Ovalbumin lipid core peptide vaccines and their CD4+ and CD8+ T cell responses

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Ovalbumin lipid core peptide vaccines and their CD4+ and CD8+ T cell responses Pavla Simerska a,∗ , Tittaya Suksamran a , Zyta Maria Ziora a , Fabian de Labastida Rivera b , Christian Engwerda b , Istvan Toth a,c,∗∗ a

School of Chemistry and Molecular Biosciences, The University of Queensland, Cooper Road, St. Lucia 4072, Queensland, Australia Immunology and Infection Laboratory, Queensland Institute of Medical Research, Herston, QLD 4029, Australia c School of Pharmacy, The University of Queensland, Pharmacy Australia Centre of Excellence, Cornwall Street, Woolloongabba, Queensland 4102, Australia b

a r t i c l e

i n f o

Article history: Received 13 December 2013 Received in revised form 30 April 2014 Accepted 11 June 2014 Available online xxx Keywords: Ovalbumin Peptide synthesis Vaccine development CD4+ CD8+ T cell

a b s t r a c t The lipid core peptide (LCP) system has successfully been used in development of peptide-based vaccines against cancer and infectious diseases (such as group A streptococcal infection). CD8+ T cells are important targets for vaccines, however developing a vaccine that activates long-lasting immunity has proven challenging. The ability of LCP vaccines to activate antigen-specific CD8+ and/or CD4+ T cell responses was tested using compounds that contained two or four copies of OVA257–264 and/or OVA323–339 peptides conjugated to LCP, which are recognised by OTI (CD8+ specific) and OTII (CD4+ specific) T cells, respectively. The LCP–ovalbumin vaccines developed in this study were synthesised in 30% yields and showed no significant haemolytic effect on red blood cells (below 4% haemolysis when tested with compounds at up to 100 ␮M concentrations). Promising in vivo data in mice suggested that this LCP–ovalbumin vaccine system could act as a novel and potent vehicle for the stimulation of robust antigen-specific CD8+ T cell responses. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Many peptides have been identified as potential new drug and vaccine targets for treatment and/or prevention of a variety of diseases. However, for the majority of peptide compounds, progression into the clinic is hampered by their unfavourable physico-chemical properties e.g. their rapid degradation in the body, problematic delivery and/or their poor inherent immunogenicity. Thus, there is a strong need for a new delivery system which overcomes all those issues [1]. A lipid core peptide (LCP) delivery system based on lipoamino acids, ␣-amino acids with long alkyl side chains, was designed and tested in variety of disease models [1]. Antigenic peptides that contained LCP elicited strong antibody production without the use of

Abbreviations: LCP, lipid core peptide; OVA1, OVA257-264 ; OVA2, OVA323-339 ; SPPS, solid phase peptide synthesis; MW, microwave; C16, (R/S)aminohexadecanoic acid. ∗ Corresponding author. Tel.: +61 7 33469892, +61 7 33654636; fax: +61 7 33654273. ∗∗ Corresponding author at: School of Chemistry and Molecular Biosciences, The University of Queensland, Cooper Road, St. Lucia 4072, Queensland, Australia. Tel.: +61 7 33469892, +61 7 33654636; fax: +61 7 33654273. E-mail addresses: [email protected] (P. Simerska), [email protected] (I. Toth).

conventional adjuvants [2]. The self-adjuvanting characteristic of this LCP system is advantageous, because many strong adjuvants used in animal models are toxic for humans and the currently available aluminium-based adjuvants used in humans are generally weak and unstable. The ability of the immune system to identify and eliminate tumour cells is established in the literature [3]. Immunotherapeutic strategies for cancer treatment require the generation of anti-tumour CD8+ T cells because the number of CD8+ T cells often correlates with a positive prognosis [4]. The CD8+ T cell response also has a central role in the host response to intracellular (viral) infections. Many research groups have focused on the production of therapeutic and prophylactic vaccines that elicit T cell responses, particularly a strong anti-tumour CD8+ T cell response. Several preclinical and clinical trials showed promising results, yet the U.S. Food and Drug Administration has only approved a few cancer vaccines for human use [5,6]. Vaccine-induced immunity also includes antigen-specific CD4+ T cells, which can influence surrounding T cells that have unrelated antigen specificities [7]. For example, parallel expansion of CD4+ T cells against tetanus toxoid was previously found during influenza virus infection [8]. Although CD8+ T cells are the principal effector cells for fighting cancer, the CD4+ T cells may help to induce, maintain and recall CD8+ T cell responses [9]. Ovalbumin peptides are

