Augmenting Influenza-Specific T Cell Memory Generation with a

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Augmenting influenza-specific T cell memory generation with a NKT cell-dependent glycolipid-peptide vaccine Regan J Anderson, Jasmine Li, Lukasz Kedzierski, Benjamin J Compton, Colin M Hayman, Taryn L Osmond, Ching-Wen Tang, Kathryn J Farrand, Hui-Fern Koay, Catarina Filipa Dos Santos Sa E Almeida, Lauren R Holz, Geoffrey M Williams, Margaret A. Brimble, Zhongfang Wang, Marios Koutsakos, Katherine Kedzierska, Dale I. Godfrey, Ian F. Hermans, Stephen J Turner, and Gavin F. Painter ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00845 • Publication Date (Web): 18 Oct 2017 Downloaded from http://pubs.acs.org on October 20, 2017

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ACS Chemical Biology

Augmenting influenza-specific T cell memory generation with a NKT cell-dependent glycolipid-peptide vaccine Regan J Anderson,1,‡ Jasmine Li,2, ‡ Lukasz Kedzierski,2, ‡ Benjamin J Compton,1 Colin M Hayman,1 Taryn L. Osmond,3 Ching-wen Tang,3 Kathryn, J. Farrand,3 Hui-Fern Koay,6,7 Catarina Filipa Dos Santos Sa E Almeida,6,7 Lauren, R. Holz,6 Geoffrey M. Williams,4 Margaret A Brimble,4,5 Zhongfang Wang,6 Marios Koutsakos,6 Katherine Kedzierska,6 Dale I Godfrey,6,7 Ian F Hermans,3,5,8 Stephen J Turner2* and Gavin F Painter1,8* 1

The Ferrier Research Institute, Victoria University of Wellington, PO Box 33436, Lower Hutt 5046, New Zealand

2

Department of Microbiology, Biomedical Discovery Institute, Monash University, Clayton, Australia

3

Malaghan Institute of Medical Research, PO Box 7060, Wellington 6242, New Zealand

4

School of Biological Sciences, The University of Auckland, 3 Symonds St, Auckland Central, 1142, New Zealand

5

Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, 3 Symonds St, Auckland Central, 1142, New Zealand

6

Department of Microbiology and Immunology, at the Doherty Institute for Infection and Immunity, The University of Melbourne, 792 Elizabeth St, Melbourne, Vic, Australia 7

Australian Research Council Centre of Excellence in Advanced Molecular Imaging, University of Melbourne, Parkville, Victoria 3010, Australia

8

Avalia Immunotherapies Limited, Gracefield Innovation Quarter, 69 Gracefield Rd, Lower Hutt 5010, New Zealand

KEYWORDS Lipophilic peptide vaccine, bioorthogonal conjugation, cell mediated immunity, influenza, crossstrain reactivity, synthetic vaccines ABSTRACT: The development of a universal vaccine for influenza A virus (IAV) that does not require seasonal modification is a long-standing health goal, particularly in the context of the increasing threat of new global pandemics. Vaccines that specifically induce T cell responses are of considerable interest because they can target viral proteins that are more likely to be shared between different virus strains and subtypes and hence provide effective cross-reactive IAV immunity. From a practical perspective, such vaccines should induce T cell responses with long-lasting memory, while also being simple to manufacture and cost-effective. Here we describe the synthesis and evaluation of a vaccine platform based on solid phase peptide synthesis and bio-orthogonal conjugation methodologies. The chemical approach involves covalently attaching synthetic long peptides from a virus-associated protein to a powerful adjuvant molecule, α-galactosylceramide (α-GalCer). Strain-promoted azide-alkyne cycloaddition is used as a simple and efficient method for conjugation, and pseudo-proline methodology is used to increase the efficiency of the peptide synthesis. α-GalCer is a glycolipid that stimulates NKT cells, a population of lymphoid-resident immune cells that can provide potent stimulatory signals to antigen-presenting cells engaged in driving proliferation and differentiation of peptide-specific T cells. When used in mice, the vaccine induced T cell responses that provided effective prophylactic protection against IAV infection, with the speed of viral clearance greater than that seen from previous viral exposure. These findings are significant because the vaccines are highly defined, quick to synthesize, and easily characterized, and are therefore appropriate for large scale affordable manufacture.

