Induction Of T-cell Responses Research Articles

VENUS, a Novel Selection Approach to Improve the Accuracy of Neoantigens’ Prediction

Neoantigens are tumor-specific antigens able to induce T-cell responses, generated by mutations in protein-coding regions of expressed genes. Previous studies demonstrated that only a limited subset of mutations generates neoantigens in microsatellite stable tumors. We developed a method, called VENUS (Vaccine-Encoded Neoantigens Unrestricted Selection), to prioritize mutated peptides with high potential to be neoantigens. Our method assigns to each mutation a weighted score that combines the mutation allelic frequency, the abundance of the transcript coding for the mutation, and the likelihood to bind the patient’s class-I major histocompatibility complex alleles. By ranking mutated peptides encoded by mutations detected in nine cancer patients, VENUS was able to select in the top 60 ranked peptides, the 95% of neoantigens experimentally validated including both CD8 and CD4 T cell specificities. VENUS was evaluated in a murine model in the context of vaccination with an adeno vector encoding the top ranked mutations prioritized in the MC38 cell line. Efficacy studies demonstrated anti tumoral activity of the vaccine when used in combination with checkpoint inhibitors. The results obtained highlight the importance of a combined scoring system taking into account multiple features of each tumor mutation to improve the accuracy of neoantigen prediction.

Integrated functional and spatial profiling of tumour immune responses induced by immunotherapy: the iPROFILER platform.

BackgroundCancer immunotherapy elicits functional activation and changes in immune cell distribution in cancer. Tumour heterogeneity is a reason for treatment failure but is difficult to capture in experimental settings. This proof-of-principle study describes the integrated functional and digital spatial profiling platform iPROFILER to capture in-situ immune activation patterns with high precision.Materials and methodsiPROFILER combines an algorithm-based image analysis approach for spatial profiling with functional analyses of patient-derived tumour fragments (PDTFs). This study utilized a folate receptor 1 (FOLR1)xCD3 bispecific antibody in dual-affinity re-targeting (DART) format as a tool for inducing T-cell responses in patient tumour samples, and an in-depth investigation of the immune perturbations induced in the tumour microenvironment was performed.ResultsEx-vivo DART stimulation induces upregulation of multiple activation markers in CD4+ and CD8+ T-cell populations and secretion of pro-inflammatory cytokines in FOLR1-positive tumour specimens. This response was reduced or absent in tissue samples that did not express FOLR1. Immunological responses were driven by a strong induction of interferon gamma (IFNγ) and IFNγ-induced chemokines suggestive of activation of cytotoxic or Th1-like T cells. Ex-vivo DART treatment led to a numerical increase in effector T cells and an upregulation of immune activation markers in the tumour microenvironment as captured by digital image analysis. Analysis of immune activation in tumour and stromal regions further supported the potential of the platform to measure local differences in cell-type-specific activation patterns.ConclusionsiPROFILER effectively combines functional and spatial readouts to investigate immune responses ex vivo in human tumour samples.

Phase I trial to determine safety and immunogenicity of amplivant, a synthetic toll-like receptor 2 ligand, conjugated to two HPV16 E6 synthetic long peptides.