http://dx.doi.org/10.1016/j.vaccine.2014.06.049 0264-410X/© 2014 Elsevier Ltd. All rights reserved.

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OVA1:

OVA2:

Fig. 1. Chemical structures of ovalbumin peptides, OVA257–264 (SIINFEKL, OVA1) and OVA323–339 (ISQAVHAAHAEINEAGR, OVA2).

often used as a model for studying vaccine applications [10,11]. We included two ovalbumin peptides to induce a CD8+ T cell response and possibly a helper CD4+ T cell response (Fig. 1). The OVA257–264 (OVA1) peptide has been shown to induce a strong cytolytic CD8+ T cell response [12], while OVA323–339 (OVA2) promotes a CD4+ T cell response [13]. Although OVA is not a vaccine antigen candidate, the availability of OVA-specific CD4+ and CD8+ T cells, allowed us to measure the ability of the LCP system to activate antigen-specific T cell responses, thereby providing proof-of-principal for the utility of this system to stimulate self-adjuvanting activity of bona fide vaccine antigen candidates. 2. Materials and methods

using a Waters Delta 600 system (Milford, Massachusetts, USA model 600 controller, 490E UV detector). Purifications were performed on Vydac® C18 or C4 preparative columns (10 ␮m, 22 mm i.d. × 250 mm) using a gradient of solvent B with 10 mL/min flow rate, and detection at 230 nm. Fractions that contained pure compound were pooled and lyophilised overnight. Mass spectra (MS) were recorded on a triple-quatropol electrospray instrument (Perkin Elmer Sciex API 3000) in positive ion mode. The MS Analyst 1.4 software was obtained from Applied Biosystems (MDS Sciex, Toronto, Canada). NMR spectra were recorded on a Bruker AM 300 MHz instrument at 297 K using CDCl3 as a deuterated solvent and tetramethylsilane as an internal standard, unless stated otherwise.

2.1. Materials

2.3. Solid phase peptide synthesis

Solvents and reagents for peptide synthesis including dimethylformamide (DMF), dichloromethane (DCM), methanol, trifluoroacetic acid (TFA) and N,N-diisopropylethylamine (DIPEA), were purchased from Auspep (Melbourne, VIC, Australia). (O-Benzotriazol-1-yl)-N,N,N ,N -tetramethyluronium hexafluorophosphate (HBTU) and di-tert-butyl dicarbonate (Boc2 O) were supplied by GL Biochem Ltd. (Shanghai, China). N˛ tert-Butyloxycarbonyl (Boc)- and 9-fluorenylmethoxycarbonyl (Fmoc)-protected amino acids, 4-methyl benzhydrylamine (pMBHA), and Rink amide MBHA resins were obtained from Novabiochem (Läufelfingen, Switzerland). Palladium (10% wt on carbon) was purchased from Lancaster Synthesis (Lancashire, England) and sodium dodecylsulfate (SDS) from Sigma-Aldrich (St. Louis, USA). 96-Well plates were purchased from Corning® Costar® (Tewksbury, Massachusetts, USA) in sterile condition. Ultra-pure gases (N2 , H2 and Ar) were supplied by BOC Gases (Brisbane, QLD, Australia). Deuterated solvents (d1 -CDCl3 and DMSO-d6 ) were manufactured by Cambridge Isotope Laboratories Inc. (Andover, MA, USA). All other reagents were purchased in analytical grade or higher purity from Sigma-Aldrich (Castle Hill, VIC, Australia) or Merck Pty (Kilsyth, VIC, Australia). Solvents were freshly distilled and dried and all moisture-sensitive reactions were carried out under inert atmosphere (N2 /Ar) using oven-dried glassware.

