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DOI: 10.1002/eji.201343983

Tamsin J. Garrod et al.

Eur. J. Immunol. 2014. 44: 1992–2002

DNA vaccines encoding membrane-bound or secreted forms of heat shock protein 70 exhibit improved potency Tamsin J. Garrod1 , Branka Grubor-Bauk1 , Tessa Gargett2 , Yanrui Li1 , Darren S. Miller2 , Wenbo Yu1 , Lee Major3 , Christopher J. Burrell4 , Steven Wesselingh5 , Andreas Suhrbier3 and Eric J. Gowans1 1

Department of Surgery, Virology Laboratory, Basil Hetzel Institute, University of Adelaide, Adelaide, Australia 2 Experimental Therapeutics Laboratory, University of South Australia, Adelaide, Australia 3 Inflammation Biology Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia 4 School of Molecular Biosciences, University of Adelaide, Adelaide, Australia 5 South Australian Health and Medical Research Institute, Adelaide, Australia Traditional vaccine strategies are inefficient against challenge with complex pathogens including HIV; therefore, novel vaccine technologies are required. DNA vaccines are attractive as they are relatively cheap and easy to manufacture, but a major limitation has been their lack of immunogenicity in humans, which may be overcome with the incorporation of an adjuvant. HSP70 is a recognised damage-associated molecular pattern, which is a potential adjuvant. We investigated the immunogenicity of a DNA vaccine encoding HIV gag and HSP70; the latter was genetically modified to produce cytoplasmic, secreted or membrane-bound HSP70, the expression of which was controlled by an independent promoter. The DNA was administered to C57BL/6 mice to evaluate gag-specific T-cell responses. Our results demonstrated the ability of membrane-bound and secreted HSP70 to significantly enhance gag-specific T-cell responses and increase the breadth of T-cell responses to include subdominant epitopes. Membrane-bound or secreted HSP70 also significantly improved the multifunctionality of HIV-specific T cells and T-cell proliferation, which is important for maintaining T-cell integrity. Most importantly, the inclusion of membrane-bound HSP70, secreted HSP70 or a combination significantly increased protection in mice challenged with EcoHIV, a chimeric virus that replicates in mouse leukocytes in vivo.

Keywords: Adjuvant r DNA vaccines



r

HSP70

Additional supporting information may be found in the online version of this article at the publisher’s web-site

Introduction

Correspondence: Prof. Eric J. Gowans e-mail: [email protected]  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Traditional vaccine strategies are unable to elicit protection against complex pathogens such as HIV and HCV [1, 2], and consequently, novel strategies are necessary. Current vaccine candidates include DNA vaccines, as they are stable, easy to manufacture and

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Eur. J. Immunol. 2014. 44: 1992–2002

can induce humoral and cell-mediated immune responses. Four DNA vaccines have been licensed for veterinary use, emphasising the potential of this approach [3]. In human clinical trials, DNA vaccines have demonstrated a good safety profile, but are limited by poor immune responses [4]. This limitation may be overcome with the addition of an adjuvant [5]. Damage-associated molecular patterns (DAMPs) are hostderived molecules that are released from cells as a result of stress, damage or necrosis [6]. Once released into the extracellular milieu, DAMPs bind to pattern-recognition receptors (PRRs) on dendritic cells (DCs) [7], resulting in DC activation and maturation. DC activation is important for upregulation of costimulatory molecule expression, migration to the draining LNs (DLNs) and T-cell activation [8]. DCs are crucial for vaccine efficacy due to their ability to cross-present exogenous protein via the MHC class I pathway and activate naive CD8+ T cells [9]. Molecules classified as DAMPs include HMGB1, uric acid and HSPs [10]. HSPs have been shown previously to be highly conserved [11]. HSP70 is essential for intracellular transport to maintain cell homeostasis and prevent aggregation of nascent or misfolded proteins [11]. When released from the cell, the inducible form of HSP70 can bind to TLR2 and TLR4 [12] on DCs, resulting in IL-12 expression that contributes to a Th1-type response [12] and upregulation of co-stimulatory molecules, including CD80/CD86, crucial for T-cell activation. Studies of protein vaccines containing HSP70 and DNA vaccines encoding HSP70 have focused predominantly on pathogen-derived HSP70, including Mycobacterium tuberculosis HSP70 (mtHSP70) fused to the human papillomavirus oncogene, E7 [13–15]. However, mtHSP70 is also immunogenic and thus may compete with the antigen encoded by the vaccine for the immune response. Therefore, the use of mammalian HSP70 is desirable, with mouse and human HSP70 used interchangeably due to their 98% amino acid similarity [16]. DNA vaccines that encode a tumour-associated antigen (TAA) together with either murine or human HSP70 have shown stronger T-cell responses to the TAA compared with the native DNA vaccine lacking HSP70, but failed to induce multifunctional T cells that are crucial for control of virus infections [17, 18]. Therefore, modifications to HSP70 may increase accessibility to DCs and induce strong T-cell responses, an aim of HIV vaccines [19], following the failure of antibodies alone to induce protection in clinical trials [20]. The aim of this study was to investigate the T-cell immunogenicity of a DNA vaccine encoding the HIV gag antigen and inducible human HSP70 that was genetically modified to produce V5-tagged cytoplasmic, membrane-bound or secreted forms of HSP70 and evaluate the protection afforded by the vaccine by challenging the mice with EcoHIV, a chimeric HIV that can infect mouse leukocytes.

