Antigen-specific CD8 T-cell Responses Research Articles

Determination of the factors associated with antigen-specific CD4+ T-cell responses to BNT162b2 in patients with rheumatoid arthritis

ObjectivesUnderstanding interpatient variation in CD4+T-cell responses is the bases for understanding the pathogenesis and management of rheumatoid arthritis (RA). We examined immune responses to SARS-CoV-2 vaccine in a cohort of...

Intravenous heterologous prime-boost vaccination activates innate and adaptive immunity to promote tumor regression

Therapeutic neoantigen cancer vaccines have limited clinical efficacy to date. Here, we identify a heterologous prime-boost vaccination strategy using a self-assembling peptide nanoparticle TLR-7/8 agonist (SNP) vaccine prime and a chimp adenovirus (ChAdOx1) vaccine boost that elicits potent CD8 Tcells and tumor regression. ChAdOx1 administered intravenously (i.v.) had 4-fold higher antigen-specific CD8 Tcell responses than mice boosted by the intramuscular (i.m.) route. In the therapeutic MC38 tumor model, i.v. heterologous prime-boost vaccination enhances regression compared with ChAdOx1 alone. Remarkably, i.v. boosting with a ChAdOx1 vector encoding an irrelevant antigen also mediates tumor regression, which is dependent on type I IFN signaling. Single-cell RNA sequencing of the tumor myeloid compartment shows that i.v. ChAdOx1 reduces the frequency of immunosuppressive Chil3 monocytes and activates cross-presenting type 1 conventional dendritic cells (cDC1s). The dual effect of i.v. ChAdOx1 vaccination enhancing CD8 Tcells and modulating the TME represents a translatable paradigm for enhancing anti-tumor immunity in humans.

Abstract LB097: A simian challenge model to evaluate the CD8-positive T cell response with cancer therapeutics

Abstract The demonstration that antibodies can be used to treat cancers has revolutionized the field of cancer immunotherapy and gave hope to many patients. Originally targeting tumor-specific antigen, new therapeutic antibodies target now immune checkpoint markers expressed on T cells. The advantage is to generate an active and long-lasting antitumor immunity with the achievement of a broad and polyclonal antitumor immunity, addressing better the heterogeneity of cancers and reducing the chances of immune escape. The development of such highly specific molecules is particularly challenging however when it comes to preclinical safety assessment and efficacy studies. An ideal animal model needs to possess the target so that the drug product is pharmacologically active. Moreover, such model should ideally provide further insight in determining the pharmacological action of the product and the determination of safety to maximize the usefulness in animal studies. Intriguingly, no equivalent of the traditional T-cell-dependent antibody response (TDAR) assay, which evaluates mainly the CD4+ T cells response, is currently available for the CD8+ T cell response. The goal of this study is to fill this gap with a Non-Human Primate (NHP) challenge model that will elicit a potent CD8+ T cell response. MHC I-genotyped Mauritian cynomolgus macaques (MCMs) were immunized with 3 replication incompetent recombinant adenovirus serotype 5 (Ad5) vectors encoding Gag, Nef or Pol SIV proteins. MCMs were distributed into 3 groups and received two intramuscular injections of adenoviruses 4 weeks apart. One group received the anti-PD-L1 Atezolizumab and a second received a molecule in early development targeting another immune checkpoint molecule. T cell activation was monitored with blood samples taken on a weekly basis for 8 weeks. Blood samples were used to characterize the T cell immunophenotypes and to correlate them with some functional assays. Vaccination was well tolerated and could be conducted on standard safety toxicology studies without compromising other endpoints. Adv5-SIVs modulated total T cells 2 weeks after a single vaccination, induced T-cell activation, T-cell proliferation and the expression of several checkpoint molecules. Moreover, functional assays confirmed that T-cell activation profile correlated with an antigen-specific CD8 T-cell response. Compounds administered differently enhanced the profile of T-cell activation, the upregulation of immune checkpoint molecules and antigen specific responses of their T cells. Moreover, both compounds extended the cytolytic activity of the antigen-specific CD8+ T cell up to 8 weeks. Taken together, the vaccination model we developed seems appropriate to study the pharmacology of new protein-based therapeutics targeting CD8+ T cells. Citation Format: Richard Graveline, Morad Haida, Carolyne Dumont, Dominic Poulin, Florence Poitout-Belissent, Rana Samadfan, Sven Kronenberg, Franziska Regenass-Lechner, Rodney Prell, Marie-Soleil Piche. A simian challenge model to evaluate the CD8-positive T cell response with cancer therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB097.