2614 Background: Therapeutic vaccines based on synthetic long peptides (SLPs) have a great potential for immunotherapy of cancer patients as these SLPs include both human leukocyte antigen (HLA) class I and II epitopes and no patient selection for HLA types is required. The antigen-induced immune response can be strengthened with immune stimulating additives. Amplivant (AV) is a synthetic Toll-like receptor 2 ligand which can be directly conjugated to tumor peptide antigens. In preclinical studies, AV-conjugation to antigens led to both enhanced antigen presentation by dendritic cells and T-cell priming and caused superior induction of effective anti-tumor responses. Moreover, AV-conjugated SLPs showed a 100 times higher immune response compared to unconjugated SLP. The current study is a first-in-human trial to investigate safety and immunogenicity of AV-conjugated human papillomavirus (HPV)16-SLPs. Methods: A dose escalation phase I trial was performed in 24 patients with HPV16 positive (pre-) malignant lesions. AV was conjugated to two SLPs derived from the most immunodominant regions of the HPV16 E6 oncoprotein. Four dose groups (1, 5, 20 or 50 μg of each peptide) in 6 patients each were studied. The vaccine was injected three times intradermally in DMSO / water with a three-week interval. Adverse events (AE) were collected according to CTCAE v4.0 up to 26 weeks. Peptide-specific T-cell immune responses were determined in blood samples taken before and after vaccination using complementary immunological assays (proliferation assay, IFNγ-ELISPOT and cytokine bead array). Results: Toxicity after three AV-conjugated HPV16-SLP vaccinations was limited to CTCAE grade 1 or 2, with predominantly inflammation at the vaccination site and sometimes flu-like symptoms, which generally resolved within one day. Dose increase resulted from no AE in the lowest dose group to mild/moderate AE in all vaccinated persons in the highest dose group. In the lowest dose group, minor vaccine-induced T-cell responses were observed in three of six vaccinated persons. In the highest dose group, all patients displayed a strong HPV16-specific T-cell response after vaccination. The induced T-cell response against HPV16 lasted until the end of the trial. Conclusions: This first-in-human study showed that AV conjugated to SLPs can safely be used as an intradermal therapeutic vaccine. AV-conjugated HPV16-SLP was able to induce robust HPV16-specific T-cell immunity in patients treated for HPV16 positive (pre-) malignancies without any other vaccine adjuvant or formulation. Increase in dose resulted in both a higher number of mild adverse events as well as stronger T-cell immunity. Clinical trial information: NCT02821494.

Vaccination With Recombinant Adenoviruses Expressing the Bluetongue Virus Subunits VP7 and VP2 Provides Protection Against Heterologous Virus Challenge.

Bluetongue virus (BTV) is the causative agent of a disease that affects domestic and wild ruminants and leads to critical economic losses. BTV is an arbovirus from the Reoviridae family that is typically transmitted by the bite of infected Culicoides midges. BTV possesses multiple serotypes (up to 28 have been described), and immunity to one serotype offers little cross-protection to other serotypes. The design of vaccines that provide protection across multiple serotypes is therefore highly desirable to control this disease. We previously reported that a recombinant replication-defective human adenovirus serotype 5 (Ad5) that expresses the VP7 inner core protein of BTV serotype 8 (Ad5VP7-8) induced T-cell responses and provided protection. In the present work, we evaluated as BTV vaccine the combination of Ad5VP7-8 with another recombinant Ad5 that expresses the outer core protein VP2 from BTV-1 (Ad5VP2-1). The combination of Ad5VP2-1 and Ad5VP7-8 protected against homologous BTV challenge (BTV-1 and BTV-8) and partially against heterologous BTV-4 in a murine model. Cross-reactive anti-BTV immunoglobulin G (IgG) were detected in immunized animals, but no significant titers of neutralizing antibodies were elicited. The Ad5VP7-8 immunization induced T-cell responses that recognized all three serotypes tested in this study and primed cytotoxic T lymphocytes specific for VP7. This study further confirms that targeting antigenic determinant shared by several BTV serotypes using cellular immunity could help develop multiserotype BTV vaccines.

Antibody-mediated delivery of T-cell epitopes to antigen-presenting cells induce strong CD4 and CD8 T-cell responses

Targeted delivery of antigen to antigen-presenting cells (APCs) enhances antigen presentation and thus, is a potent strategy for making more efficacious vaccines. This can be achieved by use of antibodies with specificity for endocytic surface molecules expressed on the APC. We aimed to compare two different antibody-antigen fusion modes in their ability to induce T-cell responses; first, exchange of immunoglobulin (Ig) constant domain loops with a T-cell epitope (Troybody), and second, fusion of T-cell epitope or whole antigen to the antibody C-terminus. Although both strategies are well-established, they have not previously been compared using the same system. We found that both antibody-antigen fusion modes led to presentation of the T-cell epitope. The strength of the T-cell responses varied, however, with the most efficient Troybody inducing CD4 T-cell proliferation and cytokine secretion at 10–100-fold lower concentration than the antibodies carrying antigen fused to the C-terminus, both in vitro and after intravenous injection in mice. Furthermore, we exchanged this loop with an MHCI-restricted T-cell epitope, and the resulting antibody enabled efficient cross-presentation to CD8 T cells in vivo. Targeting of antigen to APCs by use of such antibody-antigen fusions is thus an attractive vaccination strategy for increased activation of both CD4 and CD8 peptide-specific T cells.