Solid phase peptide synthesis (SPPS) was performed in manual shaker (Shaker WS/180◦ , Glas-Col) or assisted by a microwave (MW) peptide synthesiser (Discover Solid Phase Synthesis, CEM A. I. Scientific) at 70 ◦ C, 20 W. The HAAH peptide sequence present in the OVA2 peptide was synthesised by manual shaking at room temperature for 1 h to avoid racemisation. Both OVA1 and OVA2 peptides (Table 1) were prepared by Fmoc-chemistry using Rink amide MBHA resin (substitution ratio: 0.60 mmol/g). Each amino acid coupling involved MW Fmoc-deprotection with 20% piperidine in DMF (2 × 5 min), a 1 min DMF flow wash, followed by 10 min MW coupling of pre-activated amino acid. For LCP peptides (Table 1), Boc-chemistry was used with pMBHA resin (substitution ratio: 0.45 mmol/g). Each amino acid coupling cycle involved Boc-deprotection with neat TFA (2 × 1 min), a 1 min DMF flow wash, followed by 10 min MW coupling of pre-activated amino acid. Activation of both Boc- and Fmoc-amino acids (4.2 eq. per mol amino-group) was achieved using HBTU (4.0 eq.) and DIPEA (6.2 eq.). When the peptide sequence was completed, the terminal Boc or Fmoc groups were removed, the resin was washed with DMF, CH2 Cl2 and MeOH, and dried in vacuo to give a crude peptide product. The crude peptides from Fmoc-chemistry SPPS were cleaved from the resin using a TFA:triisopropyl silane (TIPS):H2 O (95:2.5:2.5) cleavage cocktail. The crude peptides from Bocchemistry SPPS were cleaved from the resin using hydrofluoric acid at −10 to −5 ◦ C, with p-cresol as scavenger. All cleaved peptides were precipitated from ice-cold diethyl ether, dissolved in CH3 CN/water/TFA and lyophilised overnight to give amorphous powders. All crude peptides (Table 2) were analysed and purified by RP-HPLC as described above.

2.2. General methods Analytical reverse phase high-performance liquid chromatography (RP-HPLC) was completed on a Shimadzu instrument (Kyoto, Japan LC-10AT liquid chromatograph, SCL -10A system controller, SPD-6A UV detector, SIL-6B auto injector with a SCL-6B system controller LabSolutions software). C18 and C4 Grace Vydac® columns (Columbia, Maryland, USA 10 ␮m, 4.6 mm internal diameter (i.d.) × 250 mm) were used with a linear gradient of 0–100% of CH3 CN/10% water/0.1% TFA (solvent B) and water/0.1% TFA (solvent A) for 30 min, with a 1 mL/min flow rate and detection at 214 nm, unless otherwise stated. Semi-preparative RP-HPLC was performed

2.4. Haemolytic assay The membrane-damaging properties of the synthesised ovalbumin peptides were quantified using an in vitro haemolytic assay. Blood collected from a healthy adult volunteer was treated with

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Table 1 Ovalbumin peptide vaccine structures. C16 = 2-(R/S)-aminohexadecanoic acid. Name

