and incubated in 100 ml of 1 assay buffer containing 5 ml annexin V-FITC and 10 ml propidium iodide (PI) for 20 minutes at room temperature in the dark, and then were analyzed by dual-color flow cytometry (Becton Dickinson FACS Calibur). Mice Immunizations BALB/c female mice (Razi Vaccine & Serum Research Institute, Mashhad, Iran) ages 6–8 weeks were used for inoculations. The mice were allowed free access to food and water and were kept and subjected to the experiments according to the guidelines of the Research Ethics Committee of Ferdowsi University of Mashhad. A total of 25 mice were divided into 5 groups (5 animals for each group of H5-P284, sH5-P284, H5-P284 + XIAP, sH5-P284 + XIAP, and empty vector control). Mice were immunized with a total of 100 mg plasmid DNA in 100 ml of PBS, injected intramuscularly in both quadriceps at day 1 and day 21 (booster immunization) of the experiment. For the single H5P284 or sH5-P284 plasmid groups, each animal received 50 mg DNA of each plasmid plus 50 mg of the empty vector DNA. For the 2 plasmid combination groups (H5-P284 + XIAP or sH5-P284 + XIAP), 50 mg of each plasmid DNA (total 100 mg) was injected.



Antibody Assay Two immunized sera were collected from each animal three weeks after the first immunization (day 21), and two weeks after the booster immunization (day 35). The HI assay was performed by standard methods. Briefly, 25 ml 4-hemagglutination units of the H5 antigen (GD, VLDIA097) was added to each well of a 96-well microtitre plate containing 25 ml serum (twofold diluted with PBS). After 1 h incubation at 4 C, 50 ml of 0.5% (v/v) chicken erythrocytes were added to each well, and incubated for another 40 minutes at 4 C. The reciprocal of the highest serum dilution that completely inhibited hemagglutination was determined as the HI endpoint titer. Proliferation Assay For splenocyte proliferation assay, the spleens from 5 mice groups were taken two weeks after booster immunization and homogenized. The splenocytes were isolated aseptically and depleted of red blood cells by lysis buffer (8.3 g/l ammonium chloride in 0.01 M Tris-HCL, pH 7.5). Cells were cultured at 5 105 cells per well in 96-well flat-bottom plates in 100 ml RPMI 1640 containing 10% FCS and supplements. The splenocytes from 5 mice groups were stimulated with 1 mg H5 antigen (GD, VLDIA097). Then 5 wells of splenocytes from empty vector vaccinated group stimulated with 0.5 mg of phytohemagglutinin (PHA) as positive control for T cell proliferation. After 48 h incubation at 37 C in a humid 95% air with 5% CO2 environment, 10 ml alamarBlue (AbD Serotec) was added and incubated for another 4 h. Cell proliferation was measured by spectrophotometry at 490 nm. The proliferation responses were expressed as a stimulation index (SI) which was calculated by dividing the absorbance of the H5 antigen and PHA stimulated wells over the non-stimulated wells. Statistical Analysis SPSS statistical software version 21 (SPSS Inc, Chicago, USA) was used for data summarization and statistical analysis. Statistical analyses of experimental data were performed by using the Kruskal–Wallis analysis followed by the Mann–Whitney test. The Wilcoxon test was used to compare antibody responses between the first and booster immunizations for each group. Trend analysis for experimental groups was performed using the Jonckheere– Terpstra test. Statistical significance was defined at p 0.05.

RESULTS In vitro Protein Expression The expression of different forms of H5 protein and XIAP encoded by the constructed plasmids in CHO cells was confirmed using specific polyclonal antibodies and western blot analysis. Chinese hamster ovary cells were transfected with 2 mg of plasmid and both cell lysates and supernatants were assayed for H5 expression by western blot analysis. H5-P284 expression was detected in cell lysates showing a band of 82 kD (Figure 1b, lane 2) but no protein was detected in the supernatant culture medium, indicating that the protein is produced in the cell-associated non-secretory form (Figure 1b, lane 3).