Antibodies to neutralising epitopes synergistically block the interaction of the receptor-binding domain of SARS-CoV-2 to ACE 2.

ObjectivesA major COVID‐19 vaccine strategy is to induce antibodies that prevent interaction between the Spike protein's receptor‐binding domain (RBD) and angiotensin‐converting enzyme 2 (ACE2). These vaccines will also induce T‐cell responses. However, concerns were raised that aberrant vaccine‐induced immune responses may exacerbate disease. We aimed to identify minimal epitopes on the RBD that would induce antibody responses that block the interaction of the RBD and ACE2 as a strategy leading to an effective vaccine with reduced risk of inducing immunopathology.MethodsWe procured a series of overlapping 20‐amino acid peptides spanning the RBD and asked which were recognised by plasma from COVID‐19 convalescent patients. Identified epitopes were conjugated to diphtheria‐toxoid and used to vaccinate mice. Immune sera were tested for binding to the RBD and for their ability to block the interaction of the RBD and ACE2.ResultsSeven putative vaccine epitopes were identified. Memory B‐cells (MBCs) specific for one of the epitopes were identified in the blood of convalescent patients. When used to vaccinate mice, six induced antibodies that bound recRBD and three induced antibodies that could partially block the interaction of the RBD and ACE2. However, when the sera were combined in pairs, we observed significantly enhanced inhibition of binding of RBD to ACE2. Two of the peptides were located in the main regions of the RBD known to contact ACE2. Of significant importance to vaccine development, two of the peptides were in regions that are invariant in the UK and South African strains.ConclusionCOVID‐19 convalescent patients have SARS‐CoV‐2‐specific antibodies and MBCs, the specificities of which can be defined with short peptides. Epitope‐specific antibodies synergistically block RBD–ACE2 interaction.

Cationic Polymers for the Delivery of the Ebola DNA Vaccine Encoding Artificial T-Cell Immunogen.

Background: According to current data, an effective Ebola virus vaccine should induce both humoral and T-cell immunity. In this work, we focused our efforts on methods for delivering artificial T-cell immunogen in the form of a DNA vaccine, using generation 4 polyamidoamine dendrimers (PAMAM G4) and a polyglucin:spermidine conjugate (PG). Methods: Optimal conditions were selected for obtaining complexes of previously developed DNA vaccines with cationic polymers. The sizes, mobility and surface charge of the complexes with PG and PAMAM 4G have been determined. The immunogenicity of the obtained vaccine constructs was investigated in BALB/c mice. Results: It was shown that packaging of DNA vaccine constructs both in the PG envelope and the PAMAM 4G envelope results in an increase in their immunogenicity as compared with the group of mice immunized with the of vector plasmid pcDNA3.1 (a negative control). The highest T-cell responses were shown in mice immunized with complexes of DNA vaccines with PG and these responses significantly exceeded those in the groups of animals immunized with both the combination of naked DNAs and the combination DNAs coated with PAMAM 4G. In the group of animals immunized with complexes of the DNA vaccines with PAMAM 4G, no statistical differences were found in the ability to induce T-cell responses, as compared with the group of mice immunized with the combination of naked DNAs. Conclusions: The PG conjugate can be considered as a promising and safe means to deliver DNA-based vaccines. The use of PAMAM requires further optimization.

MERS-CoV in Africa—an enigma with relevance to COVID-19

MERS-CoV in Africa—an enigma with relevance to COVID-19

CD47/SIRPα blocking peptide identification and synergistic effect with irradiation for cancer immunotherapy

BackgroundImmunotherapy has achieved remarkable advances via a variety of strategies against tumor cells that evade immune surveillance. As important innate immune cells, macrophages play important roles in maintaining homeostasis, preventing...

Comprehensive Screening of Mouse T-Cell Epitopes in Human Herpesvirus 6B Glycoprotein H/L/Q1/Q2 Tetramer Complex.

