Antigen-specific Cytotoxic T Lymphocytes Responses Research Articles

Oligosymptomatic long-term carriers of SARS-CoV-2 display impaired innate resistance but increased high-affinity anti-spike antibodies

The vast spectrum of clinical features of COVID-19 keeps challenging scientists and clinicians. Low resistance to infection might result in long-term viral persistence, but the underlying mechanisms remain unclear. Here, we studied the immuneresponse of immunocompetent COVID-19 patients with prolonged SARS-CoV-2 infection by immunophenotyping, cytokine and serological analysis. Despite viral loads and symptoms comparable to regular mildly symptomatic patients, long-term carriers displayed weaker systemic IFN-I responses and fewer circulating pDCs and NK cells at disease onset. Type 1 cytokines remained low, while type-3 cytokines were in turn enhanced. Of interest, we observed no defects in antigen-specific cytotoxic Tcell responses, and circulating antibodies displayed higher affinity against different variants of SARS-CoV-2 Spike protein in these patients. The identification of distinct immune responses in long-term carriers adds up to our understanding of essential host protective mechanisms to ensure tissue damage control despite prolonged viral infection.

Vaccination-based immunotherapy to target profibrotic cells in liver and lung.

Fibrosis is the final path of nearly every form of chronic disease, regardless of the pathogenesis. Upon chronic injury, activated, fibrogenic fibroblasts deposit excess extracellular matrix, and severe tissue fibrosis can occur in virtually any organ. However, antifibrotic therapies that target fibrogenic cells, while sparing homeostatic fibroblasts in healthy tissues, are limited. We tested whether specific immunization against endogenous proteins, strongly expressed in fibrogenic cells but highly restricted in quiescent fibroblasts, can elicit an antigen-specific cytotoxic Tcell response to ameliorate organ fibrosis. In silico epitope prediction revealed that activation of the genes Adam12 and Gli1 in profibrotic cells and the resulting "self-peptides" can be exploited for Tcell vaccines to ablate fibrogenic cells. We demonstrate the efficacy of a vaccination approach to mount CD8+ Tcell responses that reduce fibroblasts and fibrosis in the liver and lungs in mice. These results provide proof of principle for vaccination-based immunotherapies to treat fibrosis.

600 Microarray patch-based adenovirus vaccines enable effective skin immunization

Recombinant live adenovirus (Ad) vectors represent a readily modular vaccine construct that can be engineered to include any antigen of interest, thereby enabling rapid vaccine development against a myriad of infectious pathogens, including coronaviruses. However, current approaches used for delivery, storage, and distribution of Ad vaccines hinder their full potential for effective global immunization campaigns. Here, we developed simple, effective, practical, and needle-free Ad vaccines based on microarray patches (MAPs) to enable sustainable mass vaccination against SARS-CoV-2. Rational formulation of live Ads with nonreducing sugars and mechanically strong carbohydrates into dissolvable MAPs enabled effective skin-targeted delivery of Ads and efficient cutaneous transduction as determined by in vivo live imaging of mice following skin application of MAPs integrating Ad vectors with a reporter gene. Immunogenicity assessment of dissolving MAP-based Ad vectors encoding SARS-CoV-2 proteins in mice demonstrated that skin immunization via MAP delivery of Ad-vectored COVID-19 vaccines elicited robust antigen-specific antibody responses, and these antibodies led to virus-specific neutralization activities, which were enhanced compared to those obtained with traditional intramuscular immunization. Furthermore, MAP delivery of Ad-vectored vaccines elicited potent cell-mediated immune responses, including polyfunctional virus-specific CD8+ and CD4+ T-cell responses in spleens and lungs of immunized mice, as determined by intracellular cytokine staining and flow cytometry, as well as antigen-specific cytotoxic T-cell responses in spleens of mice, as determined by lytic assay. Collectively, our results suggest that dissolvable MAPs could enable the development of skin-targeted Ad-based vaccines that may increase the effectiveness of global immunization programs for SARS-CoV-2 and other existing or future pathogens.

Heterologous Prime-Boost Vaccination with a Peptide-Based Vaccine and Viral Vector Reshapes Dendritic Cell, CD4+ and CD8+ T Cell Phenotypes to Improve the Antitumor Therapeutic Effect.