INTRODUCTION Seasonal influenza A virus (IAV) epidemics are estimated to cause 250,000 deaths worldwide with the young, old and unwell making up the high risk groups. Prophylactic seasonal vaccination is a proven strategy to prevent infection and lessen the health and economic burdens of the disease. The currently available vaccines generate neutralising antibodies against cell surface glycoproteins including hemagglutinin (HA) and neuraminidase (NA). However, due to selection pressure, viable mutant strains with changes in the glycoprotein structures develop each year that escape antibody-mediated neutralisation. A matching vaccine that generates antibody responses to the al-

tered glycoproteins therefore has to be prepared each season.1 Generally these vaccines demonstrate little crossstrain reactivity to other seasonal strains and therefore would be of limited value as prophylactic vaccines in a pandemic situation. In contrast to antibodies that recognize cell surface glycoproteins, IAV-specific T cells recognize epitopes predominantly from conserved internal viral proteins.2-4 The utility of IAV-specific T cell immunity stems from the fact that, unlike antibody responses, T cell immunity can target viral proteins that are more likely to be shared between different virus strains and subtypes and hence provide effective cross-reactive IAV immunity.5-6 This was

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evident with the 2009 H1N1 pandemic (2009 pdmH1N1) where pre-existing CD8+ T cell immunity was shown to correlate with less severe disease and protection from 2009 pdmH1N1 IAV infection.7 The development of novel vaccines that elicit broad-spectrum T cell-mediated immunity to IAV infection, particularly in the context of emerging IAV pandemics, is therefore of considerable interest. Synthetic vaccines based on antigenic peptides are attractive because they are highly defined and can be easily manufactured. When considering the design of such vaccines for the induction of T cell immunity, it is not only important that the peptides are delivered to professional antigen presenting cells (APC) and correctly processed and presented via MHC molecules to T cells, but also that the APC are correctly activated.8 Activation of APCs can be achieved by the addition of adjuvant compounds that stimulate pattern recognition receptors including the tolllike receptors (TLRs).9 Vaccine immunogenicity can be enhanced further by ensuring the vaccine components (i.e. antigen and adjuvant) are delivered to the same APCs, which has been achieved by chemically conjugating peptide antigens to various adjuvant compounds, or by formulating the vaccine components in delivery particles.10-11 In an alternative approach, APCs can be activated through stimulatory interactions with T cell populations with “innate-like” activity, such as natural killer T (NKT) cells which are found in high numbers in the lymphoid tissues, particularly the spleen and liver12 Type I NKT cells express a canonical T cell receptor (TCR) with an invariant TCR Vα chain structure and limited TCR Vβ chain diversity. They respond rapidly to specific glycolipid antigens presented by CD1d, an MHC class I-like structure that is conserved structurally in many mammals, including mice and humans.13 Injected compounds that bind CD1d and activate type I NKT cells ultimately drive the stimulatory interactions that activate APCs;14-16 these compounds can therefore be used as immune adjuvants.16 The glycolipid α-galactosylceramide (α-GalCer, Figure 1) is a prototypical synthetic antigen17 that provokes strong NKT cell activity in both humans and mice.18 In animal models, co-administering α-GalCer with protein or peptide antigens results in strong antigen-specific T cell responses that have been shown to exhibit antitumor activity15-16 and provide protection against infectious agents.19-22 Importantly, in the context of IAV infection, administration of α-GalCer together with inactivated IAV promotes the survival of long-lived memory CD8+ T cell populations capable of providing protection against heterologous IAV challenge.23-24