Human herpesvirus 6 (HHV-6) infects over 90% of people. The HHV-6 subtype, HHV-6B in particular, is often associated with exanthem subitum in early childhood. Exanthem subitum is usually self-limiting and good prognosis disease; however, some infants primarily infected with HHV-6B develop encephalitis/encephalopathy, and half of the patients developed encephalopathy reported to have neurological sequelae. Furthermore, after primary infection, HHV-6B remains in a latent state and sometimes reactivated in immunosuppressed patients, causing life-threatening severe encephalopathy. However, effective immunotherapies or vaccines for controlling HHV-6B infection and reactivation have not yet been established. Recently, we have found that the HHV-6B tetrameric glycoprotein (g) complex, gH/gL/gQ1/gQ2 is a promising vaccine candidate, and currently under preclinical development. To confirm our vaccine candidate protein complex induce detectable T-cell responses, in this study, we comprehensively screened CD4+ and CD8+ T-cell epitopes in the gH/gL/gQ1/gQ2 tetrameric complex protein in mice immunisation model. Both BALB/c and C57BL/6 mice were immunised with the tetrameric complex protein or plasmid DNA encoding gH, gL, gQ1, and gQ2, and then restimulated with 162 20-mer peptides covering the whole gH/gL/gQ1/gQ2 sequences; multiple CD4+ and CD8+ T-cell-stimulating peptides were identified in both BALB/c and C57BL/6 mice. Our study demonstrates that gH/gL/gQ1/gQ2 tetramer-targeted vaccination has potential to induce T-cell responses in two different strains of mice and supports the future development and application of T-cell-inducing vaccine and immunotherapies against HHV-6B.

Abstract IA20: Personalized whole-tumor antigen vaccines in ovarian cancer: Using tumors for therapy

Abstract Disappointing results have been observed with the first-generation therapeutic cancer vaccines. However, the renaissance of immunotherapy with the checkpoint inhibitors and the demonstration, in preclinical models, that personalized vaccines can induce efficient antitumor T-cell responses have led to a renewed interest in cancer vaccination. Personalized whole-tumor lysate vaccines can encompass all putative antigens in a patient’s tumor and can potentially induce a broader, more efficient immune response. We report here a clinical study utilizing a personalized vaccine generated by autologous dendritic cells (DC) pulsed with autologous whole-tumor cell lysate (OCDC), in platinum-treated, immunotherapy-naïve, recurrent ovarian cancer patients in combination with bevacizumab or bevacizumab plus low-dose intravenous cyclophosphamide until disease progression or vaccine exhaustion. A total of 392 vaccine doses were administered without serious adverse events. Vaccine was shown to induce T-cell responses to autologous tumor antigen, which were associated with significantly prolonged survival (PFS). Vaccine was also shown to amplify T-cell responses against mutated neoepitopes derived from nonsynonymous somatic tumor mutations, and this included priming of T cells against previously unrecognized neoepitopes as well as novel T-cell clones of markedly higher avidity against previously recognized neoepitopes. Importantly, we demonstrate that the median PFS was 11.1 months in the population receiving the combination of vaccine plus bevacizumab and low-dose cyclophosphamide as compared to only 4.1 months in a historic population of patients from the same institution who received standard bevacizumab and low-dose cyclophosphamide without vaccine. We also demonstrate that survival at 2 years was 100% in the vaccine plus bevacizumab/cyclophosphamide group while it was only 40% in control patients receiving bevacizumab and cyclophosphamide alone. This proof-of-concept clinical study gives us a new horizon for the application of whole-tumor lysate personalized vaccines. Citation Format: Lana E. Kandalaft. Personalized whole-tumor antigen vaccines in ovarian cancer: Using tumors for therapy [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr IA20.

The SARS-COV-2 T-Cell Immunity is Directed Against the Spike, Membrane, and Nucleocapsid Protein and Associated with COVID 19 Severity