Results Expression of different forms of HSP70 The inducible form of HSP70 is a recognised DAMP that binds to PRRs on DCs [21]. Therefore, we designed DNA constructs  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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encoding HIV gag and HSP70 controlled by independent promoters. This was achieved by inserting the SV40 promoter into pVAX1, to create a bicistronic plasmid, pJ, in which the upstream cistron is controlled by the CMV promoter and the downstream cistron by the SV40 promoter (Fig. 1A). We have previously demonstrated that the SV40 promoter is approximately tenfold weaker than the CMV promoter [22], consistent with previous studies [23]. Gag was expressed efficiently from all constructs (data not shown). The HSP70 gene was modified to result in expression of a cytoplasmic form of HSP70 containing a V5 tag [24], a membrane-bound HSP70 and a secreted form of HSP70. The V5-tagged HSP70 was encoded in pGag/V5-HSP70 (Fig. 1A) to identify vaccineexpressed HSP70 from constitutive HSP70. The addition of the V5 tag was shown previously to have no effect on HSP70 activity [24]. Intracellular expression of V5-tagged HSP70 was confirmed by immunoblotting (Fig. 1B). HSP70 was also modified to produce a secreted form of HSP70 from pGag/secrHSP70 and a membrane-bound form of HSP70 from pGag/memHSP70 (Fig. 1A). As constitutively expressed HSP70 is neither secreted nor expressed on the plasma membrane, the V5 tag was not required for detection of these forms of HSP70. The secreted form was detected in the supernatant of transfected cells and confirmed as a 72 kDa protein by immunoblotting (Fig. 1C), while the membrane-bound HSP70 was detected by immunofluorescence on the plasma membrane of non-permeabilised cells (Fig. 1D). These results clearly demonstrate that the different forms of HSP70 were detected in their predicted locations.

The magnitude of the gag-specific T-cell function is dependent on the cellular location of HSP70 Initially, a DNA titration was performed to identify the dose of pGag necessary to detect increased gag immunogenicity resulting from the inclusion of HSP70. Mice were vaccinated as described above with DNA doses ranging from 2 to 50 μg, and the splenocytes harvested 10 days later. IFN-γ EliSpot was performed to detect gag-specific T-cell responses as described in Materials and methods section. The results demonstrate that the 10, 25 and 50 μg doses all increased the frequency of IFN-γ-secreting T cells above that of pJ (Fig. 2A). However, the 10 μg dose produced fewer IFN-γ-secreting T cells and the tightest standard error of the mean (Fig. 2A) and consequently the 10 μg dose was chosen to evaluate the effect of encoding HSP70 in the vaccines. C57BL/6 mice were vaccinated three times at 2-week intervals with 10 μg of the different constructs and 10 days after vaccination, splenocytes were harvested and IFN-γ EliSpot was performed. Expression of the V5-tagged HSP70 from pGag/V5-HSP70 resulted in equivalent T-cell responses to that of untagged HSP70 in mice vaccinated with pGag/HSP70 (data not shown). Furthermore, mice vaccinated with pGag/V5-HSP70 showed no significant increase in the number of spot forming units (SFU) in all four gag peptide pools when compared to mice vaccinated with pGag (Fig. 2B). www.eji-journal.eu

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Figure 1. DNA vaccine constructs and protein expression. (A) Schematic of the bicistronic DNA vaccines used in this study. The DNA vaccine backbone, pJ, was used to develop the following DNA vaccines: Encoding HIV gag controlled by the CMV promoter; the different forms of HSP70 controlled by the SV40 promoter. The cytoplasmic form of HSP70 contains a V5 tag to allow protein detection, the secreted form contains the signal peptide from tissue plasminogen activator and the membrane-bound form of HSP70 contains the signal peptide and transmembrane domain from human transferrin receptor. (B) Cell lysates from HEK 293T cells transfected with DNA encoding the V5-tagged HSP70 were examined by Western blot and probed with anti-V5 antibody, with β-actin as a loading control. Blot is representative of three independent experiments performed. (C) Cell culture supernatants from HEK 293T cells transfected with DNA encoding the secreted form of HSP70 were examined by Western blot and probed with anti-HSP70 antibody. Protein loading for secreted HPS70 was controlled using a bicinchoninic acid assay. Blot is representative of two independent experiments. (D) Immunofluorescence staining with antiHSP70 was performed on non-permeabilised, DNA-transfected HEK 293T cells to identify membrane-bound HSP70, and DAPI to identify the cell nucleus. Images are representative of three independent experiments performed.