Simple SummaryDeveloping new therapeutic cancer vaccines is of paramount importance to counteract tumor escape observed after conventional therapies in certain types of cancer. We have previously shown that the combination of two different vaccine platforms, targeting tumor-specific antigens, resulted in potent immune responses in preclinical models. Here, we show that the heterologous prime-boost combination with a protein vaccine and a viral vector vesicular stomatitis virus immunologically reshapes the immune-excluded TC-1 tumor model as well as the inflamed MC-38 tumor model, leading to beneficial therapeutic efficacy. Furthermore, the treatment with a multi-epitope vaccine allowed us to appreciate the various repartition among three antigen-specific cytotoxic T-cell responses combined with the viral boost. The combination leads to improved efficacy in all animals and highlights the importance of combining tumor epitopes. Our vaccine strategy could be further extended to prophylactic cancer vaccines and beyond, for infectious diseases.Heterologous prime-boost settings with a protein vaccine and the viral vector vesicular stomatitis virus, both expressing tumor-associated antigens (KISIMA-TAA and VSV-GP-TAA), have been previously shown to generate potent antitumor immunity. In the cold TC-1 model (HPV antigen) and the immune-infiltrate MC-38 model (Adpgk, Reps1 and Rpl18 neo-antigens), we further investigated pivotal immune cells that educate CD8+ T cells. Heterologous prime-boost vaccination induced a superior antitumor response characterized by the increase in number and functionality of antigen-specific CD8+ T cells, recruitment of cross-presenting dendritic cells, and polarization of CD4+ T cells towards an antitumor Th1 phenotype within the tumor and tumor-draining lymph nodes, turning the cold TC-1 tumor into a hot, inflamed tumor. In the inflamed MC-38 tumor model, treatment combination markedly prolonged the overall survival of mice. Treatment with multi-epitope vaccines also induced high frequencies of multiple antigen specificities in the periphery and in the tumor. Prime-boost treatment reduced tumor-infiltrating regulatory CD4+ T cells whilst increasing cross-presenting dendritic cells in tumor-draining lymph nodes. In conclusion, heterologous prime-boost vaccination possesses the ability to induce a potent anti-tumor response in both immune-excluded and immune-infiltrated mouse tumor models. Additionally, this study highlights the design of a multi-epitope vaccine for cancer immunotherapy.

Broadly Protective CD8+ T Cell Immunity to Highly Conserved Epitopes Elicited by Heat Shock Protein gp96-Adjuvanted Influenza Monovalent Split Vaccine.

Currently, immunization with inactivated influenza virus vaccines is the most prevalent method to prevent infections. However, licensed influenza vaccines provide only strain-specific protection and need to be updated and administered yearly; thus, new vaccines that provide broad protection against multiple influenza virus subtypes are required. In this study, we demonstrated that intradermal immunization with gp96-adjuvanted seasonal influenza monovalent H1N1 split vaccine could induce cross-protection against both group 1 and group 2 influenza A viruses in BALB/c mouse models. Vaccination in the presence of gp96 induced an apparently stronger antigen-specific T cell response than split vaccine alone. Immunization with the gp96-adjuvanted vaccine also elicited an apparent cross-reactive CD8+ T cell response that targeted the conserved epitopes across different influenza virus strains. These cross-reactive CD8+ T cells might be recalled from a pool of memory cells established after vaccination and recruited from extrapulmonary sites to facilitate viral clearance. Of note, six highly conserved CD8+ T epitopes from the viral structural proteins hemagglutinin (HA), M1, nucleoprotein (NP), and PB1 were identified to play a synergistic role in gp96-mediated cross-protection. Comparative analysis showed that most of conservative epitope-specific cytotoxic T lymphocytes (CTLs) apparently induced by heterologous virus infection were also activated by gp96-adjuvanted vaccine, thus resulting in broader protective CD8+ T cell responses. Our results demonstrated the advantage of adding gp96 to an existing seasonal influenza vaccine to improve its ability to provide better cross-protection.IMPORTANCE Owing to continuous mutations in hemagglutinin (HA) or neuraminidase (NA) or recombination of the gene segments between different strains, influenza viruses can escape the immune responses developed by vaccination. Thus, new strategies aimed to efficiently activate immune response that targets to conserved regions among different influenza viruses are urgently needed in designing broad-spectrum influenza vaccine. Heat shock protein gp96 is currently the only natural T cell adjuvant with special ability to cross-present coupled antigen to major histocompatibility complex class I (MHC-I) molecule and activate the downstream antigen-specific CTL response. In this study, we demonstrated the advantages of adding gp96 to monovalent split influenza virus vaccine to improve its ability to provide cross-protection in the BALB/c mouse model and proved that a gp96-activated cross-reactive CTL response is indispensable in our vaccine strategy. Due to its unique adjuvant properties, gp96 might be a promising adjuvant for designing new broad-spectrum influenza vaccines.

