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In the fast-evolving landscape of targeted cancer therapies, the revolutionary class of biotherapeutics known as antibody-drug conjugates (ADCs) are taking center stage. Most clinically approved ADCs utilize cleavable linkers to temporarily attach potent cytotoxic payloads to antibodies, allowing selective payload release under tumor-specific conditions. In this study, we explored the utilization of 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde), a cyclic ß-diketone featuring an active alkylidene group, to develop a novel chemically labile linker. This linker was designed to exploit the difference in reduction potential between the intracellular compartment and plasma. Upon reduction of an azido trigger strategically installed neighboring the cyclic ß-diketone, the resulting nucleophilic primary amine reacts with the alkylidene group facilitated by a favorable ring closure reaction in accordance with Baldwin's rules. Consequently, this reaction enables the simultaneous release of the attached cytotoxic payload. The therapeutic utility of this novel linker strategy was demonstrated by separate conjugation of the linker to two epidermal growth factor receptor (EGFR)-targeting ligands to afford a peptide-drug conjugate and an ADC. This work comprises a significant contribution to the bioconjugation field by introducing the alkylidene cyclic ß-diketone as a tunable scaffold used for the temporary conjugation of therapeutic agents to peptides and proteins.
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AIM: We have previously reported that polyfunctional T cell responses can be induced to the cancer testis antigen NY-ESO-1 in melanoma patients injected with mature autologous monocyte-derived dendritic cells (DCs) loaded with long NY-ESO-1-derived peptides together with α-galactosylceramide (α-GalCer), an agonist for type 1 Natural Killer T (NKT) cells. OBJECTIVE: To assess whether inclusion of α-GalCer in autologous NY-ESO-1 long peptide-pulsed DC vaccines (DCV + α-GalCer) improves T cell responses when compared to peptide-pulsed DC vaccines without α-GalCer (DCV). DESIGN, SETTING AND PARTICIPANTS: Single-centre blinded randomised controlled trial in patients ≥ 18 years old with histologically confirmed, fully resected stage II-IV malignant cutaneous melanoma, conducted between July 2015 and June 2018 at the Wellington Blood and Cancer Centre of the Capital and Coast District Health Board. INTERVENTIONS: Stage I. Patients were randomised to two cycles of DCV or DCV + α-GalCer (intravenous dose of 10 × 106 cells, interval of 28 days). Stage II. Patients assigned to DCV + α-GalCer were randomised to two further cycles of DCV + α-GalCer or observation, while patients initially assigned to DCV crossed over to two cycles of DCV + α-GalCer. OUTCOME MEASURES: Primary: Area under the curve (AUC) of mean NY-ESO-1-specific T cell count detected by ex vivo IFN-γ ELISpot in pre- and post-treatment blood samples, compared between treatment arms at Stage I. Secondary: Proportion of responders in each arm at Stage I; NKT cell count in each arm at Stage I; serum cytokine levels at Stage I; adverse events Stage I; T cell count for DCV + α-GalCer versus observation at Stage II, T cell count before versus after cross-over. RESULTS: Thirty-eight patients gave written informed consent; 5 were excluded before randomisation due to progressive disease or incomplete leukapheresis, 17 were assigned to DCV, and 16 to DCV + α-GalCer. The vaccines were well tolerated and associated with increases in mean total T cell count, predominantly CD4+ T cells, but the difference between the treatment arms was not statistically significant (difference - 6.85, 95% confidence interval, - 21.65 to 7.92; P = 0.36). No significant improvements in T cell response were associated with DCV + α-GalCer with increased dosing, or in the cross-over. However, the NKT cell response to α-GalCer-loaded vaccines was limited compared to previous studies, with mean circulating NKT cell levels not significantly increased in the DCV + α-GalCer arm and no significant differences in cytokine response between the treatment arms. CONCLUSIONS: A high population coverage of NY-ESO-1-specific T cell responses was achieved with a good safety profile, but we failed to demonstrate that loading with α-GalCer provided an additional advantage to the T cell response with this cellular vaccine design. CLINICAL TRIAL REGISTRATION: ACTRN12612001101875. Funded by the Health Research Council of New Zealand.