Development and Characterization of an HCMV Multi-Antigen Therapeutic Vaccine for Glioblastoma Using the UNITE Platform.

Glioblastoma multiforme (GBM) is an aggressive form of brain cancer with a median survival of 15 months that has remained unchanged despite advances in the standard of care. GBM cells express human cytomegalovirus (HCMV) proteins, providing a unique opportunity for targeted therapy. We utilized our UNITE (UNiversal Intracellular Targeted Expression) platform to develop a multi-antigen DNA vaccine (ITI-1001) that codes for the HCMV proteins pp65, gB, and IE-1. The UNITE platform involves lysosomal targeting technology, fusing lysosome-associated membrane protein 1 (LAMP1) with target ntigens. We demonstrate evidence of increased antigen presentation by both MHC-I and -II, delivering a robust antigen-specific CD4 and CD8 T-cell response in addition to a strong humoral response. Using a syngeneic orthotopic GBM mouse model, therapeutic treatment with the ITI-1001 vaccine resulted in ~56% survival of tumor-bearing mice. Investigation of the tumor microenvironment showed significant CD4 infiltration as well as enhanced Th1 and cytotoxic CD8 T activation. Regulatory T cells were also upregulated after ITI-1001 vaccination. In addition, tumor burden negatively correlated with activated interferon (IFN)γ+ CD4 T cells, reiterating the importance of CD4 activation in ITI-1001 efficacy and in identifying treatment responders and non-responders. Further characterization of these two groups showed high infiltration of CD3+, CD4+, and CD8+ T cells in responders compared to non-responders. Thus, we show that vaccination with HCMV antigens using the ITI-1001-UNITE platform generates strong cellular and humoral immune responses, triggering significant antitumor activity, leading to enhanced survival in a mouse model of GBM.

RNA origami as a vaccine assembly platform to promote CD8 T Cell mediated anti-tumor immunity

Abstract CD8 T cell mediated immunity is critical for controlling tumor growth. However, constructing synthetic vaccines that can effectively induce this arm of the immunity has been challenging. With the advancement of nanotechnologies, various nanostructures have been explored to link antigens and adjuvants together for increased stability, lymph node delivery, cellular uptake, and activation of innate immunity, all of which may help elicit potent T cell responses against tumors. Here, we explored a self-assembling RNA origami (RNA-OG) nanostructure as a vaccine platform to assemble tumor antigens. In addition to acting as an antigen carrier, RNA-OG is an intrinsic TLR3 agonist and therefore functions as a potent adjuvant. Thus, these RNA-OG-antigen vaccines offer simultaneous delivery of antigens and adjuvants for better initiation of T-cell mediated immunity. We demonstrated enhanced antigen cross-presentation of RNA-OG-linked antigens by dendritic cells and subsequent induction of antigen-specific CD8-T cell responses. Using B16-OVA (ovalalbumin) murine melanoma tumor model, RNA-OG-OVA vaccines induced an anti-tumor immune response that contributed to long-term tumor immunity and increased survival of the animals. Overall, our data demonstrates that RNA-OG can function as a potent anti-tumor vaccine platform.

Photochemically-Mediated Inflammation and Cross-Presentation of Mycobacterium bovis BCG Proteins Stimulates Strong CD4 and CD8 T-Cell Responses in Mice.

