A recombinant human HLA-class I antigen linked to dextran elicits innate and adaptive immune responses

Journal of Immunological Methods 360 (2010) 1–9

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Journal of Immunological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i m

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A recombinant human HLA-class I antigen linked to dextran elicits innate and adaptive immune responses Jorgen Schøller a, Mahavir Singh b, Lesley Bergmeier c, Katja Brunstedt a, Yufei Wang c, Trevor Whittall c, Durdana Rahman c, J. Pido-Lopez c, T. Lehner c,⁎ a b c

Immudex, Copenhagen, Denmark Lionex GmbH and Helmholtx Center for Infection Research, 38124 Braunschweig, Germany Kings College London, Mucosal Immunology Unit, Guy's Hospital, London, England, United Kingdom

a r t i c l e

i n f o

Article history: Received 23 February 2010 Received in revised form 18 May 2010 Accepted 25 May 2010 Available online 10 June 2010 Keywords: HSP Dendritic cells HIV SIV

a b s t r a c t The objective of this study was to produce and evaluate the immunogenic potential of a recombinant HLA-class I antigen linked to dextran. The HLA-A*0201 heavy chain and β2 microglobulin were cloned by PCR amplification of overlapping oligonucleotides and produced in E. coli. These were assembled with a CMV binding peptide motif, the HLA complex was biotinylated and bound by streptavidin coated dextran at a ratio of 24 HLA to 1 dextran molecule (termed Dextramer). Allostimulation of human PBMC in vitro and in vivo immunization of Balb c mice with the HLA-A*0201 construct elicited CD4+ and CD8+ T cell proliferative responses, IgG specific antibodies in mice and in human T cell proliferation and APOBEC3G mRNA. These adaptive and innate immune responses induced by a novel recombinant HLA construct in human cells and mice suggest their application as a potential vaccine candidate against HIV infection. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Cloning and production of HLA-A*0201 heavy chain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Cloning and production of human β2 microglobulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Assembling the HLA complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Construction of expression vectors and production of biotinylated C-terminal peptide of M. tuberculosis HSP70 (HSP70359–608) 2.5. Preparation of the Dextramer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Immunization of mice with the Dextramer and HIVgp120 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Mouse T cell proliferative assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8. Antibody analysis by ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.9. Human T cell proliferative assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.10. APOBEC3G mRNA assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: A3G, APOBEC3G;CMV, cytomegalovirus;DC, dendritic cells;FITC, fluorescein isothiocyanate;HIV, human immunodeficiency virus;HLA, human leukocyte antigens;SIV, simian immunodeficiency virus;HSP, heat shock protein;TH1, T helper 1. ⁎ Corresponding author. Kings College London, Mucosal Immunology Unit, Tower Wing Floor 28, Guy's Hospital, London SE1 9RT, England, United Kingdom. Tel.: +44 207188 3072; fax: +44 207188 4375. E-mail address: [email protected] (T. Lehner). 0022-1759/$ – see front matter. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2010.05.008

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3. 4.

Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Stimulation of human T cell proliferation with the Dextramer . 4.2. Stimulation of A3GmRNA. . . . . . . . . . . . . . . . . . 4.3. Splenic T cell proliferative responses in mice immunized with 4.4. IgG antibodies to HIVgp120 and to HLA-A*02 Dextramer . . . 5. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Immunization of macaques with SIV grown in human CD4+ T cells (Desrosiers et al., 1989; Murphey-Corb et al., 1989; Stott et al., 1990; Carlson et al., 1990; Hunsmann et al., 1995; Biberfeld and Putkonen, 1995) or with the cells alone (Stott, 1991; Stott et al., 1994; Arthur et al., 1995; Chan et al., 1995) yielded about 85% and 55% protection against SIV infection, respectively. The protection was dependent on HLA antigens acquired by the virions in the process of budding through the human CD4+ T cell membrane in which the SIV was grown. Despite the reproducibility of preventing SIV infection in macaques, this approach was abandoned largely because of the potential adverse effects of immunization with HLA+ cells, and using CD4+ T cell lines propagated with oncogenic viruses. There is also evidence in humans that alloimmune responses can prevent HIV-1 infection. This was demonstrated in vitro by inducing cytotoxic lymphocytes and soluble factors (Shearer et al., 1993). Systemic in vivo alloimmunization of women revealed that HIV-1 replication in CD4+ T cells ex vivo is inhibited (Wang et al., 1999). Epidemiological evidence suggests that transmission of HIV from mother to baby occurs more frequently among uniparous women and mother–child HLA-class I concordance increases perinatal HIV-1 transmission (McDonald et al., 1998). Furthermore, sera from multiparous women may contain alloantibodies and CCR5 antibodies which inhibit ex vivo HIV-1 replication (Wang et al., 2002a). Indeed, alloimmunization has been proposed as a strategy for inducing immune protection against HIV infection (Lehner et al., 2000). The objective of this study was to evaluate the immunogenic potential of recombinant HLA-class I antigen (A*0201) linked by the biotin–streptavidin method to dextran (Dextramer) and to use HSP70 as an adjuvant to elicit immune responses. Immunization of Balb c mice with the HLA-A*0201 Dextramer construct elicited an increase in both CD4+ and CD8+ T cell proliferation and IgG antibodies to the HLAA*0201 dextramer. Human CD4+ and CD8+ T cell proliferative responses and an innate immune response were induced in vitro by the Dextramer stimulating upregulation of APOBEC3G mRNA in human CD4+ T cells. 2. Materials and methods 2.1. Cloning and production of HLA-A*0201 heavy chain The human MHC-class 1 HLA-A*0201 coding sequence minus the signal peptide and transmembrane regions was

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. . . . . . . . . . . . . . . . . . . . HIVgp120 . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dextramer construct . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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obtained from GenBank (acc # M84379) by backtranslation. Codon usage was optimized to E. coli using www.entelechon. com before the gene was synthesized by PCR using 10 overlapping DNA primers (DNA Technology, Denmark) and KOD polymerase (EMD Chemicals, Novagen). The sequence was verified by repeated DNA sequencing (MWG Biotech, Ebersberg, Germany) and base errors introduced by the PCR were corrected using Quick Change multi site-directed mutagenesis kit (Stratagene, La Jolla, CA) before cloning in pGarboczi (Garboczi et al., 1992). Recombinant HLA-A*0201 was produced by E. coli batch fermentation. Bacteria were harvested by centrifugation and resuspended in ice cold buffer (50 mM Tris–HCl pH 8.3, and 150 mM NaCl). Proteinase inhibitor AEBSF was added to a final concentration of 0.5 mM before lyses of the cells by cell disruption. Inclusion bodies were isolated from the cell lysate by centrifugation (20 g rpm/4 °C) and washed thoroughly three times in ice cold buffer (2 M urea, 2% Triton X-100, 0.5 M NaCl, and 20 mM Tris–HCl pH 8.0). After the final wash the inclusion body pellet was resuspended 8 M Urea, 150 mM NaCl, and 20 mM Tris–HCl pH 8.0). Undissolved material was removed by centrifugation and the supernatant was filtered through 0.2 μm filter before loading on to an ion exchange column (Q Sepharose fast flow). The HLA heavy chain was eluted by a 0–100% gradient of 8 M Urea, 500 mM NaCl, and 20 mM Tris–HCl pH 8.0. Relevant fractions were identified by SDS-PAGE and concentrated. 2.2. Cloning and production of human β2 microglobulin Cloned human β2 microglobulin (GenBank acc # CAG33347.1) was a kind gift from L. Østergaard (Danish Cancer Society). Recombinant β2M was produced by E. coli batch fermentation. Bacteria were harvested by centrifugation and resuspended in ice cold buffer (50 mM Tris–HCl pH 8.3, and 150 mM NaCl). Proteinase inhibitor AEBSF (Sigma, St. Louis) was added to a final concentration of 0.5 mM before lyses of the cells by cell disruption. Inclusion bodies were isolated as described above for the heavy chain. Undissolved material was removed by centrifugation and the supernatant was filtered through 0.2 μm filter before loading on to an IMAC column. The β2M preparation was then washed in 125 mM NaCl, and 20 mM Tris–HCl pH 8.0 before elution by an imidazole gradient (0–100%, 500 mM imidazole, 125 mM NaCl, and 20 mM Tris– HCl pH 8.0). The relevant β2M fractions were identified by SDSPAGE, sterile filtered and concentrated before gel filtration (column Hi-Load 26/60 Superdex 75) was performed to isolate the monomeric β2M from dimeric and multimeric forms.