Identification of immunogenic targets of SARS-CoV-2 is crucial for monitoring of antiviral immunity and vaccine design. Currently, mainly anti-spike (S)-protein adaptive immunity is investigated. However, also the nucleocapsid (N)- and membrane (M)-proteins should be considered as diagnostic and prophylactic targets.The aim of our study was to explore and compare the immunogenicity of SARS-CoV-2 S-, M- and N-proteins in context of different COVID-19 manifestations. Analyzing a cohort of COVID-19 patients with moderate, severe, and critical disease severity, we show that overlapping peptide pools (OPP) of all three proteins can activate SARS-CoV-2-reactive T-cells with a stronger response of CD4 + compared to CD8 + T-cells. Although interindividual variations for the three proteins were observed, M‑protein induced the highest frequencies of CD4 + T-cells, suggesting its relevance as diagnostic and vaccination target. Importantly, patients with critical COVID-19 demonstrated the strongest T-cell response, including the highest frequencies of cytokine-producing bi- and trifunctional T-cells, for all three proteins. Although the higher magnitude and superior functionality of SARS-CoV-2-reactive T-cells in critical patients can also be a result of a stronger immunogenicity provided by severe infection, it disproves the hypothesis of insufficient SARS-CoV-2-reactive immunity in critical COVID-19. To this end, activation of effector T-cells with differentiated memory phenotype found in our study could cause hyper-reactive response in critical cases leading to immunopathogenesis.Conclusively, since the S-, M-, and N-proteins induce T-cell responses with individual differences, all three proteins should be evaluated for diagnostics and therapeutic strategies to avoid underestimation of cellular immunity and to deepen our understanding of COVID-19 immunity. Funding: This work was supported by grants of Mercator Foundation, the BMBF e:KID (01ZX1612A), and BMBF NoChro (FKZ 13GW0338B). Declaration of Interest: Missing Ethical Statement: We feel deep gratitude to the patients who donated their blood samples and clinical data for this project.

Evaluation of local and systemic immune responses in pigs experimentally challenged with porcine reproductive and respiratory syndrome virus

The host-associated defence system responsible for the clearance of porcine reproductive and respiratory syndrome virus (PRRSV) from infected pigs is currently poorly understood. To better understand the dynamics of host–pathogen interactions, seventy-five of 100 pigs infected with PRRSV-JA142 and 25 control pigs were euthanized at 3, 10, 21, 28 and 35 days post-challenge (dpc). Blood, lung, bronchoalveolar lavage (BAL) and bronchial lymph node (BLN) samples were collected to evaluate the cellular immune responses. The humoral responses were evaluated by measuring the levels of anti-PRRSV IgG and serum virus-neutralizing (SVN) antibodies. Consequently, the highest viral loads in the sera and lungs of the infected pigs were detected between 3 and 10 dpc, and these resulted in moderate to mild interstitial pneumonia, which resolved accompanied by the clearance of most of the virus by 28 dpc. At peak viremia, the frequencies of alveolar macrophages in infected pigs were significantly decreased, whereas the monocyte-derived DC/macrophage and conventional DC frequencies were increased, and these effects coincided with the early induction of local T-cell responses and the presence of proinflammatory cytokines/chemokines in the lungs, BAL, and BLN as early as 10 dpc. Conversely, the systemic T-cell responses measured in the peripheral blood mononuclear cells were delayed and significantly induced only after the peak viremic stage between 3 and 10 dpc. Taken together, our results suggest that activation of immune responses in the lung could be the key elements for restraining PRRSV through the early induction of T-cell responses at the sites of virus replication.

Abstract B62: Evaluation of systemic RNA-based cancer vaccine induced T-cell responses via mouse T-cell receptor (TCR) profiling

Abstract Identification of biomarkers is pivotal towards the proper development of cancer immunotherapies, in particular for designing the most effective regimens and identifying patient subgroups most likely to benefit from a given treatment. T-cell receptor (TCR) repertoire emerged as a promising biomarker in this respect, as it allows measurement of the dynamics of the T cell response both in time and in different compartments. In this work, we developed and evaluated an NGS-based mouse TCR profiling method (TCR alpha and TCR beta) for discovery and validation of biomarkers related to the antitumoral T-cell response in different compartments upon systemic administration of an RNA-based cancer vaccine (RNA-lipoplex). The in-house established mouse TCR profiling protocol was able to detect blood-spiked known-TCR sequences down to a frequency of 0,001% spiked cells for both TCR alpha and TCR beta chains and the relative frequencies of detected TCRs in the tumor correlated well with the degree of T-cell infiltration into the same tumors assessed by flow cytometry. Comparative analysis of changes in the TCR repertoire between blood and tumor upon repetitive vaccination with RNA-lipoplexes encoding immunodominant epitope of chicken ovalbumin (OVA257-264) revealed that richness as well as diversity of endogenous TCRs were generally lower in B16-OVA tumors compared to blood and decreased with further vaccination. Interestingly, top clones overlapped increasingly in blood and tumor until after the second vaccination, but drifted apart after the third vaccination, with the TCR repertoire in the tumor narrowing down. This overlap was less evident when mice were inoculated with B16-F10 tumors. Top clones within the same compartment were found to be highly variable between individual animals, complicating comparative analyses. Monitoring intra-individual TCR repertoire changes in the blood of a given mouse over time, however, indicated a tendency for decreasing richness and diversity following a single RNA vaccination, which may be pointing at repertoire adaptation and antigen focus. Supporting this notion, tumors isolated at late time-points exhibited a lower diversity than those investigated at earlier time-points and at baseline. In contrast to RNA vaccination, richness and diversity were reduced in response to treatment with an agonistic anti-CD40 antibody in blood and spleen but were similar among tumors. Consequently, richness and diversity in the periphery may not necessarily be predictive of corresponding repertoire changes in the tumor microenvironment. Citation Format: Lena Mareen Kranz, Barbara Schrörs, Thomas Bukur, Christian Albrecht, Lorieta Leppin, Martin Suchan, Marta Faryna, Eileen Powalsky, Rainer Hipfel, Özlem Türeci, Ugur Sahin, Mustafa Diken. Evaluation of systemic RNA-based cancer vaccine induced T-cell responses via mouse T-cell receptor (TCR) profiling [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B62.