Conventional vaccines are very efficient in the prevention of bacterial infections caused by extracellular pathogens due to effective stimulation of pathogen-specific antibodies. In contrast, considering that intracellular surveillance by antibodies is not possible, they are typically less effective in preventing or treating infections caused by intracellular pathogens such as Mycobacterium tuberculosis. The objective of the current study was to use so-called photochemical internalization (PCI) to deliver a live bacterial vaccine to the cytosol of antigen-presenting cells (APCs) for the purpose of stimulating major histocompatibility complex (MHC) I-restricted CD8 T-cell responses. For this purpose, Mycobacterium bovis BCG (BCG) was combined with the photosensitiser tetraphenyl chlorine disulfonate (TPCS2a) and injected intradermally into mice. TPCS2a was then activated by illumination of the injection site with light of defined energy. Antigen-specific CD4 and CD8 T-cell responses were monitored in blood, spleen, and lymph nodes at different time points thereafter using flow cytometry, ELISA and ELISPOT. Finally, APCs were infected and PCI-treated in vitro for analysis of their activation of T cells in vitro or in vivo after autologous vaccination of mice. Combination of BCG with PCI induced stronger BCG-specific CD4 and CD8 T-cell responses than treatment with BCG only or with BCG and TPCS2a without light. The overall T-cell responses were multifunctional as characterized by the production of IFN-γ, TNF-α, IL-2 and IL-17. Importantly, PCI induced cross-presentation of BCG proteins for stimulation of antigen-specific CD8 T-cells that were particularly producing IFN-γ and TNF-α. PCI further facilitated antigen presentation by causing up-regulation of MHC and co-stimulatory proteins on the surface of APCs as well as their production of TNF-α and IL-1β in vivo. Furthermore, PCI-based vaccination also caused local inflammation at the site of vaccination, showing strong infiltration of immune cells, which could contribute to the stimulation of antigen-specific immune responses. This study is the first to demonstrate that a live microbial vaccine can be combined with a photochemical compound and light for cross presentation of antigens to CD8 T cells. Moreover, the results revealed that PCI treatment strongly improved the immunogenicity of M. bovis BCG.

Effectiveness of ChAdOx1 nCoV-19 vaccine against SARS-CoV-2 infection during the delta (B.1.617.2) variant surge in India: a test-negative, case-control study and a mechanistic study of post-vaccination immune responses

BackgroundSARS-CoV-2 variants of concern (VOCs) have threatened COVID-19 vaccine effectiveness. We aimed to assess the effectiveness of the ChAdOx1 nCoV-19 vaccine, predominantly against the delta (B.1.617.2) variant, in addition to the cellular immune response to vaccination.MethodsWe did a test-negative, case-control study at two medical research centres in Faridabad, India. All individuals who had a positive RT-PCR test for SARS-CoV-2 infection between April 1, 2021, and May 31, 2021, were included as cases and individuals who had a negative RT-PCR test were included as controls after matching with cases on calendar week of RT-PCR test. The primary outcome was effectiveness of complete vaccination with the ChAdOx1 nCoV-19 vaccine against laboratory-confirmed SARS-CoV-2 infection. The secondary outcomes were effectiveness of a single dose against SARS-CoV-2 infection and effectiveness of a single dose and complete vaccination against moderate-to-severe disease among infected individuals. Additionally, we tested in-vitro live-virus neutralisation and T-cell immune responses to the spike protein of the wild-type SARS-CoV-2 and VOCs among healthy (anti-nucleocapsid antibody negative) recipients of the ChAdOx1 nCoV-19 vaccine.FindingsOf 2379 cases of confirmed SARS-CoV-2 infection, 85 (3·6%) were fully vaccinated compared with 168 (8·5%) of 1981 controls (adjusted OR [aOR] 0·37 [95% CI 0·28–0·48]), giving a vaccine effectiveness against SARS-CoV-2 infection of 63·1% (95% CI 51·5–72·1). 157 (6·4%) of 2451 of cases and 181 (9·1%) of 1994) controls had received a single dose of the ChAdOx1 nCoV-19 vaccine (aOR 0·54 [95% CI 0·42–0·68]), thus vaccine effectiveness of a single dose against SARS-CoV-2 infection was 46·2% (95% CI 31·6–57·7). One of 84 cases with moderate-to-severe COVID-19 was fully vaccinated compared with 84 of 2295 cases with mild COVID-19 (aOR 0·19 [95% CI 0·01–0·90]), giving a vaccine effectiveness of complete vaccination against moderate-to-severe disease of 81·5% (95% CI 9·9–99·0). The effectiveness of a single dose against moderate-to-severe disease was 79·2% (95% CI 46·1–94·0); four of 87 individuals with moderate-to-severe COVID-19 had received a single dose compared with 153 of 2364 participants with mild disease (aOR 0·20 [95% CI 0·06–0·54]). Among 49 healthy, fully vaccinated individuals, neutralising antibody responses were lower against the alpha (B.1.1.7; geometric mean titre 244·7 [95% CI 151·8–394·4]), beta (B.1.351; 97·6 [61·2–155·8]), kappa (B.1.617.1; 112·8 [72·7–175·0]), and delta (88·4 [61·2–127·8]) variants than against wild-type SARS-CoV-2 (599·4 [376·9–953·2]). However, the antigen-specific CD4 and CD8 T-cell responses were conserved against both the delta variant and wild-type SARS-CoV-2.InterpretationThe ChAdOx1 nCoV-19 vaccine remained effective against moderate-to-severe COVID-19, even during a surge that was dominated by the highly transmissible delta variant of SARS-CoV-2. Spike-specific T-cell responses were maintained against the delta variant. Such cellular immune protection might compensate for waning humoral immunity.FundingDepartment of Biotechnology India, Council of Scientific and Industrial Research India, and Fondation Botnar.