A DNA Nanodevice for Cancer Vaccination

To realize effective cancer immunotherapy, Ding et al. constructed a structurally well-defined DNA-based nanodevice to quantitatively assemble cancer cell-specific antigen and multiple adjuvants as a cancer vaccine. This nanodevice vaccine can efficiently accumulate in the draining lymph nodes and respond to the endosomal acidic environment of dendritic cells to release the antigen and adjuvants. These active payloads stimulate dendritic cells maturation and antigen presentation to elicit a robust, antigen-specific cytotoxic T-lymphocyte response to kill cancer cells. This work has been published online in the Nature Materials on Sept.7, 2020.

P2X receptor agonist enhances tumor-specific CTL responses through CD70+ DC-mediated Th17 induction.

Extracellular ATP is known to promote Th17 cell differentiation in the intestinal lamina propria by stimulating CD70+CD11clow dendritic cells (DCs) via P2X receptors (P2XRs). Recent studies have also shown that Th17 cells enhance antitumor immunity by directly promoting proliferation of cytotoxic T lymphocytes (CTLs). These finding led us to test a P2XR agonist, αβ-methylene ATP (αβ-ATP), as a mucosal vaccine adjuvant to promote CTL responses through Th17 induction. We demonstrated that (i) CD70+CD11clow DCs were present in the nasal lamina propria and expressed P2X1R, P2X2R and P2X4R; (ii) CD70+CD11clow DCs isolated from the nasal lamina propria enhanced Th17 cell differentiation of cocultured splenic CD4+ T cells upon stimulation with αβ-ATP; (iii) mice intranasally immunized with ovalbumin (OVA) and αβ-ATP had increased OVA-specific Th17 cells and CTLs in the nasal lamina propria and regional lymph nodes; (iv) mice intranasally immunized with OVA and αβ-ATP also had elevated resistance to E.G7-OVA tumor growth compared with those intranasally immunized with OVA alone; (v) suramin, a broad-range inhibitor of P2 receptors, suppressed the increases of OVA-specific Th17 cells and CTLs in mice intranasally immunized with OVA and αβ-ATP; and (vi) suramin also abrogated the enhanced antitumor immunity of mice intranasally immunized with OVA and αβ-ATP against E.G7-OVA. Collectively, αβ-ATP may be a promising mucosal adjuvant that promotes antigen-specific CTL responses via CD70+CD11clow DC-mediated Th17 induction.

An RNA vaccine drives immunity in checkpoint-inhibitor-treated melanoma

Treating patientswho havecancer with vaccines that stimulate a targeted immune response is conceptually appealing, but cancer vaccine trials have not been successful in late-stage patients with treatment-refractory tumours1,2. We are testing melanoma FixVac (BNT111)-an intravenously administered liposomal RNA (RNA-LPX) vaccine, which targets four non-mutated, tumour-associated antigens that are prevalent in melanoma-in an ongoing, first-in-human, dose-escalation phase I trial in patients with advanced melanoma (Lipo-MERIT trial, ClinicalTrials.gov identifier NCT02410733). We report here data from an exploratory interim analysis that show that melanoma FixVac, alone or in combination with blockade of the checkpoint inhibitor PD1, mediates durable objective responses in checkpoint-inhibitor (CPI)-experienced patients with unresectable melanoma. Clinical responses are accompanied by the induction of strong CD4+ and CD8+ T cell immunity against the vaccine antigens. The antigen-specific cytotoxic T-cell responses in some responders reach magnitudes typically reported for adoptive T-cell therapy, and are durable. Our findings indicate that RNA-LPX vaccination is a potent immunotherapy in patients with CPI-experienced melanoma, and suggest the general utility of non-mutant shared tumour antigens as targets for cancer vaccination.