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Eur. J. Immunol. 2014. 44: 1992–2002

Figure 2. The frequency of gag-specific IFN-γ-secreting T cells in mice vaccinated with DNA, determined by IFN-γ EliSpot. C57BL/6 mice were vaccinated (intradermal (i.d.)) three times with 10 μg DNA at 2-week intervals. Gag-specific T-cell responses were measured by IFN-γ production using EliSpot. Splenocytes from vaccinated animals were restimulated with four pools of overlapping peptides covering the complete gag protein. The results from mice vaccinated with (A) different doses of pGag DNA and (B) 10 μg doses of pJ, pGag, pGag/V5-HSP70, pGag/secrHSP70 or pGag/memHSP70 are shown. Data are shown as mean ± SEM of SFU per 106 splenocytes and each symbol represents an individual animal. n = 5–7 pooled from two separate experiments. *p < 0.05, **p < 0.01 (Mann–Whitney test).

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In contrast, mice vaccinated with pGag/secrHSP70 or pGag/memHSP70 demonstrated statistically significant increase in IFN-γ-secreting T cells compared with mice vaccinated with pGag (Fig. 2B). Interestingly, these results were observed in mice vaccinated with either of the above constructs after stimulation of splenocytes with different peptide pools. Mice vaccinated with pGag/memHSP70 demonstrated a significant increase in the levels of IFN-γ-secreting T cells in pools 3 and 4 (Fig. 2B), while mice vaccinated with pGag/secrHSP70 demonstrated significant increases in T-cell responses to pools 1 and 4 (Fig. 2B). A majority of the immunodominant peptides reside in pool 3. Thus, vaccination with pGag/memHSP70 led to an improved response to immunodominant and subdominant peptides, and vaccination with pGag/secrHSP70 only improved T-cell responses to subdominant peptide pools.

Secreted and membrane-bound forms of HSP70 increase gag-specific responses in vitro The above data demonstrated an increase in T-cell functionality in vivo with the inclusion of genes encoding membranebound or secreted HSP70 but not V5-tagged cytoplasmic HSP70. Therefore, to examine the mechanism by which these forms of HSP70 increased T-cell responses, naive bone marrow derived DCs (BM-DCs) were incubated with NIH 3T3 cells transfected with pGag, pGag/V5-HSP70, pGag/secrHSP70 or pGag/memHSP70, and then used to activate CD8+ T cells from vaccinated mice. The ability of the BM-DCs to engulf and cross-present gag to restimulate gag-specific CD8+ T cells was then analysed by IFN-γ EliSpot (Fig. 3A). IFN-γ was not secreted from CD8+ T cells incubated with transfected NIH 3T3 cells only (Fig. 3B). In contrast, stimulation of the gag-specific CD8+ T cells for 12 h resulted in a significant increase in the SFU after stimulation with BM-DCs previously incubated with 3T3 cells transfected with pGag/secrHSP70 or pGag/memHSP70, compared with pGag or pGag/V5-HSP70 (Fig. 3B). Interestingly, at 24 h, BM-DC previously exposed to the pGag/memHSP70-transfected 3T3 cells were able to stimulate the CD8+ T cells and this also resulted in a significant increase in the SFU; although there was an increased trend with pGag/secrHSP70-transfected 3T3 cells, this was not significant (Fig. 3B). Nevertheless, it is clear that the secreted and membranous forms of HSP70 increased the cross-presentation abilities of BM-DCs. Since the pGag/V5-HSP70 construct showed no increased activity compared with pGag, no further work was performed with it.