J. Schøller et al. / Journal of Immunological Methods 360 (2010) 1–9

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Fig. 1. (A) Elution profile of folded MHC complex HLA-A*0201(NLVPMVATV). Absorbance at 280 nm (solid line), conductivity (dotted line). 2.5 ml fractions are indicated on the X-axis. Fractions 86–89 were isolated. (B) SDS-PAGE of the folded and purified HLA-A*0201 peptide complex used for vaccine preparation. (C) SDS gel of HSP70359–609 and anti-his blot with mouse IgG HRP *Pep3 ± HSP359–608.

2.3. Assembling the HLA complex The HLA-class 1 complex is a trimeric complex consisting of the HLA heavy chain, β2M, and a peptide with an amino acid sequence corresponding to the binding motif of the HLA molecule. The peptide used was the CMV epitope NLVPMVATV and N95% pure peptide was obtained from Neosystems (France). Assembly of the complex was performed at 10 °C by continuous mixing in refolding buffer, adjusted to 0.1 mM DTT, and containing the protease inhibitors, PMSF 1 mM, Pepstatin A (20 μg/ml) and Leupeptin (20 μg/ml). The folded MHC complex was biotinylated using d-biotin, Bir-A enzyme in the presence of Pepstatin A and Leupeptin. The resulting folded and biotinylated HLA complex was isolated from excess biotin, β2M and aggregated heavy chain by gel filtration on Superdex using 50 mM NaCl, and 20 mM Tris pH 8.0 as elution buffer. The elution profile (Fig. 1A) clearly distinguishes the folded MHC complex. SDSPAGE of MHC complex elution peak shows the HLA heavy chain and β2M bands. The bound peptide is too small to be seen on the gel (Fig. 1B). Correct assembly was verified by a conformational ELISA using the conformational sensitive mouse anti-HLA ABC monoclonal W6/32 antibody (Dako MO736) as primary catchment antibody. Maxisorb ELISA plates (Nunc, Denmark) were coated with 5 μg/ml of W6/32 overnight, at 2–4 °C and blocked using 1% skimmed milk powder. The folded MHC was diluted to approx 0.3 mg/ml before application in two-fold serial dilutions. As positive

control the human myeloid cell line KG-1 was used. Negative control was the THT ELISA assay buffer (100 mM NaCl, 50 mM Tris pH 7.2, and 0.01% Bronidox). Secondary rabbit anti-β2M antibody (Dako P0174) was used to detect binding of the HLA complex. Amplification was performed using HRP conjugated polyclonal goat-anti-rabbit immunoglobulin. The ELISA assay (Table 1) confirms correct folding of the MHC complex with an absorbance value for the undiluted complex in five-fold excess of the positive control cell absorbance. The complex stays together as the tri-molecular complex with the peptide bound in the MHC grove is inherently stable, which is enhanced by conjugation to the dextran backbone (Zhu et al., 2010).

Table 1 MHC ELISA folding assay. Two-fold serial dilution of folded MHC was applied in duplicate. 1×THT buffer shows background staining. The positive control was of MHC-class 1 expressing KG-1 human myeloma cells. Sample

HLA-A*0201(NLVPMVATV)

Undiluted 1:2 1:4 1:8 1:16 1:32 1:64 Controls

0.593 0.510 0.476 0.249 0.272 0.170 0.108 1×THT 0.041

0.513 0.554 0.453 0.405 0.303 0.184 0.118 Positive control 0.110

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2.4. Construction of expression vectors and production of biotinylated C-terminal peptide of M. tuberculosis HSP70 (HSP70359–608)