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Melanoma , Neoplasias Cutáneas , Masculino , Humanos , Adolescente , Neoplasias Cutáneas/terapia , Neoplasias Cutáneas/metabolismo , Péptidos/metabolismo , Anticuerpos/metabolismo , Citocinas/metabolismo , Células Dendríticas , Antígenos de Neoplasias , Melanoma Cutáneo MalignoRESUMEN
Agonists of Toll-like Receptor 2 (TLR2) are attractive synthetic targets due to their use as adjuvants in immunotherapies to treat various diseases notably, cancer. An indepth understanding of TLR2 agonist structure-activity relationships is therefore advantageous for the methodical design of vaccines targetting the TLR2 machinery. This review aims to collate and discuss the literature regarding synthetic studies towards TLR2 agonists and the structure-activity relationships thereof. It is hoped that interested readers will gain a holistic understanding of this topic, and will prompt further efforts towards finding effective agonists of TLR2.
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Receptor Toll-Like 2/agonistas , Adyuvantes Inmunológicos/farmacología , Humanos , Ligandos , Lipopéptidos/química , Lipopéptidos/farmacología , Relación Estructura-Actividad , Vacunas/síntesis químicaRESUMEN
Activated NKT cells can stimulate antigen-presenting cells leading to enhanced peptide antigen-specific immunity. However, administration of potent NKT cell agonists like α-galactosylceramide (α-GalCer) can be associated with release of high levels of cytokines, and in some situations, hepatotoxicity. Here we show that it is possible to provoke sufficient NKT cell activity to stimulate strong antigen-specific T cell responses without these unwanted effects. This was achieved by chemically conjugating antigenic peptides to α-galactosylphytosphingosine (α-GalPhs), an NKT cell agonist with very weak activity based on structural characterisation and biological assays. Conjugation improved delivery to antigen-presenting cells in vivo, while use of a cathepsin-sensitive linker to release the α-GalPhs and peptide within the same cell promoted strong T cell activation and therapeutic anti-tumour responses in mice. The conjugates activated human NKT cells and enhanced human T cell responses to a viral peptide in vitro. Accordingly, we have demonstrated a means to safely exploit the immunostimulatory properties of NKT cells to enhance T cell activation for virus- and tumour-specific immunity.
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Células Presentadoras de Antígenos/inmunología , Vacunas contra el Cáncer/administración & dosificación , Células T Asesinas Naturales/efectos de los fármacos , Células T Asesinas Naturales/inmunología , Neoplasias Experimentales/inmunología , Péptidos/administración & dosificación , Adyuvantes Inmunológicos , Animales , Antígenos CD1d/química , Vacunas contra el Cáncer/inmunología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Epítopos/química , Glucolípidos/química , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Neoplasias Experimentales/tratamiento farmacológico , Péptidos/química , Péptidos/inmunologíaRESUMEN
Vaccines that elicit targeted tumor antigen-specific T-cell responses have the potential to be used as adjuvant therapy in patients with high risk of relapse. However, the responses induced by vaccines in cancer patients have generally been disappointing. To improve vaccine function, we investigated the possibility of exploiting the immunostimulatory capacity of type 1 Natural killer T (NKT) cells, a cell type enriched in lymphoid tissues that can trigger improved antigen-presenting function in dendritic cells (DCs). In this phase I dose escalation study, we treated eight patients with high-risk surgically resected stage II-IV melanoma with intravenous autologous monocyte-derived DCs loaded with the NKT cell agonist α-GalCer and peptides derived from the cancer testis antigen NY-ESO-1. Two synthetic long peptides spanning defined immunogenic regions of the NY-ESO-1 sequence were used. This therapy proved to be safe and immunologically effective, inducing increases in circulating NY-ESO-1-specific T cells that could be detected directly ex vivo in seven out of eight patients. These responses were achieved using as few as 5 × 105 peptide-loaded cells per dose. Analysis after in vitro restimulation showed increases in polyfunctional CD4+ and CD8+ T cells that were capable of manufacturing two or more cytokines simultaneously. Evidence of NKT cell proliferation and/or NKT cell-associated cytokine secretion was seen in most patients. In light of these strong responses, the concept of including NKT cell agonists in vaccine design requires further investigation.