Expression of membrane-bound and secreted HSP70 increases the frequency of multifunctional T cells Long-term non-progressors (LTNPs) are defined as HIV-infected individuals who remain asymptomatic with a CD4+ T cell count of over 500 cells per millilitre. This cohort is critical in evaluating potential correlates of HIV protection, as no individual  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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has naturally recovered from HIV infection. An increased frequency of multifunctional CD4+ and CD8+ T cells expressing IL-2, TNF-α and IFN-γ has been detected in LTNPs compared with other HIV-positive individuals and this may represent a potential correlate of protection against HIV infection [25, 26]. The immune responses described above after vaccination with pGag/memHSP70 or pGag/secrHSP70 highlighted the potential of modified HSP70 as an adjuvant. Therefore, we examined the frequency of multifunctional T cells able to secrete IL-2, TNF-α and IFN-γ induced by these DNA vaccines. Splenocytes from vaccinated mice were harvested 10 days post-vaccination and re-stimulated with a peptide pool containing the immunodominant HIV gag peptides, then stained for intracellular cytokines IL-2, TNF-α and IFN-γ, and surface markers CD3, CD44, CD4 and CD8, and examined by flow cytometry. Splenocytes from mice vaccinated with pGag/memHSP70 or pGag/secrHSP70 showed significantly increased frequencies of IFN-γ-producing CD4+ T cells and a trend for increased frequency of IFN-γ-producing CD8+ T cells compared with mice vaccinated with pGag (Fig. 4A). Furthermore, the pGag/memHSP70 vaccinated mice showed significantly higher frequencies of CD8+ and CD4+ TNF-α-producing T cells (Fig. 4B), while mice vaccinated with pGag/secrHSP70 showed significantly increased frequencies of double cytokine (TNF-α/IL-2) producing T cells compared with mice vaccinated with pGag (Fig. 4C). A significant increase was also observed in triple cytokine-producing CD4+ T cells following vaccination with pGag/secrHSP70 or pGag/memHSP70 relative to vaccination with pGag (Fig. 4D). Collectively, these results demonstrate improved T-cell multifunctionality in mice vaccinated with pGag/memHSP70 or pGag/secrHSP70 when compared with mice vaccinated with pGag.

Inclusion of membrane-bound or secreted HSP70 significantly increase T-cell proliferation Strong T-cell proliferation has been correlated with HIV LTNPs, and progression to AIDS is associated with decreased proliferation of CD8+ T cells [27]. Therefore, robust T-cell proliferation is an aim for any HIV vaccine strategy [28]. Consequently, to examine this parameter after vaccination, an ex vivo CFSE proliferation assay was performed on splenocytes from vaccinated mice that were re-stimulated with immunodominant HIV gag peptides to evaluate the proliferation of CD4+ and CD8+ T cells. Mice vaccinated with pGag/memHSP70 showed a significant increase in CD8+ T-cell proliferation (Fig. 5A), a vaccination outcome essential for effector T-cell function. CD4+ T cells from mice vaccinated with pGag/secrHSP70 or pGag/memHSP70 showed significantly increased levels of proliferation compared with CD4+ T cells from pGag-vaccinated mice (Fig. 5B). As CD4+ T cells are important for amplification of CD8+ T-cell clones and to maintain the function of memory CD8+ T cells [29], these results provide additional support for the inclusion of HSP70 in DNA vaccines. www.eji-journal.eu

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Figure 3. The secreted and membranous forms of HSP70 increase the cross-presentation abilities of BM-DCs. NIH 3T3 cells were transfected with pGag, pGag/V5-HSP70, pGag/secrHSP70 or pGag/memHSP70 and apoptosis induced by the addition of 3 μM doxorubicin for 16 h. BM-DCs were incubated with the apoptotic, transfected 3T3 cells for 36 h and subsequently incubated with CD8+ T cells harvested from pGag-vaccinated C57BL/6 mice, and CD8+ T cell activation was determined by IFN-γ EliSpot for 12 or 24 h. (A) Schematic of the strategy used to examine the cross-presentation of gag. (B) IFN-γ EliSpot results used to determine the different levels of cross-presentation of gag to gag-specific T cells. Results show mean ± SEM from seven replicates per group pooled from two separate experiments. *p < 0.05 (Mann–Whitney non-parametric test).

Higher DC activation and T-cell frequency in DLNs contribute to improved responses after vaccination To analyse the mechanism by which the T-cell responses are improved by vaccination with pGag/secrHSP70 or pGag/memHSP70, we investigated DC activation in the DLN. DC activation is crucial to generate an effective vaccine-induced immune response. The levels of DC activation and T-cell frequency were evaluated by harvesting the parotid LNs at baseline and day 7 post-intradermal (i.d.) vaccination with pGag/secrHSP70,

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pGag/memHSP70 or pGag. The activation status of LN-resident DCs (CD3− CD11c+ ) was analysed by CD86 expression. C57BL/6 mice vaccinated with pGag/secrHSP70 or pGag/memHSP70 demonstrated a significant increase in the frequency of activated CD3− CD11c+ CD86+ DCs compared with that induced by vaccination with pGag (Fig. 6A). Furthermore, there was a significant increase in the frequency of T cells after vaccination with pGag/memHSP70 and a trend toward increased frequency in pGag/secrHSP70-vaccinated mice (Fig. 6B). These results indicate that the mechanism of HSP70-induced immunogenicity may

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Figure 5. Ex vivo T-cell proliferation in vaccinated mice. Proliferation of CD4+ and CD8+ T cells harvested from splenocytes of mice vaccinated with the indicated constructs was assessed by CFSE staining and then analysed by flow cytometry by gating on the lymphocyte population, then CD3+ and finally CD4+ or CD8+ populations. The graphs demonstrate the percent proliferation of (A) CD8+ T cells and (B) CD4+ T cells. Each symbol represents an individual mouse and data show mean ± SEM of eight samples from two pooled experiments. *p < 0.05 (Mann–Whitney test).