known number of molecules of SA was mixed with a known number of molecules of dextran in the conjugation process. The conjugate was then purified on a Superdex 200, Hi-Load 16/60 and the fractions were collected. All SA-coated dextran was isolated and the bound and unbound amount was measured. The percentage of bound SA was then determined and the average number of SA per dextran was calculated. The number of MHC was then determined by the available dextran backbone which on average showed 8 streptavidin binding sites per dextran molecule. For each SA site a maximum of three biotinylated MHC molecules were bound due to steric constraint. Thus, 24 MHC molecules was the optimum number per dextran. For the multimeric HLA/HSP70 Dextramer equimolar amounts of HLA and HSP70 was attached resulting in a molar ratio of 12/12/1 HLA complex/HSP70/dextran. The attachment of HLA complexes and HSP70 to the dextran backbone was verified by solid-phase ELISA using again the conformational sensitive mouse anti-HLA ABC monoclonal W6/32 (Dako MO736) described above or anti-HSP70 as catchment antibody. In both cases secondary rabbit anti FITC (Dako P5100) was used to detect binding of the dextramer construct. Amplification was performed using HRP conjugated polyclonal goat-anti-rabbit immunoglobulin. The recombinant HIVgp120 (IIIB, EVA607) (NIBSC, Potters Bar, UK) was conjugated to HSP70 using N-succinimidyl(3[2-pyridyl]-ditio (SPDP) as previously described (Bogers et al., 2004).

To enable in vivo biotinylation of the C-terminal peptide of M. tuberculosis HSP70 (aa 359–608) was subcloned. A primer of 76 bases for PCR was designed that allows fusion of the recognition site for in vivo biotinylation in E. coli to the Cterminal end of HSP70(359–608). Together with a forward cloning primer the HSP70(359–608) fusion gene was amplified and after restriction with NdeI and XhoI inserted into the expression vector pET26. The resulting plasmid pLEXWO1674 was transformed into E. coli BL21 (DE3). The insert of plasmid pLEXWO167-4 was confirmed by sequencing. For rapid purification metal chelate chromatography biotinylated HSP70(359–609) was fused to a 6-fold N-terminal histidine tag. HSP70(359–609) gene was excised from plasmid pLEXWO167-4 using restriction endonucleases NdeI and XhoI and inserted into pET28 which provides the sequence for the N-terminal histidine tag. The resulting plasmid pLEXWO1754 was transformed into E. coli BL21 (DE3). This strain was used for production of biomass of biotinylated HSP70(359–608) with N-terminal histidine tag. The cell mass was suspended in 20 mM tris, 100 mM NaCl, and 10 mM imidazole, pH 8.0 and then disrupted by homogenization and sonication at 4 °C. After centrifugation the clear supernatant was applied on Ni-NTA resin (Qiagen, Hilden). After washing the bound protein was eluted in a linear gradient from 20 mM tris, 100 mM NaCl, and 10 mM imidazole, pH 8.0 to 20 mM tris, 100 mM NaCl, and 500 mM imidazole, pH 8.0. Fractions containing the target protein were pooled, diluted tenfold with 20 mM tris, pH 8.0 and applied on a Q Sepharose High Perfomance resin (GE Healthcare). After washing the protein was eluted in a linear gradient from 20 mM tris, pH 8.0 to 20 mM tris, and 1 M NaCl, pH 8.0. Fractions containing the pure target protein were pooled and underwent the final buffer exchange step. The protein was applied on a Sephadex G25 fine resin (GE Healthcare) and eluted with PBS, pH 7.4. The purified N-his HSP359–608-bio was subjected to final quality control and a Western blot is shown in Fig. 1C. The protein pool was filter sterilized through 0.2 μM PES filters, aliquoted and stored frozen at −26 to −28 °C.

Animals were housed according to UK Home Office guidelines for animal experimentation. Three groups of 6 mice/group of 6 week old Balb C mice were immunized subcutaneously in the base of the tail with 10 or 20 μg/mouse at day 0 and 21, and an unimmunized control group presented in Fig. 4C. Group 1 received 10 μg of Dextramer-HSP70 mixed with 20 μg of HIVgp120 chemically conjugated to HSP70; Group 2 received 20 μg of HIVgp120 conjugated to HSP70; and Group 3 received 10 μg of dextran per mouse. Blood samples were taken from the tail before and 10 days after the second immunization. The animals were killed by cervical dislocation 10 days after the second immunization. Spleens were removed for T cell proliferative studies.