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humans, vaccine strategies that rely on minimal MHCbinding sequences will require patient selection based on appropriate HLA expression. An approach that avoids the need to stratify patients is to use a series of synthetic long peptides (SLPs) from the target protein covering the entire sequence, thereby ensuring CD4+ and CD8+ T cell epitopes are presented in all individuals.27 For proteins where sufficient immunogenicity data is available, it is likely that fewer peptides covering only the most immunogenic regions of the protein will be required. We present here a first step towards this goal in which we investigate a vaccine design that incorporates a SLP containing a known immunogenic MHC class I-binding epitope so that protection could be assessed against challenge with recombinant IAV expressing the same epitope. Long peptides are revealing substrates for conjugation, being intermediate in complexity between “small molecules” and “large molecules”. While their size and diverse functionality makes selective coupling a challenge, they are small enough that side-products with even minor modifications can be typically resolved and identified by LCMS. They therefore represent useful substrates to probe the limits of a given conjugation chemistry. To facilitate conjugation of SLPs to the α-GalCer derivative 1 we explored both copper-catalyzed azide alkyne cycloaddition (CuAAC) and strain-promoted alkyne azide cycloaddition (SPAAC) protocols.28-29 We found that using CuAAC conditions, conjugate formation was accompanied by significant product degradation, the extent of which was related to peptide length. On the other hand, SPAAC methodology using the bicyclo[6.1.0]nonyne (BCN) scaffold30 proved to be an excellent tool for the coupling of long peptides, providing exceptionally clean conjugate products. We show that vaccines based on this design augmented IAVspecific memory cytotoxic T lymphocyte (CTL) generation and this resulted in rapid control of IAV infection. Importantly, immunity induced by this novel vaccine strategy was superior to the cross-protective response induced by prior exposure to a serologically distinct recombinant IAV strain expressing the same antigen. Such vaccines therefore have potential to generate crossprotective immunological memory against conserved IAV proteins2-4 that is potentially superior to that induced after natural IAV exposure.

We have previously reported that NKT cell-dependent antigen-specific T cell responses can be boosted, and antitumor activity enhanced, by conjugation of MHC class Ibinding CD8+ T cell epitopes to an α-GalCer derivative, 1, via a cleavable linker (Figure 1).25-26 Enzymatic cleavage of the linker in APCs releases amine 1, which subsequently rearranges to give α-GalCer, and peptide antigen. However, due to the high polymorphism of HLA molecules in


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ACS Chemical Biology thesis that incorporated a leucine-threonine-derived oxazolidine dipeptide (“pseudoproline”)34 in the place of residues Leu5-Thr4 significantly improved the synthesis (SI Figure 1B), which could be further improved by installation of a second pseudoproline at Glu13-Ser12. However these changes led to very slow couplings of His25 and His22, and the extended reaction times resulted in significant racemisation (SI Figure 1C). Fortunately this could be largely avoided by use of collidine as the base instead of N-methylmorpholine (SI Figure 1D). With these combined modifications, a much-improved HPLC profile of crude peptide was obtained (SI Figure 1D), and the desired peptides 2 and 3 could be readily prepared in high purity

Figure 1. Vaccine design and in vivo metabolism. (XY = motif resulting from chemoselective ligation). Recombinant IAVs containing an immunogenic peptide from chicken ovalbumin (OVA257; amino acid sequence SIINFEKL, presented by H2Kb) in the neuraminidase stalk were available to assess vaccine efficacy.31 Therefore, to investigate the utility of vaccines based on α-GalCeradjuvanted SLPs, peptides were prepared that contained OVA257 within a significantly longer sequence including a known CD4+ T cell epitope (although CD4+ T cell responses were not specifically explored here).32 The SLPs also contained the known protease cleavage sequence FFRK towards the N-terminus,33 a modification considered useful for a general vaccine platform to ensure release of epitopes at (or near to) the N-terminus of SLPs (see schemes 1 and 2 for peptide sequences). The vaccine design relies critically on chemoselective ligation methods to bring together the SLP and glycolipid-linker components. We have previously reported on the use of both oxime ligation and CuAAC for the construction of similar vaccines with short peptides, in reaction conditions that required considerable optimisation.25 Here we elected to focus initially on CuAAC methodology, due to the ease of synthesis of the required N-terminal alkynyl/azido peptides, compared to the N-terminal aminooxyacetyl peptides required for oxime ligation. These studies reveal that this novel vaccine adjuvant provides superior cross strain protection against IAV compared to immunisation with the virus itself.