Figure 6. DC activation and T-cell frequency in the draining LNs postvaccination. Superficial cervical lymph nodes from mice vaccinated with the indicated constructs were excised at baseline or 7 days post single vaccination. Cells were analysed by flow cytometry and gated on CD3, CD11c and CD86, and show (A) DC activation (as a percentage of activated CD3− CD11c+ CD86+ DCs) and (B) T-cell frequency. Data show mean ± SEM from three samples from one experiment. *p < 0.05 (Student’s t-test). Figure 4. Cytokine profile of the gag-specific CD4+ and CD8+ T cells in C57BL/6 mice. Splenocytes from mice vaccinated with the indicated constructs were harvested 10 days post-vaccination. Intracellular cytokine staining was performed by flow cytometry and cells were gated on CD3, CD44 and then CD4 or CD8 to assess the frequency of (A) IFN-γ-positive CD4+ /CD8+ T cells, (B) TNF-α-positive CD4+ /CD8+ T cells, (C) TNF-α/IL-2 double-positive CD4+ /CD8+ T cells and (D) IFN-γ/TNF-α/IL-2 triple-positive CD4+ /CD8+ T cells in vaccinated mice, with pJ-vaccinated mice used as the control. Each symbol represents an individual mouse and data show mean ± SEM of eight samples pooled from two experiments. *p < 0.05 (Mann–Whitney test).

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be attributed to increased DC activation and improved T-cell influx and priming in the LNs draining the site of injection.

Including membrane-bound or secreted HSP70 in a vaccine improves protection against EcoHIV infection Although EliSpot is acknowledged as a suitable measure of cell-mediated immunity, protection against viral challenge represents the most appropriate manner to measure protective immunity. EcoHIV, in which the gp120 env protein is replaced with gp80 from the murine leukaemia virus (MLV) glycoprotein [30], was used to challenge mice vaccinated with pGag or www.eji-journal.eu

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Discussion

Figure 7. Protection after EcoHIV challenge. Ten days after vaccination with the indicated constructs, mice were challenged with EcoHIV or mock challenged. Seven days later, PECs were harvested and the EcoHIV viral load was assessed by qPCR relative to RPL13a mRNA levels. Each symbol represents an individual mouse and data show mean ± SEM of six samples pooled from two experiments. Data in panels (A) and (B) were generated in independent experiments. *p < 0.05 (Mann–Whitney test).

pGag/memHSP70 (Fig. 7A) or pGag/secrHSP70 or a combination of pGag/secrHSP70 and pGag/memHSP70 (Fig. 7B). The peritoneal cavity was the primary site of infection, and consequently, peritoneal exudate cell (PECs) were collected 7 days post-infection and quantitative real-time PCR was performed to measure the levels of EcoHIV RNA relative to ribosomal protein L13A (RPL13a) as described [22]. The results demonstrated a significant decrease in the levels of EcoHIV RNA in the PEC of mice vaccinated with pGag/memHSP70, pGag/secrHSP70 or the combination compared with the pGag-vaccinated mice (Fig. 7A and B), and thus showed increased protection from EcoHIV challenge.  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