2.5. Preparation of the Dextramer

2.7. Mouse T cell proliferative assay

The dextramer backbone consisting of a 270 kDa dextran (Pharmacosmos, Denmark) was activated by divinyl sulfone acid by incubation of 1 g dextran and 5 g divinyl sulfone acid in 0.2 M Na2CO3 for 20 min with heavy stirring. Reaction was stopped by concentrated acetic acid and then thoroughly dialyzed against water (Lihme and Boenisch, 1994). Strepavidin in heavy excess was reacted with the activated dextran in the presence of a minor amount of FITC for 3 h resulting in 8% incorporation of streptavidin. Biotinylated HLA complex alone or together with biotinylated HSP70 molecules was bound to a divinyl sulfone acid activated Streptavidin coated 270 kDa dextran-FITC backbone (Lihme and Boenisch, 1994). For the present study a Dextramer with the molar ratio of 24 HLA molecules to 1 dextran was used. The number of streptavidin (SA) per dextran molecule was determined after the conjugation of SA to the activated dextran. A

The CFSE (carboxyfluorescein diacetate succinimidyl ester) cell labelling method of dye dilution, which is halved with each cell division has been used for both the murine and human lymphocyte proliferation assays. Mouse spleens were collected and teased apart to release splenocytes. The cell suspension was filtered through BD Falcon 100 μm Nylon cell strainer (BD Biosciences) to remove the remaining tissues. The cells were then washed and resuspended in RPMI 1640 medium supplemented with 10% FCS, 100 μg/ml of penicillin and streptomycin, and 2 mM glutamine. 20×107 of the splenocytes were washed once with PBS containing 1% BSA and cell pellets were resuspended in 1 ml of PBS and labelled with CFSE (Molecular Probes, Invitrogen) according to the manufacturer's instruction. CFSE-labelled cells were washed twice, resuspended in the culture medium and 3×105 cells in 100 μl were plated into 96 well plates. Two to three concentrations of HSP70, HLA-dextramer, 5 μg/ml of Con A

2.6. Immunization of mice with the Dextramer and HIVgp120

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as positive control and 10 μg/ml of BSA as negative control were added to the cultures. After 7 days of incubation, proliferation was assayed by flow cytometry (BD Canto II) and proliferation was expressed as percent proliferated cells of the total population. Proliferated CD4+ and CD8+ T cells were assayed by labelling cells with PE conjugated antibodies to murine CD4 and CD8 (Immunotool, UK).

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2.8. Antibody analysis by ELISA A solid-phase enzyme-linked immunosorbent assay (ELISA) method was used for the detection of serum IgG antibodies to the Dextramer and HIVgp120. Microtitre plates (Dynatech M 129B, Billingshurst, Kent, U.K.) were coated with 1 μg/ml of Dextramer or 1 μg/ml HIVgp120 (100 μl per

Fig. 2. (A) Stimulation of human PBMC, CD4+ and CD8+ human T cell proliferation stimulated by HLA-A*02 Dextramers, which was compared with irradiated allogeneic cells and presented as mean (±sem) (n= 3). (B) Gating of the lymphocytes examined (P1) and double staining for CD4+ and CD8+ T cells. (C) Representative flow cytometry of proliferation of CD4+ and (D) CD8+ T cells in response to the Dextramer without or with HSP70, dextran, HSP70 alone or allogeneic stimulation.