Conjugation of SLP with modified α-GalCer using CuAAC methodology. In the initial studies the previously reported azide 4 was used as the coupling partner (Scheme 1). This compound contains a migrated α-GalCer component (the pro-adjuvant), a self immolative paraaminobenzyl carbamate (PAB) linker connected to the Val-Cit cathepsin recognition motif and an azido group for chemical conjugation. LCMS was used to monitor progress of the reaction of azide 4 with alkyne 3. No product was formed in the first 4 h, due to the need to generate sufficient quantities of catalytically active Cu(I) from metallic Cu (SI Figure 2a). After this time, the reaction progresses rapidly, however in addition to the TM 5, side-products develop over time. MS analysis of these peaks indicates they are products of oxidation35 (TM+16, TM+32 Da) and peptide cleavage (TM-2850 Da = loss of HAAHAEINEAGRESIINFEKLTEWT) (SI Figure 2b-d). Plotting conversion of starting material and yield of 5 over time (SI Figure 2b) indicated that at 85% conversion (8 h), the yield of TM was only 46% and gradually declined from this maximal value with longer reaction times. Reactions could be shortened by using a greater excess of the peptide component, however even small amounts of sideproducts severely impacted the yield and final purity of the product obtained after preparative HPLC. Alternative conditions similar to those developed by Hong et al.,28 which are designed to minimize oxidative side-reactions, did not result in any triazole formation.[§] Other reported conditions, involving the use of excess Cu(II), TCEP and GnHCl, were also ineffective – with or without the inclusion of TBTA, and in a range of solvent systems at rt-50 °C.36 Scheme 1. The synthesis of vaccine 5 via CuAAC methodology

RESULTS AND DISCUSSION Peptide Synthesis. The initial approach to peptides 2 and 3 entailed a conventional synthetic pass of the SLP using standard Fmoc coupling conditions (HCTU/NMM/DMF) for each amino acid residue followed by a capping step. Cleavage from the resin and HPLC-MS analysis revealed a disappointingly complex peptide profile (SI Figure 1A), which included assorted minor deletion products and a number of significant Nacetylated truncations at several points. A modified syn-



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Conjugation of SLP with modified α-GalCer using SPAAC methodology Due to the difficulties encountered in applying the CuAAC methodology to SLPs we turned to a metal free approach that utilizes a bicyclo[6.1.0]nonyne (BCN) ringstrained alkyne30 that reacts with azide groups to relieve ring strain– a bioorthogonal methodology commonly referred to as SPAAC. Due to the extra complexity in the alkyne it was decided to incorporate this component in the glycolipid linker and install the azide group at the peptide N-terminus. To install the BCN group on the modified glycolipid, we developed a one-pot procedure in which amine 637 and BCN reagent 7 (1.1 equiv) were allowed to react in DMF for 24 h, followed by the addition of bis(4-nitrophenyl) carbonate to derivatize the hydroxyl group (Scheme 2). Activated carbonate 8 was thus obtained in 55% yield. Reaction with amine 1 afforded the cyclooctyne-modified glycolipid 9 in 66% yield after purification by silica chromatography.

Scheme 2 The chemical synthesis of vaccine 10 via SPAAC methodology

Conjugation with azido peptide 2 (1.2 equiv) was simply achieved by mixing the reactants in DMSO (10 mM) at room temperature. Whereas the analogous CuAAC reaction required a ternary solvent system to solubilize the reactants and promote the cycloaddition, neat DMSO proved effective for the SPAAC reaction and allowed higher reactant concentrations to be achieved. HPLC

analysis of the SPAAC reaction indicated full conversion to a single product after 28 h. The reaction solution was used directly for HPLC purification without workup affording conjugate 10 in 58% yield and >99% purity (HPLC-CAD). Importantly, in contrast to the CuAAC reaction conditions we explored and although generally not required, SPAAC reaction mixtures could be left for ex-