Neutrophil subtypes shape HIV-specific CD8 T-cell responses after vaccinia virus infection

Neutrophils are innate immune cells involved in the elimination of pathogens and can also induce adaptive immune responses. Nα and Nβ neutrophils have been described with distinct in vitro capacity to generate antigen-specific CD8 T-cell responses. However, how these cell types exert their role in vivo and how manipulation of Nβ/Nα ratio influences vaccine-mediated immune responses are not known. In this study, we find that these neutrophil subtypes show distinct migratory and motility patterns and different ability to interact with CD8 T cells in the spleen following vaccinia virus (VACV) infection. Moreover, after analysis of adhesion, inflammatory, and migration markers, we observe that Nβ neutrophils overexpress the α4β1 integrin compared to Nα. Finally, by inhibiting α4β1 integrin, we increase the Nβ/Nα ratio and enhance CD8 T-cell responses to HIV VACV-delivered antigens. These findings provide significant advancements in the comprehension of neutrophil-based control of adaptive immune system and their relevance in vaccine design.

T-cells modified with CD70 as an alternative cellular vaccine for antitumor immunity

Abstract Successful tumor eradication primarily depends on generation and maintenance of a large population of tumor-reactive CD8 T-cells. Dendritic cells (DCs) have applied to clinics as potent antitumor therapeutic agents. However, high cost and difficulty in obtaining sufficient amounts for clinical use are the crucial drawbacks of DC-based vaccines. Here, antigenic peptide-loaded T-cells transfected with retrovirus encoding costimulatory ligands CD70, CD80, OX40L, or 4-1BBL were assessed for antigen-specific CD8 T-cell responses, and evaluated antitumor effects along with immunization of a mixture of synthetic peptides, poly-IC and anti-CD40 antibodies (TriVax). T-cells expressing CD70 (CD70-T) exhibited similar level of stimulatory functionality and therapeutic efficacy as DCs. Moreover, CD70-T prime followed by TriVax booster heterologous vaccination elicited therapeutic antitumor effect against B16 melanoma where mediated by CD8 T-cells but not CD4 T-cells or NK cells. The combination with PD-L1 blockade led to potent therapeutic efficacy which exhibited increased tumor infiltrating CD8 T-cells. CD70-T pulsed with multi-antigenic peptide generated multiple antigen-specific polyvalent CD8 T-cells that were capable of inhibiting tumor growth effectively. Moreover, CD70-T vaccination resulted in higher expansion and migration of adoptively transferred T-cells into tumor sites, and elicits enhanced therapeutic effects with peptide-based booster immunization. These results imply that T-cells endowed with CD70 enable the design of effective vaccination strategies against solid cancer, which may overcome current limitations of DC-based vaccines.

T Cells Modified with CD70 as an Alternative Cellular Vaccine for Antitumor Immunity.