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sH5-P284 expression was detected in both of cell lysate and supernatant culture medium showing a band of 77 kD which was consistent with the deletion of the 42 amino acids corresponding to the transmembrane and cytoplasmic domains of H5, and confirming secretion of the expressed protein (Figure 1b, lanes 4-5). Recombinant 6Myc-XIAP and XIAP (DRING) were detected in cell lysates showing bands of 66 kD and 37 kD, respectively (Figure 1b, lanes 6-7). Since an anti-XIAP polyclonal antibody was used, a band of 57 kD corresponding to the endogenous XIAP was detected in both of the XIAP transfected cells (Figure 1b, lanes 6-7). In vitro Suppression of H5-Induced Apoptosis by XIAP Co-expression of full-length or ring-deleted mutant forms of XIAP with H5 antigen significantly decreased the H5-induced apoptosis during a 24-h period of cell culture (p 0.05; Figure 2). To explore the possible pro-apoptotic effects of H5 expression, CHO cells were transfected with the construct encoding H5P284 (pCAGGS-H5-P284). Using green fluorescent protein (GFP) encoding construct, transfection efficiency estimated to be about 60% (Fig. 2a-I). After 24 h, pCAGGS-H5-P284transfected cells were incubated with FITC labeled annexin V, which binds with high affinity to phosphatidylserine exposed on the outer membrane leaflet in the early apoptotic cells, and PI which accumulates in the necrotic cells. Before detection and quantification of apoptosis by flow cytometry, FITC staining pattern and morphology of apoptotic cells were assessed using fluorescence microscopy (Figure 2a-II). Cells in later stages of apoptosis stained positive for both FITC and PI as detected by flow cytometry (Figure 2b). We observed that H5 expression significantly increased the fraction of the early and late apoptotic cells during 24 h compared to the empty vector transfected group as a control (p 0.05; Figure 2c). However, by overexpression of H5, and co-expression of XIAP, the fraction of PI-labeled (necrotic) cells was not significantly different from the control group of cells. To assess the ability of XIAP protein in the protection of cells against H5induced apoptosis, CHO cells were transfected to co-express H5 and two different versions of XIAP. We observed that both forms of XIAP, full-length XIAP and XIAP (DRING), were able to significantly inhibit apoptosis mediated by H5 expression (p 0.05; Figure 2c). Co-expression of XIAP decreased the fraction of early apoptotic cells more efficiently than the mutant XIAP (DRING) (p 0.05; Figure 2c). In fact, the co-expression of full-length XIAP was able to bring down the level of apoptosis to that in control cells (Figure 2c). However, the level of apoptosis of XIAP (DRING) co-expressing cells was significantly higher than that in empty vector transfected control cells (p 0.05; Figure 2c). These results suggest that the H5-induced cell death might go through apoptotic processes, rather than necrotic cell death pathways, and that the XIAP expression is effective enough to inhibit the induced apoptotic cell death in early stages. Enhancement of HI Antibody Production by Co-administration of XIAP Encoding DNA In many of the cases, HI assay has been the most reliable and a specific indicator of immunity against avian influenza viruses, including H5N1 subtype



Figure 2. Analysis of the XIAP suppressive effects on H5-induced apoptosis in cell culture using fluorescence microscopy and flow cytometry: (a) I: Assessment of transfection efficiency using Turbofect transfection reagent and 2 mg of plasmid encoding GFP fused to SP100 nuclear antigen in CHO cells. The image captured by fluorescence microscopy at 24 h after transfection represents the nuclear localization of GFP-SP100 fusion protein. II: CHO cells transfected with H5 for 24 h, were stained with annexin V-FITC and visualized by fluorescence microscopy. Annexin V-FITC binds to phosphatidylserine exposed on the outer membrane leaflet in the early and late apoptotic cells. (b) CHO cells were transfected with H5 and XIAP encoding constructs. After 24 h, cells were stained with annexin V-FITC and PI and analyzed by flow cytometry. The lower right quadrant (region 2; FITC+/PI-) represents the early apoptosis. The upper right quadrant (region 1; FITC+/PI+) represents the late apoptosis. Regions 1 and 2 together represent the total count of apoptotic cells. (c) Co-expression of XIAP or XIAP (DRING) significantly inhibited the H5induced apoptosis. Asterisks indicate the level of statistical significance (p 0.05). The middle horizontal line of the box indicates median of the data set. The whiskers represent the highest and lowest values.

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Figure 3. Dependence of the HI antibody responses to the secretory form of H5 antigen and XIAP co-expression. The HI assay is read as the endpoint dilution of serum that completely inhibits hemagglutination. Five individual serum samples were harvested at two time points of 3 weeks after the first immunization and 2 weeks after the booster immunization. Mice vaccinated with the empty vector as the control group failed to show detectable HI titers, while the H5 injected mice exhibited a significantly higher level of HI antibodies. XIAP co-administration further enhanced the antibody responses against the H5 antigen encoded by DNA vaccine. The highest level of HI titers was observed in the group injected twice with the secretory form of H5 and XIAP. The HI titers in the latter group were significantly higher than that in the group injected with only the wild-type H5 (p = 0.02). The Jonckheere-Terpstra analysis revealed a significantly increasing levels of HI titers from H5 to secretory H5, and from secretory H5 to secretory H5 plus XIAP groups (p = 0.01). The asterisk indicates the level of statistical significance (p50.05). The middle horizontal line of the boxes indicates median of the data set. The whiskers represent the highest and lowest values.