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Antígenos de Neoplasias/genética , Células Dendríticas/inmunología , Galactosilceramidas/inmunología , Melanoma/inmunología , Proteínas de la Membrana/genética , Antígenos de Neoplasias/metabolismo , Humanos , Proteínas de la Membrana/metabolismoRESUMEN
Peptide and protein aberrant lipidation patterns are often involved in many diseases including cancer and neurological disorders. Peptide lipidation is also a promising strategy to improve pharmacokinetic and pharmacodynamic profiles of peptide-based drugs. Self-adjuvanting peptide-based vaccines commonly utilise the powerful TLR2 agonist PamnCys lipid to stimulate adjuvant activity. The chemical synthesis of lipidated peptides can be challenging hence efficient, flexible and straightforward synthetic routes to access homogeneous lipid-tagged peptides are in high demand. A new technique coined Cysteine Lipidation on a Peptide or Amino acid (CLipPA) uses a 'thiol-ene' reaction between a cysteine and a vinyl ester and offers great promise due to its simplicity, functional group compatibility and selectivity. Herein a brief review of various synthetic strategies to access lipidated peptides, focusing on synthetic methods to incorporate a PamnCys motif into peptides, is provided.
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Aminoácidos/química , Cisteína/química , Lípidos/química , Péptidos/química , Adyuvantes Inmunológicos/química , Secuencia de Aminoácidos , Modelos Químicos , Estructura Molecular , Péptidos/síntesis química , Vacunas/síntesis química , Vacunas/químicaRESUMEN
Two analogues of insulin glargine containing a 1,4-disubstituted 1,2,3-triazole group in place of the CysA7-CysB7 disulfide bond were prepared using CuAAC click chemistry to efficiently join the peptide chains. The resulting insulin analogues were analysed by circular dichroism spectroscopy to assess whether this modification compromised the folding pattern of the native form. Investigations, including an in vivo murine study, revealed that these analogues were not biologically active and that the structures were significantly unfolded, an outcome which suggests that maintaining a precise inter-chain distance is critical to the structure of the insulin hormone.
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Cisteína/química , Disulfuros/química , Insulina Glargina/química , Desplegamiento Proteico , Triazoles/química , Secuencia de Aminoácidos , Animales , Glucemia/metabolismo , Insulina Glargina/síntesis química , Insulina Glargina/farmacología , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de ProteínaRESUMEN
A considerable quantity of an alkylation by-product is observed when using 3,6-dioxa-1,8-octanedithiol as a scavenger during acidic release of peptides containing the thioether amino acid methionine from the solid support. Adjustment of the cleavage conditions by replacement of 3,6-dioxa-1,8-octanedithiol with ethane dithiol or by using methionine sulfoxide as an alternative to methionine resulted in no such impurity. The by-product was detectable by liquid chromatography and mass spectrometry and characterised by NMR spectroscopy of an isolated model peptide. It could be effectively removed in a separate post cleavage step by treatment with dilute aqueous acid at 37 °C.
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Éteres de Etila/química , Fluorenos/química , Técnicas de Síntesis en Fase Sólida/métodos , Compuestos de Sulfhidrilo/química , Espectrometría de MasasRESUMEN
Spiropins for SPPS: The rigid structure of an anomerically stabilised spiroketal motif enables the appendage of substituents in a fixed conformation. To assess the ability of a spiroketal motif to induce a turn structure and participate in solid-phase peptide synthesis (SPPS), an Fmoc-spiroketal amino acid was synthesised and incorporated into a spiroketal-containing cyclic peptide.