Endogenous TLR2 ligand embedded in the catalytic region of human cysteinyl-tRNA synthetase 1

BackgroundThe generation of antigen-specific cytotoxic T lymphocyte (CTL) responses is required for successful cancer vaccine therapy. In this regard, ligands of Toll-like receptors (TLRs) have been suggested to activate adaptive...

Role of Dendritic Cells in Exposing Latent HIV-1 for the Kill.

The development of effective yet nontoxic strategies to target the latent human immunodeficiency virus-1 (HIV-1) reservoir in antiretroviral therapy (ART)-suppressed individuals poses a critical barrier to a functional cure. The ‘kick and kill’ approach to HIV eradication entails proviral reactivation during ART, coupled with generation of cytotoxic T lymphocytes (CTLs) or other immune effectors equipped to eliminate exposed infected cells. Pharmacological latency reversal agents (LRAs) that have produced modest reductions in the latent reservoir ex vivo have not impacted levels of proviral DNA in HIV-infected individuals. An optimal cure strategy incorporates methods that facilitate sufficient antigen exposure on reactivated cells following the induction of proviral gene expression, as well as the elimination of infected targets by either polyfunctional HIV-specific CTLs or other immune-based strategies. Although conventional dendritic cells (DCs) have been used extensively for the purpose of inducing antigen-specific CTL responses in HIV-1 clinical trials, their immunotherapeutic potential as cellular LRAs has been largely ignored. In this review, we discuss the challenges associated with current HIV-1 eradication strategies, as well as the unharnessed potential of ex vivo-programmed DCs for both the ‘kick and kill’ of latent HIV-1.

Biodegradable Microneedle Patch Delivering Antigenic Peptide-Hyaluronate Conjugate for Cancer Immunotherapy.

Antigenic peptide-delivery systems have been extensively investigated to harness the immune system for cancer therapy. Cytotoxic T-cell epitope peptide can induce an antigen-specific CD8+ T-cell response, which subsequently inhibits the growth of antigen-bearing tumors. However, there are only a few facile tailored delivery systems of antigenic peptide for effective cancer immunotherapy. Here, we developed a biodegradable microneedle patch delivering a hyaluronate (HA)-antigenic peptide conjugate for prophylactic cancer immunotherapy. Cytotoxic T-cell epitope peptide (SIINFEKL) was conjugated to HA, which was loaded into a biodegradable HA microneedle (MN) patch to efficiently deliver an antigen to the immune system in the skin. HA could act as a transdermal vaccine carrier eliciting strong immune responses by the efficient stimulation of immunocompetent cells. The HA-SIINFEKL conjugates loaded into biodegradable MNs were localized near the MN administration site, exhibiting long-term residence for more than 24 h post-administration. Remarkably, a single transdermal vaccination with the MN patch containing HA-SIINFEKL conjugates resulted in a statistically significant inhibition of tumor growth in B16 melanoma model mice by enhancing antigen-specific cytotoxic T-cell responses.

Comparison of two different PEGylation strategies for the liposomal adjuvant CAF09: Towards induction of CTL responses upon subcutaneous vaccine administration