PurposeSuccessful tumor eradication primarily depends on generation and maintenance of a large population of tumor-reactive CD8 T cells. Dendritic cells (DCs) are well-known potent antigen-presenting cells and have applied to clinics as potent antitumor therapeutic agents. However, high cost and difficulty in obtaining sufficient amounts for clinical use are the crucial drawbacks of DC-based vaccines. Here, we aimed to develop T cell–based vaccine capable of eliciting potent antitumor therapeutic effects by providing effective costimulatory signals.Materials and MethodsAntigenic peptide-loaded T cells transfected with retrovirus encoding costimulatory ligands CD70, CD80, OX40L, or 4-1BBL were assessed for antigen-specific CD8 T-cell responses and evaluated antitumor effects along with immunization of a mixture of synthetic peptides, poly-IC and anti-CD40 antibodies (TriVax).ResultsT cells expressing CD70 (CD70-T) exhibited similar level of stimulatory functionality and therapeutic efficacy as DCs. Moreover, CD70-T prime followed by TriVax booster heterologous vaccination elicited therapeutic antitumor effect against B16 melanoma where mediated by CD8 T cells but not CD4 T cells or natural killer cells. The combination with programmed death-ligand 1 blockade led to potent therapeutic efficacy which exhibited increased tumor-infiltrating CD8 T cells. CD70-T pulsed with multi-antigenic peptide generated multiple antigen-specific polyvalent CD8 T cells that were capable of inhibiting tumor growth effectively. Moreover, CD70-T vaccination resulted in higher expansion and migration of adoptively transferred T cells into tumor sites and elicits enhanced therapeutic effects with peptide-based booster immu-nization.ConclusionThese results imply that T cells endowed with CD70 enable the design of effective vaccination strategies against solid cancer, which may overcome current limitations of DC-based vaccines.

Viral and tumor antigen-specific CD8 T-cell responses in Merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare and aggressive cutaneous cancer, which is immunogenic, regardless of the presence of MCPyV (80% of cases). The identification of MCC-specific epitopes recognized by CD8 T cells is crucial to expand the arsenal of immunotherapeutic treatments. Until now, most efforts focused on the identification of virus-specific epitopes, whereas immune responses directed against shared cellular tumor-specific antigens have not been evidenced. In this study, we measured T-cell responses against viral (n = 3) and tumor antigens (n = 47) from TILs derived from 21 MCC tumors. Virus-specific CD8 T-cell responses dominated MCC-specific immune responses, and we identified two new HLA-peptide complexes derived from the LT antigen, located in a region encompassing 3 previously identified epitopes. Finally, we show that MAGE-A3 antigen, frequently expressed by MCC tumors, was recognized by CD8 TILs from a virus-negative MCC tumor and thus could be a target for immunotherapy in this setting.

High-throughput Screening of Human Tumor Antigen-specific CD4 T Cells, Including Neoantigen-reactive T Cells.

Characterization of tumor antigen-specific CD4 T-cell responses in healthy donors and malignant melanoma patients using an in vitro amplified T-cell library screening procedure. A high-throughput, human leukocyte antigen (HLA)-independent approach was used to estimate at unprecedented high sensitivity level precursor frequencies of tumor antigen- and neoantigen-specific CD4 T cells in healthy donors and patients with cancer. Frequency estimation was combined with isolation and functional characterization of identified tumor-reactive CD4 T-cell clones. In healthy donors, we report frequencies of naïve tumor-associated antigen (TAA)-specific CD4 T cells comparable with those of CD4 T cells specific for infectious agents (Tetanus toxoid). Interestingly, we also identified low, but consistent numbers of memory CD4 T cells specific for several TAAs. In patients with melanoma, low frequencies of circulating TAA-specific CD4 T cells were detected that increased after peptide-based immunotherapy. Such antitumor TAA-specific CD4 T-cell responses were also detectable within the tumor-infiltrated tissues. TAA-specific CD4 T cells in patients displayed a highly polyfunctional state, with partial skewing to Type-2 polarization. Finally, we report the applicability of this approach to the detection and amplification of neoantigen-specific CD4 T cells. This simple, noninvasive, high-throughput screening of tumor- and neoantigen-specific CD4 T cells requires little biologic material, is HLA class II independent and allows the concomitant screening for a large number of tumor antigens of interest, including neoantigens. This approach will facilitate the immunomonitoring of preexisting and therapy-induced CD4 T-cell responses, and accelerate the development of CD4 T-cell-based therapies.

Dose Optimization of H56:IC31 Vaccine for Tuberculosis-Endemic Populations. A Double-Blind, Placebo-controlled, Dose-Selection Trial.