Neoadjuvant PD-1 Immune Checkpoint Blockade Reverses Functional Immunodominance among Tumor Antigen-Specific T Cells.

Surgical resection of primary tumor with regional lymphadenectomy remains the treatment of choice for patients with advanced human papillomavirus-negative head and neck squamous cell carcinoma. However, even when pathologic disease-free margins can be achieved, locoregional and/or distant disease relapse remains high. Perioperative immunotherapy may improve outcomes, but mechanistic data supporting the use of neoadjuvant or adjuvant treatment clinically are sparse. Two syngeneic models of oral cavity carcinoma with defined T-cell antigens were treated with programmed death receptor 1 (PD-1) mAb before or after surgical resection of primary tumors, and antigen-specific T-cell responses were explored with functional and in vivo challenge assays. We demonstrated that functional immunodominance developed among T cells targeting multiple independent tumor antigens. T cells specific for subdominant antigens expressed greater levels of PD-1. Neoadjuvant, but not adjuvant, PD-1 immune checkpoint blockade broke immunodominance and induced T-cell responses to dominant and subdominant antigens. Using tumors lacking the immunodominant antigen as a model of antigen escape, neoadjuvant PD-1 immune checkpoint blockade induced effector T-cell immunity against tumor cells lacking immunodominant but retaining subdominant antigen. When combined with complete surgical excision, neoadjuvant PD-1 immune checkpoint blockade led to formation of immunologic memory capable of preventing engraftment of tumors lacking the immunodominant but retaining subdominant antigen. Together, these results implicate PD-1 expression by T cells in the mechanism of functional immunodominance among independent T-cell clones within a progressing tumor and support the use of neoadjuvant PD-1 immune checkpoint blockade in patients with surgically resectable carcinomas.

Microfluidic-prepared DOTAP nanoparticles induce strong T-cell responses in mice.

For the induction of antigen-specific T-cell responses by vaccination, an appropriate immune adjuvant is required. Vaccine adjuvants generally provide two functions, namely, immune potentiator and delivery, and many adjuvants that can efficiently induce T-cell responses are known to have the combination of these two functions. In this study, we explored a cationic lipid DOTAP-based adjuvant. We found that the microfluidic preparation of DOTAP nanoparticles induced stronger CD4+ and CD8+ T-cell responses than liposomal DOTAP. The further addition of Type-A CpG D35 in DOTAP nanoparticles increased the induction of T-cell responses, particularly in CD4+ T cells. Further investigations revealed that the size of DOTAP nanoparticles, prepared buffer conditions, and physicochemical interaction with vaccine antigen are important factors for the efficient induction of T-cell responses with a relatively small antigen dose. These results suggested that microfluidic-prepared DOTAP nanoparticles plus D35 are a promising adjuvant for a vaccine that induces therapeutic T-cell responses for treating cancer and infectious diseases.