Peptide structure

SIINFEKL K

ISQAVHAAHAEINEAGR OVA1–OVA2–Lys

K

SIINFEKL

K

ISQAVHAAHAEINEAGR

SIINFEKL K

SIINFEKL OVA1–LCP

K

SIINFEKL

C16

G

C16

C16

G

NH2

K

SIINFEKL

SIINFEKL ISQAVHAAHAEINEAGR OVA1–OVA2–LCP

SIINFEKL ISQAVHAAHAEINEAGR

heparin prior the isolation of human erythrocytes by centrifugation at 3000 rpm for 15 min (Sigma 2-5 centrifuge, Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany). The erythrocyte pellet was washed four times with an isotonic phosphate buffer saline, pH 7.4 (PBS Gibco-BRL, Grand Island, NY, USA) then centrifuged at 3000 rpm for 15 min and resuspended in PBS to a final concentration of 4% (v/v) erythrocytes. The ovalbumin peptides (100 ␮L) were prepared in three concentrations (10 ␮M, 50 ␮M and 100 ␮M) in PBS, transferred into the 96-well flat-bottom microtiter plates (TPP, Zurich, Switzerland), and incubated at 37 ◦ C for 1 h on a Titramax 1000 Vibrating Shaker (Heidolph, Schwabach, Germany). The erythrocyte suspension (100 ␮L) was added to each well on the plate and incubated for 1 h at 37 ◦ C with constant shaking. The plates were centrifuged at 2400 rpm for 15 min and the supernatant (75 ␮L) was transferred into a new 96-Well plate to record the absorbance of each sample at 540 nm using a Spectramax 250-microplate reader (Molecular Devices, Sunnyvale, California, United States). Each experiment was performed in triplicate SDS (10 mM) in PBS, and PBS alone, were used as positive and negative controls, respectively. This in vitro haemolytic assay was performed with the approval of the University of Queensland Ethics Committee (No. 2009000661) and all procedures were completed using a laminar flow bench (Nuaire) in sterile conditions. 2.5. OTI and OTII expansion and IFN production Inbred female C57BL/6 and B6.SJL.Ptprca (B6.CD45.1) mice were purchased from the Australian Resource Centre (Canning Vale,

K K

C16

G

C16

C16

G

NH2

K

Western Australia) and maintained under conventional conditions. B6.SJL.Ptprca x OT I [14] and B6.SJL.Ptprca x OT II [15] mice were bred and maintained at the Queensland Institute of Medical Research (QIMR). All mice used were age- and sex-matched and housed under specific-pathogen free conditions. All animal procedures were approved and monitored by the QIMR Animal Ethics Committee under ethics approval number A0512-619M, in accordance with the Australian code of practice for the care and use of animals for scientific purposes (Australian National Health & Medical Research Council). To assess antigen-specific T cell proliferation in vivo, congenic (CD45.1) ovalbumin specific OTI and OTII T cells were isolated from the spleen and 104 OTI and 2 × 104 OTII cells were adoptively transferred into C57BL/6 mouse 2 h prior to challenge with ovalbumin peptides. OTI and OTII cell numbers were determined at indicated times by FACS. Allophycocyanin (APC)-conjugated anti-TCR␤ chain (H57-597), phycoerythrin (PE)-Cy5-conjugated anti-CD4 (GK1.5), fluorescein isothiocyanate (FITC)-conjugated anti-CD45.1 (clone A20), brilliant violet 421-conjugated anti-IFN-␥ (clone XMG1.2), Alexa fluor 700-conjugated anti-CD8␣ (clone 53-6.7) and Pacific Blue-conjugated anti-CD45R/B220 (clone RA3-6B2), were purchased from Biolegend (San Diego, CA) or BD Biosciences (Franklin Lakes, NJ). Dead cells were excluded from the analysis using Aqua Live/dead fixable stain or near infra-red live/dead fixable stain (Invitrogen-Molecular Probes, CA), in accordance with manufacturer instructions. Cell surface antigen and intracellular cytokine staining was carried out as previously described [16]. Briefly, cells were incubated for 2 h in vitro with 10 ␮g/mL brefeldin A

Table 2 ESI-MS and RP-HPLC data of ovalbumin peptides. Compound

Purif. yield (%)

ESI-MS m/z:

RP-HPLC retention time (min)