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Aminoácidos/síntesis química , Furanos/síntesis química , Péptidos/síntesis química , Técnicas de Síntesis en Fase Sólida/métodos , Compuestos de Espiro/síntesis química , Secuencia de Aminoácidos , Aminoácidos/química , Furanos/química , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Compuestos de Espiro/químicaRESUMEN
Diabetes mellitus, characterised by hyperglycemia and altered ß-cell function, is an increasingly common disorder affecting millions of individuals world-wide. While therapeutic regimens exist to manage the condition, diabetic individuals remain prone to complications that are detrimental to both their length and quality of life. An improved understanding of the disease which may then enable development of new treatments is therefore a desirable goal. Vesiculin, a novel IGF-II-like protein was recently isolated from the secretory granules of murine ß-cells, and preliminary studies indicate it is capable of signalling via the insulin receptor (IR)/insulin-like growth factor receptor 1(IGF1R) family giving it the potential to elicit both metabolic and mitogenic responses in the beta-cell. In order to facilitate further studies on this new member of the insulin-family of hormones, we undertook a chemical synthesis of the protein using regioselective disulfide bond formation.
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Disulfuros/síntesis química , Factor II del Crecimiento Similar a la Insulina/química , Proteínas del Tejido Nervioso/síntesis química , Animales , Disulfuros/química , Relación Dosis-Respuesta a Droga , Glucógeno/biosíntesis , Glucógeno/química , Masculino , Ratones , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/farmacología , Ratas , Ratas Sprague-Dawley , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
A radical lipidation: Application of a novel thiol-ene lipidation enables the one-step synthesis of self-adjuvanting antigenic peptides as vaccine candidates. The resultant monoacyl lipopeptides are shown to activate monocytes in a robust manner.
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Lipopéptidos/síntesis química , Lipopéptidos/inmunología , Vacunas/síntesis química , Antígenos/química , Antígenos/inmunología , Humanos , Péptidos/química , Péptidos/inmunología , Estereoisomerismo , Compuestos de Sulfhidrilo/química , Vacunas/farmacologíaRESUMEN
Society is confronted by interconnected threats to ecological sustainability. Among these is the devastation of forests by destructive non-native pathogens and insects introduced through global trade, leading to the loss of critical ecosystem services and a global forest health crisis. We argue that the forest health crisis is a public-good social dilemma and propose a response framework that incorporates principles of collective action. This framework enables scientists to better engage policymakers and empowers the public to advocate for proactive biosecurity and forest health management. Collective action in forest health features broadly inclusive stakeholder engagement to build trust and set goals; accountability for destructive pest introductions; pooled support for weakest-link partners; and inclusion of intrinsic and nonmarket values of forest ecosystems in risk assessment. We provide short-term and longer-term measures that incorporate the above principles to shift the societal and ecological forest health paradigm to a more resilient state.
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Ecosistema , Médicos , Humanos , Bosques , Bioaseguramiento , Medición de RiesgoRESUMEN
The efficient synthesis of multivalent neoglycoconjugates of MUC1 is reported, which utilizes Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuACC) of azide-functionalized GlcNAc-centered neoglycotetrasaccharide clusters to the MUC1 peptide sequence that was equipped with a propargylglycine residue for "click chemistry". In turn the azido-GlcNAc-centered neoglycoclusters were assembled by reaction of a GlcNAc core containing peripheral propargyl functionalities with an appropriate azido-functionalized monosaccharide. The resulting suitably substituted tetrasaccharyl triazole cluster can be easily appended to a range of acetylene-functionalized peptides to produce neoglycoconjugates of biologically important glycopeptides. As proof of principle, the click neoglycoclusters prepared herein were ligated to the MUC1 peptide sequence.
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Carbohidratos/química , Glicoconjugados/química , Mucina-1/química , Péptidos/síntesis química , Triazoles/química , Alquinos/química , Azidas/química , Catálisis , Química Clic , Cobre/química , Ciclización , Conformación Molecular , Péptidos/químicaRESUMEN
The synthesis of naturally occurring glycosylated (2S,5R)-hydroxylysine still remains a challenge. This perspective highlights the importance of this post-translationally modified amino acid residue in the observed bioactivity of collagen and related collagen-like proteins such as adiponectin, an important target for the treatment of type II diabetes. Strategies employed to date for the syntheses of (2S,5R)-hydroxylysine and the methods to effect glycosylation of this modified amino acid are also summarized herein.