1181PD - Personal antigen selection calculator (PASCal) for the design of personal cancer vaccines

BackgroundThe current challenge in developing effective cancer vaccines is the accurate prediction of epitopes that induce CD8+ cytotoxic T-cell responses. Recent technological advances have enabled development of patient-specific therapeutic vaccines. However, in these vaccines only about 10-20% of the predicted neoepitopes induced CD8+ T-cell responses in patients. To overcome this limitation, we developed PASCal for improved selection of peptides (epitopes) that induce T-cell responses. MethodsPASCal operates by 3 moduls: (1) a validated epitope database containing 108 true HLA-epitope pairs derived from 1300 tumor antigens and HLA class I and II molecules covering the HLA genotype of 26000 subjects. (2) Expression frequency-based shared tumor antigen database established for 19 indications based on>96000 tumor biopsies. (3) Algorithm for the dentification of immunogenic peptides by the selection of personal epitopes (PEPIs) binding to≥3 autologous HLA alleles. Using PASCal, personal 20mer peptide vaccines were designed for 3 HLA-genotyped cancer patients (with ovarian-, breast- and colorectal cancer). Immunogenicity of the vaccines was tested by ELISPOT and Intracellular Citokine Staining (ICS). ResultsPersonalized cancer vaccines contained PEPIs from 12 disease specific tumor-antigens most frequently expressed in the patients disease. T-cell responses were induced by 100% of peptides. An average of 11/12 PEPIs induced CD8+ T-cell responses and 12/12 induced CD4+ T-cell responses in each patient. Pre-existing antigen specific T-cell reactivities were detectable against 25% of vaccine antigens (demonstrating the expression of the target vaccine antigens by the patient’s tumor), the others were induced de novo. Both CD8+ and CD4+ T-cells were polyfunctional, as evident by secretion of multiple cytokines determined by ex vivo ICS. ConclusionsWe used the largest validated database of tumor epitopes reported to-date along with an algorithm successfully selecting immunogenic peptides to develop personalized cancer vaccines. PEPIs outperform reported immunogenicity of personalized neoantigen vaccines and induced unprecedented immune responses in cancer patients. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureE. Somogyi: Shareholder / Stockholder / Stock options: Treos Bio Ltd. Z. Csiszovszki: Shareholder / Stockholder / Stock options: Treos Bio Ltd. O. Lorincz: Shareholder / Stockholder / Stock options: Treos Bio Ltd. J. Toth: Shareholder / Stockholder / Stock options: Treos Bio Ltd. L. Molnar: Shareholder / Stockholder / Stock options: Treos Bio Ltd. W. Schönharting: Shareholder / Stockholder / Stock options: PMCR GmbH. S. Urban: Shareholder / Stockholder / Stock options: PMCR GmbH. K. Pántya: Shareholder / Stockholder / Stock options: Treos Bio Ltd. M. Megyesi: Shareholder / Stockholder / Stock options: Treos Bio Ltd. E.R. Tőke: Shareholder / Stockholder / Stock options: Treos Bio Ltd.; Leadership role: Treos Bio Zrt. All other authors have declared no conflicts of interest.

Vaccination with viral vectors expressing NP, M1 and chimeric hemagglutinin induces broad protection against influenza virus challenge in mice

Seasonal influenza virus infections cause significant morbidity and mortality every year. Annual influenza virus vaccines are effective but only when well matched with circulating strains. Therefore, there is an urgent need for better vaccines that induce broad protection against drifted seasonal and emerging pandemic influenza viruses. One approach to design such vaccines is based on targeting conserved regions of the influenza virus hemagglutinin. Sequential vaccination with chimeric hemagglutinin constructs can refocus antibody responses towards the conserved immunosubdominant stalk domain of the hemagglutinin, rather than the variable immunodominant head. A complementary approach for a universal influenza A virus vaccine is to induce T-cell responses to conserved internal influenza virus antigens. For this purpose, replication deficient recombinant viral vectors based on Chimpanzee Adenovirus Oxford 1 and Modified Vaccinia Ankara virus are used to express the viral nucleoprotein and the matrix protein 1. In this study, we combined these two strategies and evaluated the efficacy of viral vectors expressing both chimeric hemagglutinin and nucleoprotein plus matrix protein 1 in a mouse model against challenge with group 2 influenza viruses including H3N2, H7N9 and H10N8. We found that vectored vaccines expressing both sets of antigens provided enhanced protection against H3N2 virus challenge when compared to vaccination with viral vectors expressing only one set of antigens. Vaccine induced antibody responses against divergent group 2 hemagglutinins, nucleoprotein and matrix protein 1 as well as robust T-cell responses to the nucleoprotein and matrix protein 1 were detected. Of note, it was observed that while antibodies to the H3 stalk were already boosted to high levels after two vaccinations with chimeric hemagglutinins (cHAs), three exposures were required to induce strong reactivity across subtypes. Overall, these results show that a combinations of different universal influenza virus vaccine strategies can induce broad antibody and T-cell responses and can provide increased protection against influenza.