MW

Found

C-18 column

C-4 column

OVA1 OVA2 OVA1–OVA2–Lys

68 54 49

962.14 1772.92 5971.70

19.20 11.70 17.21

18.10 13.60 21.19

OVA1–LCP OVA1–OVA2–LCP

30 31

5056.37 6846.07

962.7 [M + H]+ ; 482.0 [M + 2H]2+ 1774.4 [M + H]+ ; 887.5 [M + 2H]2+ ; 592.1 [M + 3H]3+ 1494.0 [M + 4H]4+ ; 1195.7 [M + 5H]5+ ; 996.6 [M + 6H]6+ ; 854.3 [M + 7H]7+ ; 747.6 [M + 8H]8+ 1012.6 [M + 5 M]5+ ; 843.9 [M + 6 M]6+ ; 723.5 [M + 7]7+ 1712.9 [M + 4 M]4+ ; 1370.3 [M + 5H]5+ ; 1142.5 [M + 6H]6+ ; 979.5 [M + 7H]7+ ; 857.1 [M + 8H]8+

– –

35.44 34.66

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Fig. 2. Optimisation of peptide cleavage from resin after Boc-SPPS. Crude RP-HPLC (A) and ESI-MS (C) of the cleaved product at 0 ◦ C crude RP-HPLC (B) and ESI-MS (D) of the product cleaved at −10 to −5 ◦ C.

(Sigma-Aldrich). After cell surface antibody labelling, cells were fixed and permeabilised with Cytofix/Cytoperm buffer (BD Biosciences) and incubated with PE-conjugated IFN-␥ (XMG1.2) or PE-conjugated rat IgG1 (RTK2071) (isotype-matched control) on ice for 30 min. FACS was performed using a FACS Canto II or LSR FORTESSA (BD Biosciences), and data was analysed using FlowJo software (TreeStar, Oregon, USA). 2.6. Statistical analysis Statistical differences between groups were determined using the Mann–Whitney U test using GraphPad Prism version 4.03 for Windows (GraphPad Software, San Diego, CA) and p < 0.05 was considered statistically significant. All data are presented as the mean values plus or minus standard error.

(Table 1). The purified vaccines OVA1–LCP and OVA1–OVA2–LCP were obtained in around 30% yields and their structure and purity were confirmed by ESI-MS and RP-HPLC (Table 2). A control compound, OVA1–OVA2–Lys, was synthesised in almost 50% purified yield and was used in the in vitro and in vivo assays to determine the role of the lipid moiety. Peptides synthesised via Boc-chemistry were generally cleaved from the resin using hydrofluoric acid at 0 ◦ C in the presence of p-cresol as a scavenger. However, impurities (Fig. 2A and C) necessitated optimisation. Lower temperatures (−10 to −5 ◦ C) prevented the formation of by-products between p-cresol and the free carboxylic moieties of the peptides and allowed formation of the required peptides in higher yields (Fig. 2B and D).

3. Results 3.1. Synthesis of LCP–ovalbumin vaccines The desired ovalbumin peptides were synthesised from Fmocand Boc-protected amino acids via conventional SPPS with MBHA resins. Two ovalbumin peptide fragments, OVA257–264 peptide (SIINFEKL, OVA1) and OVA323–339 peptide (ISQAVHAAHAEINEAGR OVA2) were synthesised in over 50% purified yields and used as a control peptides for in vitro and in vivo studies. Vaccine candidates with a built-in adjuvant comprised of four copies of OVA1 or two copies of the OVA1 and two copies of OVA2, which were coupled to the self-adjuvanting LCP system that contained a lipoamino acid with 14-carbon long side chain

Fig. 3. Haemolytic effect of ovalbumin peptide derivatives (10, 50 and 100 ␮M) in PBS on human red blood cells. All measurements were made in triplicate and are shown as mean ± SD.

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Fig. 4. The expansion kinetics of OTI and OTII cells in vivo in response to challenge with ovalbumin peptides and LPS. C57BL/6 were administered 1 × 104 OTI cells and 2 × 104 OTII cells 2 h before challenge with ovalbumin peptides mixed with LPS. The gating strategy to identify OTI and OTII cells is shown in (A). The number of OTI (B) and OTII (C) cells in the spleen was measured at days 4 and 7 post-challenge, as indicated. Control mice received the same number of OTI and OTII cells but were injected with saline and cell numbers measured at day 7 after challenge. Each bar represents the mean ± SEM. These data are representative of two independent experiments (n = 5 mice per group). Statistical differences of p < 0.01 (**) are indicated.