Global tuberculosis (TB) control requires effective vaccines in TB-endemic countries, where most adults are infected with Mycobacterium tuberculosis (M.tb). We sought to define optimal dose and schedule of H56:IC31, an experimental TB vaccine comprising Ag85B, ESAT-6, and Rv2660c, for M.tb-infected and M.tb-uninfected adults. We enrolled 98 healthy, HIV-uninfected, bacillus Calmette-Guérin-vaccinated, South African adults. M.tb infection was defined by QuantiFERON-TB (QFT) assay. QFT-negative participants received two vaccinations of different concentrations of H56 in 500 nmol of IC31 to enable dose selection for further vaccine development. Subsequently, QFT-positive and QFT-negative participants were randomized to receive two or three vaccinations to compare potential schedules. Participants were followed for safety and immunogenicity for 292 days. H56:IC31 showed acceptable reactogenicity profiles irrespective of dose, number of vaccinations, or M.tb infection. No vaccine-related severe or serious adverse events were observed. The three H56 concentrations tested induced equivalent frequencies and functional profiles of antigen-specific CD4 T cells. ESAT-6 was only immunogenic in QFT-negative participants who received three vaccinations. Two or three H56:IC31 vaccinations at the lowest dose induced durable antigen-specific CD4 T-cell responses with acceptable safety and tolerability profiles in M.tb-infected and M.tb-uninfected adults. Additional studies should validate applicability of vaccine doses and regimens to both QFT-positive and QFT-negative individuals. Clinical trial registered with www.clinicaltrials.gov (NCT01865487).

Interleukin-6 signalling mediates Galectin-8 co-stimulatory activity of antigen-specific CD4 T-cell response.

Galectin-8 (Gal-8) is a mammalian lectin endowed with the ability to co-stimulate antigen-specific immune responses. We have previously demonstrated that bone-marrow-derived dendritic cells produce high levels of interleukin-6 (IL-6) in response to Gal-8 stimulation. As IL-6 is a pleiotropic cytokine that has a broad effect on cells of the immune system, we aimed to elucidate whether IL-6 was involved in Gal-8-dependent co-stimulatory signals during antigen recognition by specific CD4 T cells. With this aim, splenocytes from DO11.10 mice were incubated with a low dose of the cognate ovalbumin peptide in combination with Gal-8. Interleukin-6 was found significantly increased in cultures stimulated with Gal-8 alone or Gal-8 plus cognate peptide. Moreover, IL-6 signalling was triggered during Gal-8-induced co-stimulation, as determined by phosphorylation of signal transducer and activator of transcription 3. Interleukin-6 blockade by neutralizing monoclonal antibody precluded Gal-8 co-stimulatory activity but did not affect the antigen-specific T-cell receptor activation. Different subsets of dendritic cells, as well as macrophages and B cells, were identified as the cellular source of IL-6 during Gal-8-induced co-stimulation. To confirm that IL-6 mediated the Gal-8 co-stimulatory effect, antigen-presenting cells from IL-6-deficient or wild-type mice were co-cultured with purified CD4 T cells from OTII mice in the presence of cognate peptide and Gal-8. Notably, Gal-8-induced co-stimulation, but not the antigen-specific response, was significantly impaired in the presence of IL-6-deficient antigen-presenting cells. In addition, exogenous IL-6 fully restored Gal-8-induced co-stimulation. Taken together, our results demonstrate that IL-6 signalling mediates the Gal-8 immune-stimulatory effect.

Abstract 724: A novel heterologous prime boost vaccine system drives tumor specific and potent CD8 T cell responses for cancer immunotherapy