In this study, we demonstrated that the inclusion of novel genes encoding membrane-bound or the secreted form of HSP70 in a DNA vaccine encoding HIV gag significantly improved the frequency of gag-specific T cells, which secreted IFN-γ, as well as T-cell multifunctionality and proliferation. Furthermore, we showed that the mechanism of enhanced vaccine-induced immunogenicity was attributed to an increase in cross-presentation of exogenous gag by DCs, as well as an increase in DC activation and T-cell frequency within the DLNs. Most importantly, protection against challenge with EcoHIV was significantly higher in mice which were vaccinated with DNA encoding either membranebound or secreted HSP70 compared with mice vaccinated with gag DNA alone. Expression of the gag protein and the different forms of HSP70 were controlled by separate promoters in the same plasmid construct, ensuring that HSP70-enhanced immune responses were specific to gag-positive cells. The mice were vaccinated via the i.d. route as this has been shown to be more efficacious for vaccine-induced immune responses compared with the intramuscular route [31]. Furthermore, we chose to vaccinate the mice with a low dose of DNA compared with other studies [32, 33]. This lowdose vaccination regimen resulted in low levels of SFU by EliSpot and enhanced the likelihood of detecting significant increases in the frequency of IFN-γ-secreting T cells by the addition of secreted or membrane-bound HSP70. Moreover, this low-dose vaccination regimen more closely mimics the poor immunogenicity shown in DNA vaccine clinical trials [34, 35]. Gag-specific antibody titres were examined in mice vaccinated with pGag, pGag/secrHSP70 or pGag/memHSP70, and resulted in similar titres to the mockvaccinated mice (data not shown). This may have resulted from the low DNA dose and is consistent with a previous study [36]. Previous studies of HSP70 incorporated into a vaccine strategy focused predominantly on cancer DNA vaccines, which encoded pathogen-derived HSP70 fused to a TAA expressed in the cytoplasm [14, 37] or modified to be secreted as a fusion protein [38]. However, the use of human HSP70 rather than pathogen-derived HSP70 is likely to be an advantage as pathogen-derived HSP70 may compete with the target antigen for the immune response. A direct comparison between DNA vaccines that encoded mtHSP70 and the human form of HSP70 in a cancer vaccine demonstrated a stronger CD8+ T-cell response in mice vaccinated with the human form [18]. In our DNA vaccines, HSP70 was modified to produce a V5tagged cytoplasmic HSP70, membrane-bound HSP70 or secreted HSP70. Mice vaccinated with the DNA construct encoding V5cytoplasmic HSP70 demonstrated no significant increase in the frequency of gag-specific T cells. However, mice vaccinated with DNA that encoded the membrane-bound or the secreted forms of HSP70 showed significant increases in the frequency of IFN-γsecreting T cells and increased responses to subdominant epitopes. This broadening of the immune response may be beneficial for protection against HIV challenge and is therefore an aim of HIV vaccines [39]. An increase in immune responses targeting www.eji-journal.eu

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subdominant peptides has been previously shown to rely on the increased stability of the normally short-lived peptide-MHC I complex [40]. Therefore, in addition to its role as a DAMP, which facilitated the uptake of antigenic material by DCs, it is possible that HSP70, known to aid in MHC presentation of peptides, may also increase the stability of subdominant peptide MHC complexes and thus increase their presentation to naive T cells [41, 42]. Furthermore, the inclusion of secretion and membrane-bound HSP70 significantly increased T-cell multifunctionality, and it is thought to be crucial for HIV protection as LTNPs have higher levels of multifunctional T cells than individuals who develop AIDS [25, 26]. Significant increases in T-cell proliferation were also detected in mice vaccinated with these constructs compared with mice vaccinated with gag alone. Our results suggest that the membrane-bound and secreted forms of HSP70 were more accessible to receptors on DCs, facilitated binding to TLR2 and TLR4 and increased co-stimulatory molecule expression on DCs, as shown by increased frequency of activated CD11c+ CD86+ DCs in vaccinated mice. This, in turn, facilitated the recruitment of T cells to LNs, and this was shown to be increased after vaccination with constructs encoding membrane-bound or secreted HSP70. Most importantly, our results demonstrate a significant decrease in the levels of EcoHIV RNA after challenge when membrane-bound HSP70, secreted HSP70 or a combination were included in the vaccine. Therefore, the inclusion of these novel forms of HSP70 may increase the efficacy of DNA vaccines in large animals and opens up the possibility of the use of human HSP70 as an effective adjuvant in DNA clinical trials.

Materials and methods Plasmid constructs All constructs were based on pVAX1 (Life Technologies) and are shown schematically in Figure 1A. The SV40 promoter and polyadenylation sequence and a novel multiple cloning site were inserted into pVAX1 to generate a novel plasmid, pJ. The primer and synthetic oligonucleotide sequences used to generate the DNA vaccines are shown in Supporting Information Table 1. The codonoptimised clade B HIV gag gene was inserted downstream of the CMV promoter using the restriction enzyme sites NheI and EcoRI to create pGag. The human HSP70 gene (HSPA1A from Addgene, plasmid 15215) [43] was amplified by PCR and inserted downstream of the SV40 promoter using restriction enzyme sites MfeI and PacI. To generate pGag/V5-HSP70, the V5 tag [24] was produced using synthetic oligonucleotides and inserted at the 5 end of the HSP70 gene using the AscI and MfeI restriction enzyme sites. pGag/secrHSP70 was constructed by inserting the signal sequence from tissue plasminogen activator (a gift from A/Prof Heidi Drummer), at the 5 end of the HSP70 gene using the restriction sites AscI and MfeI. pGag/memHSP70 was produced by PCR amplification of the signal and transmembrane domain  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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of the human transferrin receptor [44] (a gift from Dr. John Martyn) and inserted directly upstream of HSP70 using the AscI and MfeI restriction enzyme sites. All DNA constructs were verified by restriction enzyme digestion and DNA sequencing.