Intranodal administration of mRNA encoding nucleoprotein provides cross-strain immunity against influenza in mice

BackgroundCurrent human influenza vaccines lack the adaptability to match the mutational rate of the virus and therefore require annual revisions. Because of extensive manufacturing times and the possibility that antigenic alterations occur during viral vaccine strain production, an inherent risk exists for antigenic mismatch between the new influenza vaccine and circulating viruses. Targeting more conserved antigens such as nucleoprotein (NP) could provide a more sustainable vaccination strategy by inducing long term and heterosubtypic protection against influenza. We previously demonstrated that intranodal mRNA injection can induce potent antigen-specific T-cell responses. In this study, we investigated whether intranodal administration of mRNA encoding NP can induce T-cell responses capable of protecting against a heterologous influenza virus challenge.MethodsBALB/c mice were immunized in the inguinal lymph nodes with different vaccination regimens of mRNA encoding NP. Immune responses were compared with NP DNA vaccination via IFN-γ ELISPOT and in vivo cytotoxicity. For survival experiments, mice were prime-boost vaccinated with 17 µg NP mRNA and infected with 1LD50 of H1N1 influenza virus 8 weeks after boost. Weight was monitored and viral titers, cytokines and immune cell populations in the bronchoalveolar lavage, and IFN-γ responses in the spleen were analyzed.ResultsOur results demonstrate that NP mRNA induces superior systemic T-cell responses against NP compared to classical DNA vaccination. These responses were sustained for several weeks even at low vaccine doses. Upon challenge infection, vaccination with NP mRNA resulted in reduced lung viral titers and improved recovery from infection. Finally, we show that vaccination with NP mRNA affects the immune response in infected lungs by lowering immune cell infiltration while increasing the fraction of T cells, monocytes and MHC II+ alveolar macrophages within immune infiltrates. This change was associated with altered levels of both pro- and anti-inflammatory cytokines.ConclusionsThese findings suggest that intranodal vaccination with NP mRNA induces cross-strain immunity against influenza, but also highlight a paradox of influenza immunity, whereby robust immune responses can provide protection, but can also transiently exacerbate symptoms during infection.

Human model to predict the outcome of cancer vaccine clinical trials.

e14298 Background: Cancer vaccines activate T-cells with peptides presented by HLA alleles. We hypothesized that peptides binding to multiple HLA alleles of a patient are more likely to induce T-cell responses than epitopes presented by a single HLA. To prove this hypothesis we predicted the outcome of vaccine clinical trials in a Model Population of subjects with complete HLA genotype. Methods: We collected the immune- and objective response rate (IRR, ORR) results from 64 clinical trials conducted in 1,790 patients treated with 42 different vaccines. From these 42 vaccines, we identified personal epitopes (PEPIs) with strong binding affinity to at least 3 HLA alleles of each subject in the Model Population and calculated the proportion of subjects with PEPIs (PEPI Score), multiple PEPIs (MultiPEPI-Score) and PEPIs against multiple vaccine antigens (MultiAg PEPI-Score). Results: Highly significant correlation between the PEPI Score and IRR (p = 0.001) suggests that peptides binding to at least 3 HLA alleles of the subject induce T-cell responses. MultiPEPI-Score correlated with ORR when the tumor of patients expressed the target antigen (p = 0.01). MultiAg PEPI-Score correlated with ORR of clinical trials conducted with vaccines containing multiple antigens (p = 0.01). Conclusions: Our results suggest that (i) a patient’s HLA genotype is the main determinant of vaccine response and (ii) T-cell responses against multiple antigens expressed in the patient’s tumor favors tumor shrinkage. We introduced a novel pre-clinical human model that accurately predicts the outcome of clinical trials, will reduce the risk, cost and time of vaccine development for the prevention and treatment of cancer.