Abstract Tumor-specific neoantigens (TSNAs) are present in a majority of tumor types and are key targets for T cells released by immune checkpoint blockade therapy. Given that TSNAs are non-self antigens, they are particularly attractive cancer vaccine targets. We have developed a potent heterologous prime/boost immunization approach to deliver predicted TSNAs to patients, which is comprised of a replication incompetent chimpanzee adenoviral vector (ChAdV) for the prime vaccination and a self-replicating, synthetic viral vector (srRNA) for repeated boost vaccinations. The ChAdV vector is similar in design to other adenoviral vectors that have demonstrated induction of high titer, polyfunctional and durable CD4 and CD8 T-cell responses against non-self antigens that protected humans against infections. The srRNA vector is based on Venezuelan equine encephalitis virus (VEE), where sequences encoding the structural proteins of VEE were deleted and replaced by a TSNA expression cassette. For delivery in vivo, the srRNA is formulated with a lipid nanoparticle (LNP), which facilitates efficient cellular uptake of the RNA and enhances antigen expression as well as the resulting immune response. We demonstrate that the srRNA vector effectively replicates in vitro and in vivo resulting in durable and high levels of antigen expression. To characterize the vectors in pre-clinical animal models, a prototypical expression cassette that encodes multiple mouse MHC class I tumor antigens as well as Mamu-A01 restricted class I antigens for monitoring immune responses in mice and non-human primates (NHPs) was introduced into both vector systems. We demonstrate that immunization of mice with either vector results in strong antigen-specific CD8 T-cell responses against the encoded murine epitopes. The heterologous prime/boost approach provided a statistically significant survival advantage to tumor bearing mice when compared to untreated mice. The potency of the immunization platform was also assessed in Indian rhesus macaques and the platform demonstrated a quick onset of T-cell responses 1 week post ChAdV prime vaccination with peak T-cell responses against all delivered Mamu-A01 restricted class I antigens at 2-3 weeks. Such ChAdV primed T-cell responses were effectively boosted by the LNP formulated srRNA vector. Co-administration of anti-CTLA4 with the vaccine demonstrated enhanced vaccine induced immune response. These data demonstrate that the heterologous prime/boost platform effectively programs robust T-cell immunity toward encoded non-self antigens in NHPs, which is a highly predictive model of vaccine responses in humans. This vaccine platform is targeted for entry into clinical trials in mid 2018. Citation Format: Wade Blair, Gijsbert Grotenbreg, Ciaran Scallan, Amy Rappaport, Renee Greer, Leonid Gitlin, Kieu Lam, James Heyes, Anne Van Abbema, Hadley Hanson, Petra Schreiner, karin Jooss. A novel heterologous prime boost vaccine system drives tumor specific and potent CD8 T cell responses for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 724.

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.

Severe Tuberculosis in Humans Correlates Best with Neutrophil Abundance and Lymphocyte Deficiency and Does Not Correlate with Antigen-Specific CD4 T-Cell Response.

It is generally thought that Mycobacterium tuberculosis (Mtb)-specific CD4+ Th1 cells producing IFN-γ are essential for protection against tuberculosis (TB). In some studies, protection has recently been associated with polyfunctional subpopulation of Mtb-specific Th1 cells, i.e., with cells able to simultaneously secrete several type 1 cytokines. However, the role for Mtb-specific Th1 cells and their polyfunctional subpopulations during established TB disease is not fully defined. Pulmonary TB is characterized by a great variability of disease manifestations. To address the role for Mtb-specific Th1 responses during TB, we investigated how Th1 and other immune cells correlated with particular TB manifestations, such as the degree of pulmonary destruction, TB extent, the level of bacteria excretion, clinical disease severity, clinical TB forms, and “Timika X-ray score,” an integrative parameter of pulmonary TB pathology. In comparison with healthy Mtb-exposed controls, TB patients (TBP) did not exhibit deficiency in Mtb-specific cytokine-producing CD4+ cells circulating in the blood and differed by a polyfunctional profile of these cells, which was biased toward the accumulation of bifunctional TNF-α+IFN-γ+IL-2− lymphocytes. Importantly, however, severity of different TB manifestations was not associated with Mtb-specific cytokine-producing cells or their polyfunctional profile. In contrast, several TB manifestations were strongly correlated with leukocyte numbers, the percent or the absolute number of lymphocytes, segmented or band neutrophils. In multiple alternative statistical analyses, band neutrophils appeared as the strongest positive correlate of pulmonary destruction, bacteria excretion, and “Timika X-ray score.” In contrast, clinical TB severity was primarily and inversely correlated with the number of lymphocytes in the blood. The results suggest that: (i) different TB manifestations may be driven by distinct mechanisms; (ii) quantitative parameters and polyfunctional profile of circulating Mtb-specific CD4+ cells play a minor role in determining TB severity; and (iii) general shifts in production/removal of granulocytic and lymphocytic lineages represent an important factor of TB pathogenesis. Mechanisms leading to these shifts and their specific role during TB are yet to be determined but are likely to involve changes in human hematopoietic system.