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well) and then incubated overnight at 4 °C. The plates were washed ×3 in PBS (pH 7.4) and blocked by adding 200 μl of PBS/BSA/T20 at 37 °C for 1 h. Dilutions of samples in duplicates starting from 1:50 to 1:64,000 (100 μl per well) were added to the plates and incubated at 37 °C for 2 h after which the plates were washed (3×) with PBS. 100 μl/well goat-anti-mouse IgG alkaline phosphatase conjugate (1:2000 Sigma) in PBS/BSA/T20 was added, the plates were incubated for 2 h at 37 °C and then washed twice in PBS and once in distilled water. One tablet of p-nitrophenol was dissolved in 5 ml of diethanolamine buffer and 100 μl was added to each well. When the colour had developed the reaction was stopped by the addition of 50 μl per well of 3 M sodium hydroxide and read at 405 nm in an ELISA reader (Anthos 2001 Labtec International UK). 2.9. Human T cell proliferative assay Dextran, Dextramer, Dextramer linked to HSP70, each at 10 μg per ml, or 104 irradiated allogeneic monocytes were incubated with 105 CFSE-labelled human peripheral blood mononuclear cells (PBMC) for seven days, avoiding HLAA*0201 cells. The cells were stained with PE anti-CD8 and APC-Cy7 anti-CD4 and analysed by flow cytometry using a BD FACSCanto II flow cytometer and FACSDiva software. Proliferation was determined by measuring the percentage of PBMC, CD4 T cells, or CD8 T cells with reduced CFSE content. 2.10. APOBEC3G mRNA assay A3G mRNA expression by 1 × 106 CD4+ MACS isolated cells was measured by real-time PCR following an 18 hour stimu-

lation with medium alone or different dilutions (1:5, 1:10 or 1:20) of either dextramer, dextran, dextramer linked to HSP70(359–608) or HSP70(359–608) alone as previously described (Pido-Lopez et al., 2007). The 18 hour stimulation period was determined after undertaking a time-course study. In addition A3G mRNA was measured following allogeneic stimulation of 1 × 106 CD4+ cells with HLA mismatched 1 × 106 C8166 T cells. A3G was calculated as fold increase in relation to unstimulated (medium alone) A3G levels. 3. Theory The rationale of linking recombinant HLA-class I molecules and HSP70 to a dextran backbone is to enable stable attachment of a number of protein molecules in proximity by using the biotin–streptavidin technique. The linked construct may be more effective in inducing innate and adaptive immune responses by two or more antigens than when used separately. 4. Results 4.1. Stimulation of human T cell proliferation with the Dextramer Human CD4+ T cell proliferation stimulated in vitro by the Dextramer construct was significantly increased from mean (±sem) 3.4(±1.6) to 7.7(±4.5)% and further enhanced when linked to HSP70 (11.0 ± 2.6%), which is not significantly different from proliferation in response to irradiated allogeneic cells (18.4 ± 2.2%; Fig. 2A). PBMC yielded similar results. However, human CD8+ T cell proliferation stimulated in vitro by the Dextramer was lower (4.6 ± 3.4%) than that of CD4+ T cells and when the Dextramer was linked to HSP70 it failed to

Fig. 3. Innate anti-HIV factor — APOBEC3G mRNA elicited by stimulating human CD4+ T cells with the Dextramer (HLA-A*0201) alone or linked with HSP70, Dextran, HSP70 or allogeneic human CD4+ T cells (n = 8).

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enhance proliferation to the same level as that stimulated with allogeneic cells (Fig. 2A). Representative flow cytometry is illustrated in Fig. 2B. The positive control, concanavalin A was consistently high (71.2–96.4%; data not presented) and the negative control, without any stimulant was low (1.7– 14.8) and subtracted from the above values. 4.2. Stimulation of A3GmRNA Stimulation of human CD4+ T cells with the Dextramer preparations clearly demonstrated a significant increase in A3G mRNA expression induced by the Dextramer linked to HSP70 at 1:5, 1:10 and 1:20 dilution (Fig. 3), compared with Dextramer alone (p = 0.02). However, stimulation of CD4+ T cells with Dextran alone failed to increase A3G mRNA, whereas HSP70 alone induced a 4.3 ± 0.7-fold increase in A3G mRNA. Allogeneic stimulation with irradiated CD4+ T cells (C8166 cell line) induced 10.7(±2.7)-fold increase in mRNA, which compares favourably with 9.0-10.5 fold increase with the Dextramer linked to HSP70 (Fig. 3). 4.3. Splenic T cell proliferative responses in mice immunized with the HLA-A*02 and HIVgp120 Dextramer construct Immunization with Dextramer linked to HSP70 yielded the highest CD4+ T cell proliferative response to the Dextramer