HSP70 protein detection To analyse HSP70 protein expression, HEK 293T cells were transfected with DNA using FuGENE 6 (Promega) following the manufacturer’s instructions. The cells were then incubated at 37°C in 5% CO2 for 3 days and either the cell supernatant or whole-cell lysates were harvested. Immunoblotting was performed to examine the expression of V5-tagged HSP70 using an anti-V5 antibody (Life Technologies), and secreted HSP70 protein was detected using an anti-HSP70 antibody (BD Biosciences). The signals were visualised using chemiluminescence. Immunofluorescence was performed to detect membrane-bound HSP70 using a primary anti-HSP70 antibody (BD Biosciences) and a FITC-conjugated secondary antibody (Millipore), and counterstained with DAPI (Invitrogen).

Animals and immunisation Six- to eight-week old female C57BL/6 mice were obtained from the University of Adelaide, Laboratory Animal Services. The mice were housed in the Women’s and Children’s Hospital animal house and used in accordance with the Women’s and Children’s Hospital Health Network and the University Animal Ethics Committee guidelines. Plasmid DNA was prepared using endotoxin-free MegaPrep kits (Qiagen). Mice were injected with DNA in PBS in a final volume of 50 μL via the i.d. route in the ear pinna, as we described [22], consisting of a single dose or three doses at 2-week intervals.

Cell preparation Spleens were harvested 10 days after vaccination, and cervical LNs were isolated at baseline or 7 days after the vaccination. The LNs and spleen were teased through a 70 μm cell strainer to create a single-cell suspension. Splenocytes were treated with ACK lysis buffer to remove erythrocytes. All cells were washed and resuspended in R10 media (RPMI 1640 media containing 10% FBS, 2 mM L-glutamine, 50 μM β-mercaptoethanol, 1 mM sodium pyruvate, 10 mM HEPES buffer, pen/strep).

In vitro cross-presentation assay NIH 3T3 cells were transfected with pGag, pGag/V5-HSP70, pGag/secrHSP70 or pGag/memHSP70 using lipofectamine LTX following the manufacturer’s instructions (Life Technologies), and then incubated for 56 h. Doxorubicin (3 μM) was added for an additional 16 h to induce apoptosis. BM-DCs were generated from www.eji-journal.eu

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BM harvested from naive C57BL/6 mice by culture in media containing murine GM-CSF for 8 days [45]. BM-DCs and transfected NIH 3T3 cells were co-cultured at a ratio of 1:1 (2 × 104 cells each) for 30 h. Six-week old mice were vaccinated as described above with 50 μg pGag, and CD8+ T cells were isolated 10 days later by MACS purification (Miltenyi Biotec). The isolated cells were rested overnight then incubated with or without BM-DCs and with 3T3 cells in an EliSpot plate at a DC:T cell ratio of 1:10 for 12 or 24 h. The IFN-γ EliSpot was performed as described below, but without peptide stimulation.

IFN-γ EliSpot assay The IFN-γ EliSpot has been described previously [22]. Briefly, multiscreen-IP filter plates (Millipore) were coated with anti-IFN-γ antibodies (clone AN18, Mabtech) (5 μg/mL) and 5 × 105 splenocytes were stimulated with 4 μg/mL HIV gag peptide pools for 40 h at 37°C. The gag peptides were divided into four pools, each containing 29–31 individual peptides (Supporting Information Table 2). Secreted IFN-γ was detected with anti-mouse IFN-γ-biotin (clone R4–6A2, MabTech), streptavidin-AP (Sigma) and SigmaFast BCIP/NBT. PHA was used as a positive control and unstimulated splenocytes were used, cultured in R10 media, as a negative control. The spots were counted automatically using an ELISPOT reader and adjusted to 106 cells (AID-EliSpot reader system).

Flow cytometry The frequency of gag-specific CD8+ and CD4+ T cells was determined by intracellular cytokine staining. Briefly, 106 cells were stimulated for 12 h with 4 μg/mL C57BL/6 immunodominant gag epitopes (http://www.hiv.lanl.gov/content/index) in the presence of GolgiStop (BD Biosciences). All antibodies were purchased from BD Biosciences unless otherwise stated. The cells were then stained for cell surface markers CD4-eFluor450 (eBioscience), CD3-PerCP-Cy5.5, CD44-allophycocyanin and CD8allophycocyanin-Cy7 and for intracellular cytokines IL-2-FITC, TNF-α-PE and IFN-γ-PE-Cy7. Samples were run on a BD FACSCanto and analysed using BD FACS Diva software. The unstimulated control reading for each mouse was subtracted from the reading in the peptide-stimulated samples. To assess DC activation and T-cell frequency, LN lymphocytes were stained with antibodies for CD3-PerCP-Cy5.5, CD11c-PE-Cy7 and CD86-PE.