Using subunit vaccines, e.g., based on peptide or protein antigens, to teach the immune system to kill abnormal host cells via induction of cytotoxic T lymphocytes (CTL) is a promising strategy against intracellular infections and cancer. However, customized adjuvants are required to potentiate antigen-specific cellular immunity. One strong CTL-inducing adjuvant is the liposomal cationic adjuvant formulation (CAF)09, which is composed of dimethyldioctadecylammonium (DDA) bromide, monomycoloyl glycerol (MMG) analogue 1 and polyinosinic:polycytidylic acid [poly(I:C)]. However, this strong CTL induction requires intraperitoneal administration because the vaccine forms a depot at the site of injection (SOI) after subcutaneous (s.c.) or intramuscular (i.m.) injection, and depot formation impedes the crucial vaccine targeting to the cross-presenting dendritic cells (DCs) residing in the lymph nodes (LNs). The purpose of the present study was to investigate the effect of polyethylene glycol (PEG) grafting of CAF09 on the ability of the vaccine to induce antigen-specific CTL responses after s.c. administration. We hypothesized that steric stabilization and charge shielding of CAF09 by PEGylation may reduce depot formation at the SOI and enhance passive drainage to the LNs, eventually improving CTL induction. Hence, the vaccine (antigen/CAF09) was post-grafted with a novel type of anionic PEGylated peptides based on GDGDY repeats, which were end-conjugated with one or two PEG1000 moieties, resulting in mono- and bis-PEG-peptides of different lengths (10, 15 and 20 amino acid residues). For comparison, CAF09 was also grafted by inclusion of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(PEG)-2000 (DSPE-PEG2000) in the bilayer structure during preparation. Grafting of CAF09 with either type of PEG resulted in charge shielding, evident from a reduced surface charge. Upon s.c. immunization of mice with the model antigen ovalbumin (OVA) adjuvanted with PEGylated CAF09, stronger CTL responses were induced as compared to immunization of mice with unadjuvanted OVA. Biodistribution studies confirmed that grafting of CAF09 with DSPE-PEG2000 improved the passive drainage of the vaccine to LNs, because a higher dose fraction was recovered in DCs present in the draining LNs, as compared to the dose fraction detected for non-PEGylated CAF09. In conclusion, PEGylation of CAF09 may be a useful strategy for the design of an adjuvant, which induces CTL responses after s.c. and i.m. administration. In the present studies, CAF09 grafted with 10 mol% DSPE-PEG2000 is the most promising of the tested adjuvants, but additional studies are required to further elucidate the potential of the strategy.

Prevention and treatment of cervical cancer by a single administration of human papillomavirus peptide vaccine with CpG oligodeoxynucleotides as an adjuvant in vivo

No licensed therapeutic human papillomavirus (HPV) vaccine is currently available, so it remains a high priority to develop a therapeutic HPV vaccine or prophylactic/therapeutic HPV vaccine for cervical cancer. In this current study, we designed an HPV vaccine including CpG oligodeoxynucleotides 1826 as an adjuvant and HPV16 E7 43-77 peptide as antigen, which contains a CD8 T cell epitope (E7 49-57), and two CD4 T cell epitopes (E7 43-77 and E7 50-62). The prophylactic and therapeutic effect on cervical cancer induced by a single administration of vaccine, were comprehensively evaluated by examining the tumor size and the percentage of tumor-free/bearing mice. The cellular immunity and modulation of immunosuppressive cells induced by the vaccine were evaluated by examining intracellular cytokine staining (ICS) of splenocytes and FCM, respectively. Antigen-specific cytotoxic T-lymphocyte (CTL) responses were investigated using in vivo cytolytic assay. The results showed that the single administration of vaccine elicited significant prophylactic as well as therapeutic effect on cervical cancer. The increased cellular immunity mediated by CD4 + IFN-γ + T cells and CD8 + IFN-γ + T cells, and the decreased numbers of immunosuppressive cells including regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) were induced by the vaccine. Antigen-specific CTL response was also induced by vaccination. These findings suggested that significant anti-tumor effect of the vaccine may result from the induction of increased cellular immunity and decreased immunosuppressive cells.

HLA-A2-Restricted Epitopes Identified from MTA1 Could Elicit Antigen-Specific Cytotoxic T Lymphocyte Response.

Overexpression of metastasis-associated protein 1 (MTA1) has been observed in many human malignancies and is significantly related to tumor invasion and metastasis, therapeutic resistance to radiation and chemotherapy, making MTA1 an ideal candidate tumor antigen. We identified several human leukocyte antigen- (HLA-) A2-restricted epitopes in MTA1 and evaluated their binding ability to HLA-A∗0201 molecules. Subsequently, a recombinant fragment encompassing the dominant epitopes, MTA1(1–283), was expressed, and the abilities of the selected epitopes of MTA1 and the MTA1(1–283) fragment to induce cytotoxic T lymphocytes (CTLs) were examined. Our results indicated that the epitopes and MTA1(1–283) fragment elicited HLA-A2-restricted and antigen-specific CTL responses both in vitro and in vivo. The new epitopes identified here may help promote the development of new therapeutic vaccines for HLA-A2+ patients expressing MTA1.