3.2. Toxicity evaluation of LCP–ovalbumin vaccines To confirm that the ovalbumin peptide vaccines synthesised in this study do not have any significant haemolytic effect on human erythrocytes, the peptides were evaluated by the in vitro human erythrocyte assay. This model is commonly used to investigate membrane interactions between the synthetic compounds and cell membrane, where the release of haemoglobin is easily measured as absorbance at 540 nm after exposure of the erythrocytes to the compound of interest. Haemolytic lysis of human red blood cells was evaluated after the 1 h incubation of the cells at 37 ◦ C with three concentrations of the test compounds. PBS and SDS were used as negative and positive controls and assigned 0% and 100% lysis, respectively. The maximum haemolysis (<4%) was detected for OVA1–LCP at 50 ␮M only (Fig. 3). Haemolysis below 10% is considered to be non-haemolytic [17].

3.3. Stimulation of potent antigen-specific CD8+ T cell responses by LCP-conjugated peptides The ability of LCP-conjugated ovalbumin peptides to activate antigen-specific CD4+ and CD8+ T cell responses was tested using compounds in which LCP was conjugated to chemically linked OVA1 and OVA2 peptides, which are known to be recognised by OTI and OTII T cells, respectively, as described above. We first established the expansion kinetics of OTI and OTII T cells in response to chemically linked OVA1 and OVA2 peptides mixed with the potent Toll-like receptor 4 agonist, liposaccharide (LPS) (Fig. 4). OTI cells have expanded significantly more in mice receiving ovalbumin peptides plus LPS (p < 0.01), than control animals, by days 4 and 7 after antigen challenge, with expansion peaking on day 4 (Fig. 4A and B). OTII cells also expanded significantly more in mice receiving ovalbumin peptides plus LPS (p < 0.01), than in control animals, by

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Fig. 5. Ovalbumin LCP vaccine is a potent adjuvant for CD8+ T cell activation and expansion. C57BL/6 were administered 1 × 104 OTI cells and 2 × 104 OTII cells 2 h before challenge with ovalbumin peptides alone, in combination with LPS, or chemically conjugated to LCP, as indicated. The gating strategy to identify OTI and OTII cells was the same as shown in Fig. 4A. T cells were further gated and IFN-␥-producing OTI and OTII cells were identified as shown in (A) and compared with an isotype control antibody (far right panels). The number of total OTI cells (B), IFN-␥+ OTI cells (C), total OTII cells (D) and IFN-␥+ OTII cells (E) in the spleen was measured at day 4 post-challenge. Control mice received the same number of OTI or OTII cells as test mice, but were injected with saline and cell numbers measured at day 4 after challenge. Each bar represents the mean ± SEM. These data are representative of two independent experiments (n = 5 mice per group). Statistical differences of p < 0.01 (**) are indicated.

days 4 and 7 after antigen challenge, but these cells continued to expand until at least day 7 after the challenge (Fig. 4A and C). Our interest in CD8+ T cell activation led us to focus our studies on day 4 after antigen challenge. We next measured OTI and OTII cell expansion and interferon gamma (IFN-␥) production after injection with OVA1–OVA2–LCP, and compared this in control animals and mice receiving

OVA1–OVA2–Lys alone and with LPS (Fig. 5). We found that the OVA1–OVA2–LCP compound generated significantly greater (p < 0.01) expansion of OTI cells (Fig. 5 B) and resultant IFN-␥ production (Fig. 5A and C) than control animals and mice that received the LPS/OVA1–OVA2–Lys mixture. In contrast, mice that received the LPS/OVA1–OVA2–Lys mixture generated significantly greater (p < 0.01) expansion of OTII cells (Fig. 5D) and IFN-␥ production

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(Fig. 5A and E), than control animals and those that received the LCP–OVA compound. Thus, the LCP conjugated peptides selectively promoted the activation and expansion of antigen-specific CD8+ T cells, while CD4+ T cells were better activated by an LPS and OVA1–OVA2–Lys mixture.