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(27.9%) but also high responses when stimulated with Dextran (16.1%) or HSP70 (19.9%) (Fig. 4A). Immunization with Dextramer alone also yielded a high response to the Dextramer (21%) and Dextran (13.2%) but no response to HSP70. CD8+ T cell proliferation was also maximal when immunized with the Dextramer linked to HSP70 (27.9%), with no increase in the CD8+ T cells of Dextramer immunized mice (Fig. 4B). Thus, Dextramer linked with HSP70 resulted in the highest CD4+ and CD8+ T cell proliferative responses to the Dextramer which, however, also yielded significant responses to Dextran and HSP70. Immunization with HIVgp120 linked to HSP70 elicited 5.4(±2.7)% CD4+ T cell proliferation (Fig. 4C), which was greatly enhanced when mixed with DextramerHSP70 (14.7 ± 7.0). However, the possibility that some of the proliferative response may have been due to the immunogenicity of streptavidin cannot be excluded, though the low CD8+ T cell proliferation with Dextramer alone makes this less likely (Fig. 4A). Negligible Dextramer or HIVgp120 stimulated proliferation was found in the Dextran immunized or unimmunized control animals (Fig. 4A,B). 4.4. IgG antibodies to HIVgp120 and to HLA-A*02 Dextramer High IgG antibody titres were elicited to HIVgp120 in mice immunized with the Dextramer, linked to HSP70 and mixed with HIVgp120-HSP70 (9600 ± 1848) (Fig. 4C). A low antibody

Fig. 4. (A) CD4+ and (B) CD8+ T cell proliferative responses to dextran, Dextramer (HLA-A*0201) and HSP70 in Balb c mice immunized with the Dextramer (HLAA*0201) and covalently linked with HSP70. (C) CD4+ T cell proliferation stimulated by HIVgp120, antibodies to HIVgp120 and the Dextramer (HLA-A*02) were measured by ELISA and the results were expressed as mean (± sem); n = 4–5).

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titre to HIVgp120 was also elicited by HIVgp120-HSP70 (350± 38). However, antibodies to HLA-A*02 were recorded only in group 1 mice immunized with Dextramer-HSP70 mixed with HIVgp120-HSP70 (1600 ± 566; Fig. 4C). This is consistent with the highest level of CD4+ T cell proliferative responses (Fig. 4C). As with T cell proliferative studies, we cannot exclude the possibility that some of the antibodies to Dextramer may have been induced by streptavidin. 5. Discussion Conventional alloimmunization with cells elicits not only immune responses to the foreign HLA molecules but also a variety of cytokines and chemokines, which may boost the immune response. In principle it was assumed that the response to recombinant alloantigens would prove to be inferior to the corresponding cell-induced alloimmune responses, especially as the cells express HLA-A, B, CW, DR and DQ molecules, in contrast to the single recombinant HLAclass I A*0201 molecule. Surprisingly, the proliferative human CD4+ and CD8+ T cell responses stimulated in vitro by the recombinant HLA antigen was comparable to allostimulated cells. In addition to the specific alloimmune response, significant T cell proliferation was induced to dextran and HSP70 to which the HLA antigens were linked. Microbial HSP70 was used as an adjuvant as there is a great deal of evidence that it is capable of internalizing exogenous antigen not only by the MHC-class 2 but also the class I (crosspresentation) pathway (Castellino et al., 2000). It stimulates production of 3 CC chemokines (CCL-3, CCL-4 and CCL-5), of which CCL5 is a potent chemoattractant of monocytes, CD4 cells and activated CD8 cells (Schall et al., 1990; Murphy et al., 1994; Meurer et al., 1993; Kim et al., 1998). CCL3 and CCL4 attract CD4+ T- and B-cells (Schall et al., 1993) and all three chemokines attract immature DC (Dieu et al., 1998). HSP70 also stimulates the production of a number of cytokines (IL12, TNF-α, IL-1β and IL-6) (Wang et al., 2002b; MacAry et al., 2004); IL-12 is one of the most potent cytokines inducing TH1 polarization (Trichieri, 1994) and the C-terminal portion of the HSP70-linked peptide elicits high serum IgG2a and IgG3 subclasses of antibodies, consistent with Th1-polarizing response (Wang et al., 2002b) and induces maturation of DC. Alloimmunization with human cells would have to utilize cell lines, which raises the problem of removing oncogenic viruses commonly used for the production of cell lines. Recombinant alloantigens would bypass this problem and restrict the immune response to a single antigen, quite apart from the superior quality control over the production of a recombinant alloantigen, compared with cell lines. In vitro allogeneic stimulation of human CD4+ and CD8+ T cell proliferation was followed by in vivo immunization of Balb c mice. Significant CD4+ and CD8+ T cell proliferative responses were elicited by the Dextramer. Surprisingly, both human and murine CD4+ T cell proliferative responses were elicited with the HLA-class I antigen, which differs from the conventional CD8+ T cell response and will need to be further investigated. Significantly raised IgG antibody titres to HLA-A*02 were found in the sera from the Dextramer immunized mice, but the former gave a smaller standard error of the mean, suggesting greater consistency of response. These results suggest that recombinant alloantigens might prove to be useful reagents in