(Swayne & Kapczynski, 2008; Treanor & Wright, 2003). Thus, HI assay was used to measure antibody titers in this investigation. Twenty-five female BALB/c mice, at the age of 6–8 weeks, were divided into five groups and immunized twice with a 3-week interval. By combining XIAP molecular adjuvant and secretory form of H5 antigen, an increasing trend in antibody response observed after booster immunization (p50.05; Figure 3). Co-administration of XIAP enhanced HI antibody titers for both of the transmembrane and secretory forms of H5. Mice vaccinated with empty vector as control group failed to show detectable HI titers. All boosted mice showed higher titers after booster immunization (p50.05; Figure 3). The highest titers were recorded for the secretory form of H5-P284 co-administered with the plasmid encoding the XIAP protein. Using the Jonckheere–Terpstra test, a significant trend was observed in combination of XIAP and the secretory form of H5 after booster immunization (H5-P2845H5-P284+XIAP5sH5-P2845sH5-P284+XIAP, p50.05). Co-administration of XIAP with the secretory form of H5 enhanced



Figure 4. T-cell proliferation after DNA immunization. The splenocytes from all H5 vaccinated groups showed a significantly higher proliferation response than the empty vector vaccinated group. Co-administration of XIAP did not enhance T cell proliferation responses in H5 DNA vaccinated mice. A single suspension of splenocytes of mice immunized with the indicated DNA constructs was harvested two weeks after the booster immunization and stimulated in vitro with H5 antigen and PHA as a positive control. Splenocytes obtained from the empty vector vaccinated group of mice considered as negative control. The ratio of the spectrophotometric reading of antigen stimulated and non-stimulated subjects were considered as ‘‘Stimulation Index.’’ The middle horizontal line of the boxes indicates median of the data set. The whiskers represent the highest and lowest values. Outliers are represented by circles.

antibody responses compared to wild-type transmembrane form of H5 after booster immunization (p50.05). These results showed an enhancement of antibody response against both of transmembrane and secretory form H5 DNA vaccine when it was co-administered with a DNA construct encoding XIAP. Lymphocyte Proliferation Response Increased for H5 DNA Vaccinated Groups The splenocytes from all H5 vaccinated groups showed a significantly higher proliferation response than empty vector vaccinated group (p50.05; Figure 4). However, co-administration of XIAP did not enhance cell proliferation responses in H5 DNA vaccinated mice. The splenocytes from mice vaccinated with the transmembrane form (H5-P284) showed a higher proliferation rate than those obtained from mice vaccinated with the secretory form (sH5-P284) (p = 0.07; Figure 4).

DISCUSSION Here, we show that by using a DNA construct encoding XIAP, an anti-apoptotic protein and stimulator of the inflammatory pathway, it is possible to hinder