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tended times without the appearance of product impurities. Compound 9 has served as a useful advanced intermediate in our wider chemical immunology program. It can be readily made on a 100-200 mg scale, stored for months without noticeable degradation (-20 °C, Ar), and is conjugated with ease to any azido peptide (> 30 examples) for biological studies. In all cases, very clean crude products are obtained and after removal of excess peptide, products are typically isolated with >95% purity. A critical feature of the vaccine design is that the rearranged α-GalCer derivative, 1 used in the conjugation process, which is inactive due to a migrated acyl chain, is cleaved from the SLPs by intracellular enzymes within APCs, and then undergoes spontaneous rearrangement to form active α-GalCer. The active compound can then be presented via CD1d to stimulate the licensing activity of NKT cells. Vaccines 5 and 10 activate mouse NKT cells in vivo. To assess whether the SLP-conjugate vaccines were indeed capable of activating NKT cells in vivo, 5 and 10 were injected into mice and then the level of accumulation of splenic NKT cells, and their phenotype, was assessed by flow cytometry with fluorescent α-GalCer-loaded CD1d tetramers. Animals injected with unmodified α-GalCer served as positive controls. Upregulation of CD69, which is indicative of NKT cell activation, was detected on tetramer+ cells 3 h after injection of α-GalCer, but this was not seen after injection of the conjugates (SI Figure 3). However, by 72 h it was possible to detect significantly expanded NKT cell populations in animals treated with the conjugates, suggesting NKT cell activation had indeed taken place (and perhaps the peak of CD69 had been missed). Furthermore, as is typically seen after α-GalCer treatment, expression of both CD69 and NK1.1 on the NKT cells was substantially reduced by 72 h, in some cases reaching levels significantly below those seen in vehicle-treated controls. These data provide strong evidence that the conjugates do release an active NKT cell agonist in vivo. Method of vaccine manufacture (i.e. CuAAC vrs SPAAC) does not influence T cell priming. To ensure that the vaccines could induce potent T cell responses, an in vivo assay of cytotoxic activity against OVA257-loaded target cells was used.38 Both vaccines induced responses that efficiently eradicated peptide-loaded target cells and this activity was lost in CD1d-deficient animals, which do not harbor NKT cells (Figure 2).38 The SLP-conjugates were therefore effectively processed in vivo to release both the MHC class I-binding CD8+ T cell epitope and a NKT cell agonist. As the T cell activity induced by the two vaccines was comparable, it was clear that the larger hydrophobic tricyclic structure formed as a result of the SPAAC chemistry did not negatively impact vaccine activity. Due to the ease of synthesis using SPAAC methodology, this form of vaccine was used for the ongoing biological studies.

Figure 2. Assessment of in vivo activity of SLP-conjugate vaccines. Assessment of cytotoxic responses assessed seven days after administration of vaccines 5 and 10 into wildtype -/or CD1d recipients. Cytotoxic activity was assessed on injected fluorescent syngeneic splenocytes that had been loaded with OVA257 peptide. An additional population of fluorescent splenocytes without peptide served as internal negative control. Lysis was assessed by flow cytometry on blood, and is expressed as percent reduction in peptide-loaded cells relative to unloaded control. Data from five mice per group are shown with mean percentage of specific lysis ± SEM indicated. * p< 0.05.

Vaccine 10 induces memory T cells. To test if vaccine 10 could induce long-term memory T cells, 1 x 104 naïve OVA257-specific TCR transgenic CD8+ T cells (OT-I cells) were adoptively transferred into recipient animals 1 day prior to vaccination. This model system ensures that OT-I CTL respond both numerically and functionally, in an equivalent way to a normal endogenous influenza CD8+ T cell response.32,39 To investigate the relevance of conjugation on memory formation, additional groups of transfer recipients were vaccinated with SLP alone, α-GalCer alone, or an admix of unconjugated SLP and α-GalCer. Furthermore, to investigate how the memory phenotype of the vaccine-induced response compared to a response known to provide protection against IAV, an additional group was challenged with A/PR8-OVA which is known to induce an OVA257-specific memory response that can provide cross-protection against challenge with a second serologically distinct OVA-expressing strain.40 Spleens and livers were harvested on days 7, 10, 21 and 60 after vaccination (or challenge) and the proportion and number of OT-I T cells (CD45.1+ CD8+) in the spleen were determined by flow cytometry (SI Figure 4). Spleen and liver samples were also examined for NKT cell status. The NKT cells in all groups showed reduced NK1.1 and CD69 expression, confirming that the α-GalCer component had worked as expected in these mice (SI Figure 5). The conjugate vaccine induced a primary CD8+ T cell response that was proportionally and numerically similar to that observed after challenge with A/PR8-OVA (Figure 3). Moreover, a similar level of both early (day 21) and late (day 60) T cell memory was also induced after vaccination. The CD8+ OT-I T cell response was below the level of detection when unconjugated vaccine components were administered (SI Fig.4).



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a measure of protection provided against secondary IAV challenge (Fig. 4, diamonds) and this correlated with a strong secondary OTI response in the BAL together with a weaker response in the spleen (SI Fig. 6). This suggests that there was priming with the α-GalCer peptide admix but it was not as efficient as vaccine 10.