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Adiponectina/química , Hidroxilisina/análogos & derivados , Hidroxilisina/síntesis química , Glicosilación , Hidroxilisina/química , Conformación Molecular , EstereoisomerismoRESUMEN
Pancreatic islet-cell function and volume are both key determinants of the maintenance of metabolic health. Insulin resistance and islet-cell dysfunction often occur in the earlier stages of type 2 diabetes (T2D) progression. The ability of the islet cells to respond to insulin resistance by increasing hormone output accompanied by increased islet-cell volume is key to maintaining blood glucose control and preventing further disease progression. Eventual ß-cell loss is the main driver of full-blown T2D and insulin-dependency. Researchers are targeting T2D with approaches that include those aimed at enhancing the function of the patient's existing ß-cell population, or replacing islet ß-cells. Another approach is to look for agents that enhance the natural capacity of the ß-cell population to expand. Here we aimed to study the effects of a new putative ß-cell growth factor on a mouse model of pre-diabetes. We asked whether: 1) 4-week's treatment with vesiculin, a two-chain peptide derived by processing from IGF-II, had any measurable effect on pre-diabetic mice vs vehicle; and 2) whether the effects were the same in non-diabetic littermate controls. Although treatment with vesiculin did not alter blood glucose levels over this time period, there was a doubling of the Proliferating Cell Nuclear Antigen (PCNA) detectable in the islets of treated pre-diabetic but not control mice and this was accompanied by increased insulin- and glucagon-positive stained areas in the pancreatic islets.
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Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Islotes Pancreáticos , Estado Prediabético , Animales , Insulina , Factor II del Crecimiento Similar a la Insulina , Ratones , Proteínas del Tejido NerviosoRESUMEN
Objectives: Metastasis is the principal cause of breast cancer mortality. Vaccines targeting breast cancer antigens have yet to demonstrate clinical efficacy, and there remains an unmet need for safe and effective treatment to reduce the risk of metastasis, particularly for people with triple-negative breast cancer (TNBC). Certain glycolipids can act as vaccine adjuvants by specifically stimulating natural killer T (NKT) cells to provide a universal form of T-cell help. Methods: We designed and made a series of conjugate vaccines comprising a prodrug of the NKT cell-activating glycolipid α-galactosylceramide covalently linked to tumor-expressed peptides, and assessed these using E0771- and 4T1-based breast cancer models in vivo. We employed peptides from the model antigen ovalbumin and from clinically relevant breast cancer antigens HER2 and NY-ESO-1. Results: Glycolipid-peptide conjugate vaccines that activate NKT cells led to antigen-presenting cell activation, induced inflammatory cytokines, and, compared with peptide alone or admixed peptide and α-galactosylceramide, specifically enhanced CD8+ T-cell responses against tumor-associated peptides. Primary tumor growth was delayed by vaccination in all tumor models. Using 4T1-based cell lines expressing HER2 or NY-ESO-1, a single administration of the relevant conjugate vaccine prevented tumor colonisation of the lung following intravenous inoculation of tumor cells or spontaneous metastasis from breast, respectively. Conclusion: Glycolipid-peptide conjugate vaccines that activate NKT cells prevent lung metastasis in breast cancer models and warrant investigation as adjuvant therapies for high-risk breast cancer.
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Cancer immunotherapy has gained increasing attention due to its potential specificity and lack of adverse side effects when compared to more traditional modes of treatment. Toll-like receptor 2 (TLR2) agonists are lipopeptides possessing the S-[2,3-bis(palmitoyloxy)propyl]-l-cysteine (Pam2Cys) motif and exhibit potent immunostimulatory effects. These agonists offer a means of providing "danger signals" in order to activate the immune system toward tumor antigens. Thus, the development of TLR2 agonists is attractive in the search of potential immunostimulants for cancer. Existing SAR studies of Pam2Cys with TLR2 indicate that the structural requirements for activity are, for the most part, very intolerable. We have investigated the importance of stereochemistry, the effect of N-terminal acylation, and homologation between the two ester functionalities in Pam2Cys-conjugated lipopeptides on TLR2 activity. The R diastereomer is significantly more potent than the S diastereomer and N-terminal modification generally lowers TLR2 activity. Most notably, homologation gives rise to analogues which are comparatively active to the native Pam2Cys containing constructs.