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pro-apoptotic effect of influenza A virus H5 protein in cell culture and enhance humoral immune responses in H5 DNA vaccinated mice. In this study, we found that H5 expression was able to induce cell death through apoptosis rather than necrosis (Figure 2). Although it is known that influenza virus infection induces caspase activation and subsequent apoptosis (Lowy, 2003; Ludwig et al., 2006), it is not clear which viral gene(s) are involved in the process of apoptotic cell death. It has been shown that ectopic expression of the nonstructural (NS) gene of influenza A virus is individually able to induce apoptosis in cultured cells (Lam et al., 2008; Schultz-Cherry et al., 2001; Zhang et al., 2010). Also, the PB1-F2 protein is characterized as a pro-apoptotic influenza A protein that induces apoptosis through mitochondrial pathway (Zamarin et al., 2005). The H5 expression increased the percentage of apoptotic cells during a 24-h period (p 0.05; Figure 2c), a mean percentage of 15.1% cells in the early stage of apoptosis. However, considering the transfection efficiency of about 60% (Figure 2a-I), it could be expected to find a higher percentage of apoptotic cells (Figure 2c). The variable level of endogenous XIAP might be the reason for the low level of observed apoptosis. In fact, a study performed on 60 tumor cell lines showed that the XIAP protein level is widely variable for different cell lines (Tamm et al., 2000). We also observed a noticeable level of endogenous XIAP protein in CHO cell line (Figure 1b, lanes 6-7), which might have an antiapoptotic effect (Figure 2c). A previous study showed that XIAP(DRING) was as efficient as full-length XIAP to inhibit Fas-induced apoptosis, and the fragment containing BIR1+2+3 domains of XIAP (XIAP(DRING) in the present study) was enough to inhibit caspases-3 and -7, whereas the RING domain containing fragment did not (Takahashi et al., 1998). In addition to XIAP (DRING), we also tested the anti-apoptotic effect of full-length XIAP and found a stronger anti-apoptotic effect for XIAP than XIAP (DRING) (p 0.05; Figure 2c). Considering the role of the RING domain of XIAP in augmentation of NF-iB activity (Hofer-Warbinek et al., 2000), it can be speculated that in addition to effector caspase inhibitory effect of BIR1+2+3 domains, RING domain mediated NF-iB activation is also required for a stronger inhibition of H5-induced apoptosis (Figure 2c). NF-iB is involved in the regulation of both pro- and anti-apoptotic activity. Ectopic expression of XIAP stimulates NF-iB-dependent expression of IAP gene family and leads to an amplification of anti-apoptotic response (HoferWarbinek et al., 2000). Avian influenza hemagglutinin activates NF-iB (Flory et al., 2000; Pahl & Baeuerle, 1995), but the activated NF-iB in the context of an influenza infection acts pro-apoptotically and induces expression of proapoptotic factors such as tumor necrosis factor related apoptosis-inducing ligand (TRAIL) and first apoptosis signal ligand (FasL)(Wurzer et al., 2004). Thus, although both hemagglutinin 5 and XIAP are able to induce NF-iB activation, but the consequence of this induction regarding apoptosis is different. We speculate that in the XIAP co-transfected cells, the stronger antiapoptotic effect of full-length XIAP than XIAP (DRING) could be related to a shift of the H5-induced NF-iB mediated pro-apoptotic response to a NF-iB dependent anti-apoptotic response. This observation shows that in addition to



caspase inhibitory effect of XIAP (DRING), RING domain mediated NF-iB stimulation is also required for a potent shift of pro-apoptotic to anti-apoptotic response. Because activation of NF-iB dependent pro-apoptotic pathway is required for efficient influenza virus propagation (Wurzer et al., 2004), exploiting molecules such as XIAP to shift the pro-apoptotic activity of NFiB to anti-apoptotic responses may have implications for antiviral therapies. Our findings obtained from in vitro apoptosis assay implicated that proapoptotic activity of a DNA vaccine encoded antigen might have a much faster effect than antigen specific adaptive immune responses in the clearance of antigen producing cells and decreasing the duration of antigen expression (Davis et al., 1997; Geiben-Lynn et al., 2008). These results motivated us further to study the effect of apoptosis inhibition of XIAP as a molecular adjuvant to hinder H5-induced apoptosis and investigate its effect on immune responses in vivo. For the mice immunization study, we used the DNA encoding full-length XIAP due to its strong anti-apoptotic activity against H5-induced apoptosis in cell culture (Figure 2c), and possession of RING domain, which is involved in NF-iB activation and proinflammatory cytokine production (Hofer-Warbinek et al., 2000; Lu et al., 2007). Statistical analysis showed a significantly increasing trend toward the concurrent use of XIAP and the secretory form of the H5 antigen (p50.05, Jonckheere–Terpstra test). Among four groups, the highest antibody titers were detected for the group containing the secretory form of H5 that was coadministered with XIAP (Figure 3). Co-administration of XIAP with the secretory form of antigen enhanced antibody responses compared to immunization with the wild-type transmembrane form (p50.05; Figure 3). Production of higher titers for the secretory H5 and XIAP co-administered mice could be related to the secretion of intact antigen from life-prolonged antigen producing cells and antigen availability in the draining lymph node to prime B cells and APCs. Co-administration of XIAP enhanced antibody responses for both of the transmembrane and the secretory forms of H5 might also be related to XIAP mediated inhibition of apoptosis in H5-producing myocytes and increasing the duration of antigen production. In support of these findings, previous studies have shown that an enhancement of the immune response occurs by prolonged survival of antigen-producing cells induced by co-delivery of anti-apoptotic genes (Kim et al., 2003, 2004). Kim and colleagues exploited intradermal co-delivery of DNA encoding different anti-apoptotic factors, including B-cell lymphomaextra large (BCL-xL), XIAP, B-cell lymphoma 2 (BCL-2), dominant negative caspase-8, or dominant negative caspase-9, and found that these factors are effective in prolonging the survival of transduced DCs and enhancement of antigen specific CD8+ T cell immune responses (Kim et al., 2003). Another study showed an enhanced antigen specific CD8+ T cell immune response, when DNA vaccine was co-administered with DNA encoding BCL-xL delivered intradermally via gene gun or intramuscularly via injection (Kim et al., 2004). According to the mentioned studies regarding the use of anti-apoptotic genes, this is the first study that employs XIAP encoding DNA via the intramuscular route to enhance the immune responses against influenza virus H5 antigen. In the present study, co-administration of XIAP enhanced antibody responses for both forms of antigen.