T-cell proliferation assay Proliferation of CD4+ and CD8+ T cells was assessed by flow cytometry after CFSE staining. Briefly, splenocytes from vaccinated mice were labelled with 10 μM CFSE following the CellTrace  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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CFSE Cell Proliferation Kit protocol (Life Technologies) and stimulated in vitro with 4 μg/mL immunodominant gag peptides or left unstimulated. After 4 days, CFSE-labelled splenocytes were surface stained with CD3-PerCP-Cy5.5, CD4-eFluor450 (eBioscience) and CD8-APC-Cy7, and analysed by flow cytometry.

EcoHIV Challenge The production of EcoHIV has been described previously [30]. Briefly, HEK 293T cells were transfected with EcoHIV NL4–3 plasmid DNA and incubated for 48 h. The supernatant was collected and cell-free virus was concentrated with a 10 000 MW cutoff filter (Millipore). The EcoHIV titre was determined indirectly by analysis of the p24 concentration using the p24 ELISA kit (Zeptometrix). Mice challenged with EcoHIV were previously vaccinated as described above and challenged 10 days post-vaccination with 1.5 μg p24 EcoHIV/NL4–3 cell-free virus via the intraperitoneal route as described [30]. Negative control mice were similarly vaccinated with 10 μg pGag and injected with mock-purified EcoHIV. The mice were culled 7 days post-EcoHIV challenge and peritoneal washes performed with R10 media to collect PECs. The PECs were pelleted, resuspended in Trizol (Life Technologies) and total RNA was isolated following the manufacturer’s instructions. The RNA was converted to cDNA using the QuantiTect reagents (Qiagen) and quantitative real-time PCR was performed using QuantiFast (Qiagen) to measure the levels of EcoHIV RNA. The primers for the PCR were MLV fwd 5 -TAGGG CCAAACCCCGTTCTG-3 and MLV rev 5 -GCCGGTGGAAGTTGGG TAGG-3 . The levels of EcoHIV RNA were normalised to levels of RPL13a (RPL13a fwd 5 -GAGGTCGGGTGGAAGTACCA-3 and RPL13a rev 5 -TGCATCTTGGCCTTTTCCTT-3 ) and were measured after calculating primer efficiency within the parameters of the CT (threshold cycle) analysis. Results were expressed as the mean normalised EcoHIV RNA expression relative to RPL13a mRNA.

Statistics The results are graphed as the mean ± SEM. Statistical analysis used the unpaired two-tailed Student’s t-test or the unpaired two-tailed non-parametric Mann–Whitney test, with p < 0.05 considered significant.

Acknowledgements: We thank the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH for the HIV-1 Con B gag peptide pools–Complete Set. We also thank Associate Professor Heidi Drummer, Dr. John Martyn and Dr. Ehud Hauben for advice and materials, and Dr. Stuart Howell for advice on www.eji-journal.eu

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statistical analysis. This work was supported by grants 543143 and APP1026293 from the National Health and Medical Research Council (NHMRC) of Australia and by grant number BF040005 from the Australia-India Biotechnology Fund awarded to E.J.G., who is a Senior Research Fellow of the NHMRC.

Immunity to infection

17 Choi, D. H., Woo, J. K., Choi, Y., Seo, H. S. and Kim, C. W., A novel chimeric DNA vaccine: enhancement of preventive and therapeutic efficacy of DNA vaccine by fusion of Mucin 1 to a heat shock protein 70 gene. Mol. Med. Rep. 2011. 4: 885–890. 18 Zong, J., Peng, Q., Wang, Q., Zhang, T., Fan, D. and Xu, X., Human HSP70 and modified HPV16 E7 fusion DNA vaccine induces enhanced specific CD8+ T cell responses and anti-tumor effects. Oncol. Rep. 2009. 22: 953– 961.

Conflict of interest: The authors declare no financial or commercial conflict of interest.

19 Korber, B. T., Letvin, N. L. and Haynes, B. F., T-cell vaccine strategies for human immunodeficiency virus, the virus with a thousand faces. J. Virol. 2009. 83: 8300–8314.

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Received: 9/8/2013 Revised: 17/2/2014 Accepted: 31/3/2014 Accepted article online: 10/4/2014

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DNA vaccines encoding membrane-bound or secreted forms of heat shock protein 70 exhibit improved potency.

Traditional vaccine strategies are inefficient against challenge with complex pathogens including HIV; therefore, novel vaccine technologies are requi...
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