Factoring in CD4 T cells during treatment of HIV.

CD4 T cells acquire many functions upon activation and this diversity is essential for good health. During infection with human immunodeficiency virus (HIV), CD4 T cells become infected and decrease in number. Furthermore, these cells become less responsive to stimuli and are known to acquire biases in function that may not be beneficial for combating HIV or other co-infections. How the diversity of the CD4 T-cell response is affected following treatment of HIV-infected individuals with antiretroviral therapy (ART) is not well understood and has been difficult to study for two reasons. First, assessing antigen-specific CD4 T-cell responses has been problematic due to a lack of reagents clearly defining these cells, such as MHC-II tetramers. Second, reliable antibodies capable of detecting the different functional subsets of CD4 T cells are lacking, as is the case for GATA-3 and ROR-γt. The study by Phetsouphanh et al.1 tackles both of these challenges head-on and reveals striking changes in the diversity of CD4 T-cell responses to HIV and Cytomegalovirus (CMV) over a 48-week course of ART (Figure 1).

Synthetic Poly(L-Glutamic Acid)-conjugated CpG Exhibits Antitumor Efficacy With Increased Retention in Tumor and Draining Lymph Nodes After Intratumoral Injection in a Mouse Model of Melanoma.

There is an urgent need for new clinically applicable drug-delivery methods to enhance accumulation of immune-activating drugs in tumors. We synthesized a poly(L-glutamic acid)-CpG ODN2216 conjugate (PG-CpG) and injected it intratumorally into C57BL/6 mice bearing subcutaneous B16-ovalbumin melanoma. PG-CpG elicited the same potent antitumoral activity as CpG with respect to reducing tumor growth and triggering antigen-specific CD8 T-cell responses in this well-established solid tumor model. Moreover, PG-CpG was retained significantly longer in both tumor and draining lymph nodes than was free CpG after intratumoral injection. Specifically, 48 hours after injection, 26.5%±16.9% of the injected PG-CpG dose versus 4.72%±2.61% of free CpG remained at the tumor, and 1.53%±1.22% of the injected PG-CpG versus 0.37%±0.33% of free CpG was retained in the draining inguinal lymph nodes. These findings indicate that PG is an effective synthetic polymeric carrier for delivery of immunostimulatory agents to tumors and lymph nodes.

Peripheral self-reactivity regulates antigen-specific CD8 T-cell responses and cell division under physiological conditions.

T-cell identity is established by the expression of a clonotypic T-cell receptor (TCR), generated by somatic rearrangement of TCRα and β genes. The properties of the TCR determine both the degree of self-reactivity and the repertoire of antigens that can be recognized. For CD8 T cells, the relationship between TCR identity—hence reactivity to self—and effector function(s) remains to be fully understood and has rarely been explored outside of the H-2b haplotype. We measured the affinity of three structurally distinct CD8 T-cell-derived TCRs that recognize the identical H-2 Ld-restricted epitope, derived from the Rop7 protein of Toxoplasma gondii. We used CD8 T cells obtained from mice generated by somatic cell nuclear transfer as the closest approximation of primary T cells with physiological TCR rearrangements and TCR expression levels. First, we demonstrate the common occurrence of secondary rearrangements in endogenously rearranged loci. Furthermore, we characterized and compared the response of Rop7-specific CD8 T-cell clones upon Toxoplasma gondii infection as well as effector function and TCR signalling upon antigenic stimulation in vitro. Antigen-independent TCR cross-linking in vitro uncovered profound intrinsic differences in the effector functions between T-cell clones. Finally, by assessing the degree of self-reactivity and comparing the transcriptomes of naive Rop7 CD8 T cells, we show that lower self-reactivity correlates with lower effector capacity, whereas higher self-reactivity is associated with enhanced effector function as well as cell cycle entry under physiological conditions. Altogether, our data show that potential effector functions and basal proliferation of CD8 T cells are set by self-reactivity thresholds.