An autologous dendritic cell vaccine polarizes a Th-1 response which is tumoricidal to patient-derived breast cancer cells

Breast cancer remains one of the leading causes of cancer-associated death worldwide. Conventional treatment is associated with substantial toxicity and suboptimal efficacy. We, therefore, developed and evaluated the in vitro efficacy of an autologous dendritic cell (DC) vaccine to treat breast cancer. We recruited 12 female patients with stage 1, 2, or 3 breast cancer and matured their DCs with autologous tumour-specific lysate, a toll-like receptor (TLR)-3 and 7/8 agonist, and an interferon-containing cocktail. The efficacy of the vaccine was evaluated by its ability to elicit a cytotoxic T-lymphocyte response to autologous breast cancer cells in vitro. Matured DCs (≥ 60% upregulation of CD80, CD86, CD83, and CCR7) produced high levels of the Th1 effector cytokine, IL12-p70 (1.2 ng/ml; p < 0.0001), compared to DCs pulsed with tumour lysate, or matured with an interferon-containing cocktail alone. We further showed that matured DCs enhance antigen-specific CD8 + T-cell responses to HER-2 (4.5%; p < 0.005) and MUC-1 (19%; p < 0.05) tetramers. The mature DCs could elicit a robust and dose-dependent antigen-specific cytotoxic T-lymphocyte response (65%) which was tumoricidal to autologous breast cancer cells in vitro compared to T-lymphocytes that were primed with autologous lysate loaded-DCs (p < 0.005). Lastly, we showed that the mature DCs post-cryopreservation maintained high viability, maintained their mature phenotype, and remained free of endotoxins or mycoplasma. We have developed a DC vaccine that is cytotoxic to autologous breast cancer cells in vitro. The tools and technology generated here will now be applied to a phase I/IIa clinical trial.

Adjuvant effect of the novel TLR1/TLR2 agonist Diprovocim synergizes with anti–PD-L1 to eliminate melanoma in mice

Successful cancer immunotherapy entails activation of innate immune receptors to promote dendritic cell (DC) maturation, antigen presentation, up-regulation of costimulatory molecules, and cytokine secretion, leading to activation of tumor antigen-specific cytotoxic T lymphocytes (CTLs). Here we screened a synthetic library of 100,000 compounds for innate immune activators using TNF production by THP-1 cells as a readout. We identified and optimized a potent human and mouse Toll-like receptor (TLR)1/TLR2 agonist, Diprovocim, which exhibited an EC50 of 110 pM in human THP-1 cells and 1.3 nM in primary mouse peritoneal macrophages. In mice, Diprovocim-adjuvanted ovalbumin immunization promoted antigen-specific humoral and CTL responses and synergized with anti-PD-L1 treatment to inhibit tumor growth, generating long-term antitumor memory, curing or prolonging survival of mice engrafted with the murine melanoma B16-OVA. Diprovocim induced greater frequencies of tumor-infiltrating leukocytes than alum, of which CD8 T cells were necessary for the antitumor effect of immunization plus anti-PD-L1 treatment.

Efficacy of a Prototype Live-vectored Multi-antigen African Swine Fever Virus Vaccine

Abstract Subunit vaccine development against some complex pathogens require inclusion of multiple targets to optimize efficacy and or broaden protection against diverse strains. To hasten vaccine development, it is rational to evaluate efficacy of a prototype vaccine in the actual outbred host that require protection. African Swine Fever Virus (ASFV) is a complex high-consequence Transboundary Animal Disease pathogen for which there is no vaccine or treatment available. Current evidence shows that protection against the ASFV can be stimulated with a vaccine since pigs that recover from infection with less virulent isolates are protected against closely related virulent isolates. In addition, there is evidence that ASFV-specific cytotoxic T lymphocytes (CTLs) are required for viral clearance. We have tested protective efficacy of several adenovirus-vectored prototype formulations containing multiple rationally selected ASFV antigens. Homologous prime-boost immunization of commercial pigs with one of the experimental vaccine induced significant and unprecedented antigen-specific CTL responses that were detectable in peripheral blood mononuclear cells (PBMCs). Significant IFN-gamma-secreting cells were also detected in PBMCs post-priming and the memory was recalled upon boost. In addition, the immunogen primed antibody responses that were recalled upon boost. More importantly, the prototype vaccine conferred protection in 5/9 of vaccinees following intranasal challenge with the ASFV Georgia 2007/1 isolate. Notably, these survivors were healthy with low clinical scores. In comparison, only 2/5 of the negative controls were alive 17 days post-challenge and they had high clinical scores that necessitated termination.