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Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/farmacología , Lipopéptidos/química , Lipopéptidos/farmacología , Receptor Toll-Like 2/agonistas , Adyuvantes Inmunológicos/síntesis química , Vacunas contra el Cáncer/farmacología , Cisteína/análogos & derivados , Cisteína/síntesis química , Cisteína/farmacología , Humanos , Lipopéptidos/síntesis química , Neoplasias/prevención & control , Estereoisomerismo , Receptor Toll-Like 2/metabolismoRESUMEN
Adrenomedullin (AM) is a 52 amino acid peptide that plays a regulatory role in the vasculature. Receptors for AM comprise the class B G protein-coupled receptor, the calcitonin-like receptor (CLR), in complex with one of three receptor activity-modifying proteins (RAMPs). The C-terminus of AM is involved in binding to the extracellular domain of the receptor, while the N-terminus is proposed to interact with the juxtamembranous portion of the receptor to activate signaling. There is currently limited information on the molecular determinants involved in AM signaling, thus we set out to define the importance of the AM N-terminus through five signaling pathways (cAMP production, ERK phosphorylation, CREB phosphorylation, Akt phosphorylation, and IP1 production). We characterized the three CLR:RAMP complexes through the five pathways, finding that each had a distinct repertoire of intracellular signaling pathways that it is able to regulate. We then performed an alanine scan of AM from residues 15-31 and found that most residues could be substituted with only small effects on signaling, and that most substitutions affected signaling through all receptors and pathways in a similar manner. We identify F18, T20, L26, and I30 as being critical for AM function, while also identifying an analogue (AM15-52 G19A) which has unique signaling properties relative to the unmodified AM. We interpret our findings in the context of new structural information, highlighting the complementary nature of structural biology and functional assays.
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Pancreatic islet-derived peptide hormones play key roles in the maintenance of systemic energy homeostasis and glucose balance and defects in their regulation are strongly implicated in the pathogenesis of obesity and diabetes. Peptides have also been used as lead compounds for therapeutics targeting metabolic disease. It is therefore important to understand the activity and function of islet hormones in both their target tissues and the whole organism. Insulin-like growth factor II (IGF-II) is an insulin homolog secreted by the islet ß-cells. Vesiculin is a newly discovered peptide hormone, processed from IGF-II and secreted from islet ß-cells in response to glucose. We postulated that vesiculin might act to regulate systemic glucose metabolism. Here we report our original investigations of vesiculin's activity in relation to glucoregulation. Vesiculin and IGF-II displayed similar dose-response relationships for lowering blood glucose in insulin-responsive FVB/n mice. By contrast, the ability of IGF-II to lower blood glucose was blunted in insulin-resistant triprolyl human-amylin transgenic mice, whereas vesiculin's ability to lower blood glucose remained unaffected. We also confirmed the ability of vesiculin to bypass insulin resistance in a second mouse model. In vitro analysis of signalling by vesiculin and IGF-II indicates that, like IGF-II, vesiculin signals through the IR/ IGF1R. Overall, we show that removal of only four amino acids from IGF-II has generated a peptide hormone with different bioactivity relevant to blood-glucose regulation. Investigating the differences among vesiculin, IGF-II and insulin signalling and activity may provide new insights into insulin resistance and potentially inform the design of novel therapeutics.
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Factor II del Crecimiento Similar a la Insulina/genética , Insulina/genética , Proteínas del Tejido Nervioso/genética , Receptor IGF Tipo 1/genética , Animales , Glucemia/genética , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patología , Metabolismo Energético/genética , Glucosa/genética , Glucosa/metabolismo , Humanos , Resistencia a la Insulina/genética , Polipéptido Amiloide de los Islotes Pancreáticos/genética , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Ratones , Ratones Transgénicos , Obesidad/genética , Obesidad/metabolismo , Obesidad/patología , Hormonas Peptídicas/genéticaRESUMEN
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 pseudoproline 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.