Figure 3. Evaluation of primary response induced by vaccine construct 10 in comparison to challenge with A/PR8-OVA. Shown are the proportion (A) and number (B) of OT-I+ CD8+ T cells isolated at day 7 (black bars), day 10 (striped bars), day 21 (grey) and day 60 (hatched) after vaccination. Shown is mean +/- SD, n=5 mice per group/per time point. Representative of 2 biological replicates.

A single administration of vaccine 10 provides effective immunity against IAV challenge. To determine whether administration of the vaccine conjugate was able to provide protective immunity against IAV challenge, mice that had received either conjugate vaccine, its components or had been primed with A/PR8-OVA were challenged intranasally with HKx31-OVA257 (H3N2), which is serologically distinct to A/PR8-OVA but expresses the same antigen (OVA257) in the neuraminidase stalk.40 Mice were weighed daily as an indirect measure of morbidity. Lungs were removed at specific time points after infection to determine viral load using MDCK plaque assay, and also to determine T cell numbers and phenotype by flow cytometry. Mice that received α-GalCer alone exhibited the greatest weight loss (Fig 4, squares) commensurate with exhibiting the greatest levels of viral load at day 4 (Fig. 5, squares). All other groups exhibited similar weight loss with maximal loss at days 4-5. Animals challenged first with either A/PR8-OVA or vaccine 10 exhibited a more rapid recovery of weight (Fig. 4). This correlated with robust and rapid secondary OTI CD8+ T cell responses highlighting the relevance of virus-induced CD8+ T cell-mediated cross-protection (SI Fig. 6).Interestingly, while mice that were vaccinated with the α-GalCer peptide admix did not exhibit measurable CD8+ T cell responses during the primary response or a readily measurable memory CTL population (SI Fig. 4), there was clearly

Fig. 4. Assessment of IAV-associated morbidity after challenge in vaccinated mice. Mice that had received naïve OT-I T cells were administered the following agents, αGalCer alone, (squares); αGalCer+OVA peptide, (diamonds); vaccine 10, (triangles) or A/PR8-OVA (circles). All 4 treated mice were then challenged with 10 plaque forming units (pfu) A/HKx-31-OVA 40 days after initial vaccination and weight loss measured daily. Results are shown as % body weight from initial starting body weight measured prior to virus challenge.

In terms of viral load, animals injected with the vaccine 10 demonstrated substantially accelerated viral clearance by day 4 compared to all other treatment groups, suggesting early control of IAV infection. All mice that had received vaccine 10 exhibited complete viral clearance whereas only 2/4 mice previously primed by exposure to A/PR8OVA exhibited clearance. By day 6 all mice in the A/PR8OVA-primed group had completely cleared the infection, whereas complete clearance was only seen after 9 days in animals that received either α-GalCer alone, or the admixed unconjugated vaccine components. Interestingly, the more rapid clearance of IAV in vaccine 10 primed mice correlated with establishment of a proportion of effector memory (TEM, CD62LloCD44hi) OTI cells in the memory pool (SI Fig. 7). While OTI numbers were too few at days 2 and 4, making any firm conclusion difficult, there did appear to be a trend for a greater proportion of CD62LloCD44hi OTI cells in the spleens of vaccine 10 vaccinated mice and A/PR8-OVA challenged mice after secondary challenge compared with the admixed or α-GalCer only groups (SI Fig. 8). This coincided with a greater proportion of effector OTIs in the BAL at day 6 (SI Fig. 6) upon re-challenge. IAV-specific TEM cells exhibit more rapid migration to inflamed lungs early after secondary challenge.39 Hence vaccine 10 appears to promote formation of a memory pool dominated by TEM phenotype capable of more rapid recruitment and hence control of secondary IAV infection.


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Overall these results indicate that the conjugate vaccine generates a cellular immune response that is at least commensurate with, if not superior to, that observed in animals with a virus-induced memory response.