Photochemical Internalization of Peptide Antigens Provides a Novel Strategy to Realize Therapeutic Cancer Vaccination.

Effective priming and activation of tumor-specific CD8+ cytotoxic T lymphocytes (CTLs) is crucial for realizing the potential of therapeutic cancer vaccination. This requires cytosolic antigens that feed into the MHC class I presentation pathway, which is not efficiently achieved with most current vaccination technologies. Photochemical internalization (PCI) provides an emerging technology to route endocytosed material to the cytosol of cells, based on light-induced disruption of endosomal membranes using a photosensitizing compound. Here, we investigated the potential of PCI as a novel, minimally invasive, and well-tolerated vaccination technology to induce priming of cancer-specific CTL responses to peptide antigens. We show that PCI effectively promotes delivery of peptide antigens to the cytosol of antigen-presenting cells (APCs) in vitro. This resulted in a 30-fold increase in MHC class I/peptide complex formation and surface presentation, and a subsequent 30- to 100-fold more efficient activation of antigen-specific CTLs compared to using the peptide alone. The effect was found to be highly dependent on the dose of the PCI treatment, where optimal doses promoted maturation of immature dendritic cells, thus also providing an adjuvant effect. The effect of PCI was confirmed in vivo by the successful induction of antigen-specific CTL responses to cancer antigens in C57BL/6 mice following intradermal peptide vaccination using PCI technology. We thus show new and strong evidence that PCI technology holds great potential as a novel strategy for improving the outcome of peptide vaccines aimed at triggering cancer-specific CD8+ CTL responses.

Nasal delivery of H5N1 avian influenza vaccine formulated with GenJet™ or in vivo-jetPEI® induces enhanced serological, cellular and protective immune responses

Avian influenza virus infection is a serious public health threat and preventive vaccination is the most cost-effective public health intervention strategy. Unfortunately, currently available unadjuvanted avian influenza vaccines are poorly immunogenic and alternative vaccine formulations and delivery strategies are in urgent need to reduce the high risk of avian influenza pandemics. Cationic polymers have been widely used as vectors for gene delivery in vitro and in vivo. In this study, we formulated H5N1 influenza vaccines with GenJet™ or in vivo-jetPEI®, and showed that these formulations significantly enhanced the immunogenicity of H5N1 vaccines and conferred protective immunity in a mouse model. Detailed analyses of adaptive immune responses revealed that both formulations induced mixed TH1/TH2 antigen-specific CD4 T-cell responses, antigen-specific cytotoxic CD8 T-cell and memory B-cell responses. Our findings suggest that cationic polymers merit future development as potential adjuvants for mucosal delivery of poorly immunogenic vaccines.

IL-12 stimulates CTLs to secrete exosomes capable of activating bystander CD8+ T cells

An effective cytotoxic T lymphocyte (CTL) response against intracellular pathogens is generally accomplished by immense CTL expansion and activation, which can destroy infected cells. Vigorous immune responses can lead to activation of bystander CD8+ T cells, but the contribution from antigen-specific CTLs is not well understood. We found that CTLs secrete extracellular vesicles following antigen stimulation. These CTL-derived vesicles contain CTL proteins and exhibit markers and size profiles consistent with exosomes. Interestingly, further stimulation of CTLs with IL-12 impacts exosome size and leads to selective enrichment of certain exosomal proteins. More important, exosomes from IL-12-stimulated CTLs directly activated bystander naïve CD8+ T cells to produce interferon-γ (IFNγ) and granzyme B (GZB) in the absence of antigens, whereas control exosomes derived from antigen-stimulated CTLs did not. In addition, IL-12 induced exosomes are able to strengthen the effects of weak antigen stimulation on CTLs. Proteomic analysis demonstrates that IL-12 stimulation alters catalytic and binding activities of proteins in CTL exosomes. Our findings indicate that the biological function and morphology of exosomes secreted by CTLs can be influenced by the type of stimulation CTLs receive. Thus, a fully functional, ongoing, antigen-specific CTL response may influence bystander CD8+ T cells through secretion of exosomes.