immunotherapy. A critical issue in utilizing this vaccination strategy is to alloimmunize the individual subject. With the help of Dr. R. Vaughan we have calculated from the frequency of the alleles found in different populations that to cover over 90% of the Caucasian, African and Chinese populations may require four HLA-class I (HLA-A*0101, *0201, *0301 and *1101) and one HLA-class II (DRB1*04) alleles. However, the potential application of alloimmunization in prevention of HIV-1 infection or the treatment of tumours may cause future problems if an organ transplant was required. Recently developed immunosuppressive agents may deal with these, but the risk: benefit ratio will no doubt have to be considered. Furthermore, the vaccine needs to be affordable, especially with a multiple allele constructs, and this may be resolved if the vaccine was to be produced on an industrial scale. Immunization with the Dextramer mixed with HIVgp120HSP70, also yielded higher CD4+ T cell proliferation to HIVgp120 than immunization with HIVgp120-HSP70 alone. This is consistent with the Dextramer mixed with HIVgp120HSP70 enhancing CD4+ T cell proliferative response and B cell antibody response. Thus, alloimmunization with the HLAclass I mixed with HIVgp120-HSP70 yields high CD4+ T cell responses and antibodies to both HLA-I and HIVgp120. It will be of interest to follow up systemic with mucosal immunization, as in both heterosexual and homosexual HIV infection the virus is transmitted by the mucosal route. Allostimulation induces the innate anti-viral A3G factor (Pido-Lopez et al., 2009) and this lead us to examine human CD4+ T cells to find out if stimulation with the recombinant HLA-class I antigen will induce A3G mRNA. Indeed, significant A3G mRNA resulted if human CD4+ T cells were stimulated with the HLA-A*0201 Dextramer, with a maximum reached when linked to HSP70. A comparative assay of the dextramer with allogeneic irradiated cells suggests that they are equally potent in stimulating A3G mRNA production. Thus, allostimulation in vitro with the recombinant HLAclass I construct elicits adaptive immune T cell proliferative responses in human CD4+ and CD8+ T cells and innate A3G mRNA expression in CD4+ T cells. HLA immunization in vivo in mice induces CD4+ and CD8+ T cell proliferative responses and IgG antibodies to the alloantigen. Early production of A3G may be important in HIV-1 vaccination, as the innate response is required to prevent early infection and destruction of CD4+ CCR5+ T cells within 2 weeks of the onset of infection. Indeed, single alloimmunization of women elicited significant A3G expression in their PBMC and inhibited ex vivo HIV replication (Wang et al., 1999; Pido-Lopez et al., 2009). 6. Conclusions A novel recombinant HLA-class I and HSP70 molecules were linked to dextran backbones using the biotin–avidin method. Stimulation of human PBMC in vitro with this construct induced CD4+ and CD8+ T cell proliferation and the innate anti-viral factor APOBEC3G. Immunization of Balb c mice with the HLA-A*0201 Dextramer also elicited an increase in CD4+ and CD8+ T cell proliferation and IgG antibodies to the HLA-A*0201. The recombinant HLA-class Idextran-HSP70 construct compared favourably with the immune responses elicited by native allogeneic stimulation. The application of this construct as a potential vaccine

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