R.J.A. was primarily responsible for the chemical synthesis with contributions from B.J.C. and C.M.H. G.W. and M.A.B. were responsible for peptide synthesis. L.K. and J.L. carried out the mouse vaccination, viral challenge and memory T cell analysis studies, with contributions from K.K., M.K., Z.W., L.H., and T.K. T.O., C.T. and K.F. carried out the NKT cell and cytotoxicity assays. C.F.D.S.S.E.A and H.F.K carried out the NKT cell analysis in the vaccination experiments. G.F.P. led the chemical component of the investigation, S.J.T. lead the influenza studies and D.I.G and I.F.H. lead the NKT cell studies. D.I.G., G.F.P., I.F.H and S.J.T conceptualised and planned the project. The manuscript was drafted with editorial contributions from all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally.

Conflict of Interest

Fig. 5. Assessment of viral load in lung after challenge in vaccinated mice. Experimental set-up as in Fig 4. Lung viral titres were measured by MDCK plaque assay. Results shown are the number of PFU per mL of lung homogenate for individual mice. The small horizontal lines represent the mean for each group.

CONCLUSION A synthetic vaccine design based on model IAV SLP antigens conjugated to a NKT cell agonist not only results in the induction of effective CTL immunity in a murine model, similar to that observed after natural IAV infection, but in fact results in more rapid viral clearance when compared to those previously exposed to a serologically distinct IAV strain known to provide cross-protection. It will therefore be of interest to develop this approach to actual IAV antigens which will, eventually, likely comprise a series of overlapping SLPs covering the most immunogenic regions of viral proteins that are the major targets of immunity. The chemical synthesis utilized pseudo-proline methodology to dramatically improve the efficiency of the SLP synthesis and SPAAC conjugation to enable facile vaccine synthesis. Based on these results, this chemistry should enable the rapid synthesis of a suite of conjugates covering an entire target-protein sequence, utilizing cyclooctyne 9 as a common advanced intermediate.

G.F.P. and I.F.H. are the CTO and CSO respectively of biotech start-up Avalia Immunotherapies Limited and D.I.G. is a member of its Scientific Advisory Board. Availa holds exclusive, world-wide license to patents related to aspects of the chemical design reported here. Avalia partially funded the study.

Notes § Conditions: alkynyl peptide (2.3 mM), BnN3 (2.8 mM), TBTA (1.3 mM), CuSO4 (0.25 mM), sodium ascorbate (2.5 mM), DMSO/MeOH/CHCl3/H2O (3:3:3:1), 20 °C, 4 d.

ACKNOWLEDGMENTS The authors wish to acknowledge the New Zealand Ministry of Business Innovation and Employment (grant RTV1603), Genesis oncology trust (grant GOT-1548-RPG), Avalia Immunotherapies and the Health Research Council of New Zealand (grant HRC 14/500) for financial support. Monomer supplied by the NIH Tetramer Core Facility was used to prepare α-GalCer-loaded CD1d tetramer. S.J.T, K.K and D.I.G. are supported by NHMRC program grants 1071916, 1013667 and 1113293). K.K is supported by an NHMRC Senior Research fellowship; S.J.T is supported by an NHMRC Principal Research Fellowship and D.I.G is supported by an NHMRC Senior Principal Research Fellowship (1117766).

Supporting Information. Synthetic procedures and analytical data for vaccine synthesis, the material and methods for the biological experiments and supplementary figures are available free of charge via the Internet at http://pubs.acs.org.

ASSOCIATED CONTENT ABBREVIATIONS

AUTHOR INFORMATION Corresponding Author

TM, target material; MDCK, Madin-Darby Canine Kidney Epithelial Cells.

*[email protected] and/or *[email protected]

REFERENCES

Present Addresses †If an author’s address is diﬀerent than the one given in the affiliation line, this information may be included here.

Author Contributions

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N N HO

OH O

HO

HN HO

enzymatic cleavage

O

O

C25H51OCO

O OH

N

Peptide Antigen

in vivo

Peptide Antigen HO

OH O

HO N Val-Cit C O H O

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HO

O

NHCOC 25H51 OH

C14H29

Adjuvant

Synthetic Vaccine

HO

C14H29


Influenzaspecific CTLs

10

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


N N HO

OH O

HO

HN HO

enzymatic cleavage

O

O

C25H51OCO

O OH

N

Peptide Antigen

Peptide Antigen in vivo

HO

OH O

HO N Val-Cit C O H O

HO

O

NHCOC25H51 OH

C14H29

Adjuvant

HO

C14H29

Synthetic Vaccine


Influenzaspecific CTLs

Augmenting Influenza-Specific T Cell Memory Generation with a

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