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However, it should be considered that in some cases, employing antiapoptotic molecular adjuvants might be ineffective or even have a detrimental effect on the immune responses. In a study where BCL-xL was used as a molecular adjuvant to enhance immune responses against a gene gundelivered DNA vaccine encoding the circum sporozoite protein (CSP) of Plasmodium berghei, it was shown that the molecular adjuvant could cause a shift in the Th profile of the immune response that is associated with a decrease in quantity and quality of the antigen-specific antibodies and reduced vaccine efficacy (Bergmann-Leitner et al., 2009). To assess cell-mediated immune responses, splenocytes were harvested 2 weeks after booster immunization and used for proliferation assay. Splenocyte proliferation rate significantly increased for H5 vaccinated groups after 48 h incubation with H5 antigen (Figure 4). We observed higher proliferation rate for the transmembrane form compared to the secretory form of H5 (p = 0.07), however, the XIAP co-administration was ineffective to change cell proliferation responses (Figure 4). It has been previously reported that the transmembrane and cytoplasmic domains of hemagglutinin contain a subpopulation of antigenic epitopes which are recognized by subtype-specific or cross-reactive hemagglutinin-specific CTL clones (Braciale et al., 1987; Kuwano et al., 1989). In the present study, we used a hemagglutinin from H5N2 subtype for stimulation of T cells in the proliferation assay. Splenocytes obtained from mice vaccinated with the secretory form of H5 may lack some of CTL clones that recognize epitopes within the transmembrane and cytoplasmic domains of H5. Thus, it could be speculated that the H5 antigen was also able to stimulate the cross-reactive CTL clones obtained from transmembrane H5 vaccinated mice, leading in higher T cell proliferation responses (p = 0.07). Although using the secretory form of H5 enhanced HI antibody responses probably due to availability of intact antigen for B cell priming, but deletion of the transmembrane and cytoplasmic domains may lead to the reduction of cell-mediated immune responses due to the loss of a subpopulation of T cell epitopes. Our findings suggest that for a given plasmid DNA vaccine, pro-apoptotic activity of encoded antigen may lead to the destruction of antigen producing cells and reduction of the duration of antigen production. Despite the effectiveness of the XIAP anti-apoptotic activity in the present study, it should be considered that the application of a molecular adjuvant should be evaluated in the context of each specific vaccine antigen. The effects of the molecular adjuvant for a given antigen might not be favorable for vaccine efficacy (Bergmann-Leitner et al., 2009; Pence et al., 2012; Xu et al., 2014). Our results on the anti-apoptotic effect of XIAP, enhancement of antibody responses, and the dependence of this response to the secretory form of antigen could have important implications for the design of new DNA vaccines.

ACKNOWLEDGEMENTS We thank M. Kohanghadr and A. Mahouti at the Faculty of Veterinary Medicine, Ferdowsi University of Mashhad for the technical assistance; Dr. H. A. Seifi at the Faculty of Veterinary Medicine, Ferdowsi University of Mashhad for statistical analysis; the Imaging Facility of the Faculty of


Sciences, Ferdowsi University of Mashhad; and the Imaging Facility of the Buali Research Institute, Mashhad University of Medical Sciences. The authors would like to thank Robert G. Korneluk at Apoptosis Research Centre, Children’s Hospital of Eastern Ontario Research Institute, Ottawa, Ontario, Canada for the generous gift of 6Myc-XIAP encoding plasmid. This work was supported by a grant (No. 56896) from Institute of Biotechnology, Ferdowsi University of Mashhad and Iran’s Headquarters for Development of Biotechnology to H.D.


DECLARATION OF INTEREST The authors declare that they have no conflict of interest. The authors alone are responsible for the content and writing of the paper.

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