4. Discussion We aimed to investigate the T cell activation properties of vaccine constructs that contained both OVA1 and OVA2 peptides incorporated in the same complexes and coupled to the LCP system. With the exception of the HAAH peptide sequence in OVA2 and the final cleavage of Boc-synthesised compounds from the resin, standard SPPS protocols were used [18]. Manual synthesis of the HAAH sequence was necessary to avoid racemisation of the histidine. The Boc-chemistry strategy was chosen for the synthesis of branched LCP peptides because the Fmoc protecting group was too bulky and caused problems during the couplings. Peptide cleavage using hydrofluoric acid under standard conditions (p-cresol as a scavenger and at 0 ◦ C) was optimised to overcome the many impurities that this step introduced. The ESI-MS spectra showed signals that correspond with the desired mass of the tested compound plus multiplets of 90 Da. Initial attempts to overcome this problem included the use of thiocresol as an alternative scavenger which resulted in the appearance of a new signal that corresponded to the desired product mass plus 96 Da. This variation agreed with the difference in molecular weights of cresol and thiocresol. Similar observations were reported for the synthesis of peptides that contained glutamic acid residues in the sequence [19]. After testing various conditions, the cleavage was found to be the most efficient, giving crude products of relatively high purity and yield, when performed with p-cresol and at −10 to −5 ◦ C. Other solutions to overcome formation of undesired products during peptide cleavage were also previously published [20]. CD8+ T cells serve many functions, including the recognition and killing of infected and transformed cells. They can also produce pro-inflammatory cytokines that help innate immune cells to eliminate numerous pathogens [21]. Therefore, CD8+ T cells are important targets for vaccines. However, the development of a vaccination that efficiently activates long-lasting immunity has proven challenging. There is a strong correlation between the number of activated antigen-specific CD8+ T cells at the peak of an immune response and the quality of the resulting memory CD8+ T cell response [22–25]. Our data indicates that LCP-conjugated ovalbumin peptides may be useful for generating long-lasting memory CD8+ T cell responses. The OVA1–OVA2–LCP peptide greatly enhanced antigen-specific CD8+ T cell expansion and IFN-␥ production. Somewhat surprisingly, antigen-specific CD4+ T cells were better activated by an LPS and OVA1–OVA2–Lys mixture than the LCP-conjugated peptides. One possible explanation is that CD4+ T cell responses were still emerging at the time point studied (day 4 after challenge), and may have developed significantly better at later time points. However, another possible explanation is that the LCP-conjugated peptides targeted a different dendritic cell population than the LPS and OVA1–OVA2–Lys mixture. Furthermore, CD8␣␣+ dendritic cells are specialised primers of antigen-specific CD8+ T cell responses [26], and it is possible that this dendritic cell subset is targeted by the LCP-conjugated ovalbumin peptides. In contrast, the LPS and OVA1–OVA2–Lys mixture is possible to target CD8− dendritic cell subsets that have been shown to efficiently prime antigen-specific CD4+ T cell responses [27]. These hypotheses remain to be tested experimentally. We believe LCP-conjugated ovalbumin peptides represent a novel and potent vehicle for the generation of robust antigen-specific CD8+ T cell responses.

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Acknowledgements We acknowledge the Australian Research Council for their support of this work with the Discovery Project Grant DP1092829, Professorial Research Fellowship to I.T. (DP110100212), and an Australian Postdoctoral Fellowship to P.S. (DP1092829). We thank Abdul Kader Shabbir for help with haemolytic assay, which was performed with the approval of the University of Queensland Ethics Committee (No. 2009000661). All institutional and national guidelines for the care and use of laboratory animals were followed (QIMR animal ethics approval number A0512-619M). We also thank Thalia Guerin for revising this manuscript. There is no conflict of interest.

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Please cite this article in press as: Simerska P, et al. Ovalbumin lipid core peptide vaccines and their CD4+ and CD8+ T cell responses. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.06.049

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Please cite this article in press as: Simerska P, et al. Ovalbumin lipid core peptide vaccines and their CD4+ and CD8+ T cell responses. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.06.049