Peptide-pulsed Target Research Articles

Abstract 8: Deep Immunomics pipeline for discovery and validation of novel cancer-specific T cell receptors

Abstract Background: T cell receptor-engineered T cell (TCR-T) therapy has been studied as a high potential approach for cancer treatment. Beyond cell-surface tumor antigens, TCR-Ts can recognize HLA-presented intracellular epitopes which allows them to address a wide range of cancer-specific targets. Existing TCR-T therapies focus on a limited number of epitopes, many of which are restricted to HLA allele HLA-A*02:01. Identification of novel anti-tumor TCRs covering a broad range of HLA alleles is necessary to address unmet clinical needs. Natural T cells undergo thymic selection and present a favorable safety profile for mining such anti-tumor TCRs for therapeutic development. However, discovery of natural anti-tumor TCRs has been challenging due to i) the absence of high-throughput methods for identifying tumor-reactive TCRs in limited quantity human samples and ii) lack of sensitivity in detecting low frequency, naturally occurring tumor-reactive T cells. Methods: We developed TargetScape® as a part of our Deep Immunomics platform for efficient discovery of natural tumor-specific TCRs in healthy donors and cancer patients. Cells from blood and tissue samples were stained with metal-barcoded peptide-MHC tetramers, containing 500+ tumor-specific antigens, alongside metal-conjugated antibodies, allowing rapid identification and high dimensional phenotypic analysis of CD8+ T cells at the millions of cells scale. Single-cell sequencing revealed paired TCR sequences. Selected TCRs were introduced into luminescent Jurkat reporter cells for validation screening with peptide-pulsed targets and the relevant HLA restriction. TCRs of interest were further integrated into primary T cells and evaluated for their effector function. Results: We utilize our sensitive, high-throughput TargetScape® platform to detect and characterize putative cancer-specific TCRs from blood and tissue samples in frequencies as low as ~0.003% of total CD8+ cells. We have functionally validated over 100 TargetScape® derived TCRs with luminescent Jurkat reporters. Validated TCRs cover high prevalence HLA alleles and target a broad range of cancer antigen classes, including tumor associated antigens (TAA), Human endogenous retroviruses (HERVs), shared splice variants, and frameshift mutations. Several natural TCRs demonstrate recognition of low concentrations of peptide, indicating high functional avidity. Further evaluations of specificity and activity of TCRs of interest transduced into primary T cells are currently in progress. Conclusions: Our Deep Immunomics platform allows us to identify and confirm activity of novel, naturally-derived TCR candidates against therapeutically relevant cancer antigens presented on six common HLA alleles. These findings validate our discovery workflow leading to a portfolio of potential clinically relevant candidates that are currently under preclinical development. Citation Format: Hannah Fields, Juliana Velez Lujan, Kan Xing Wu, Natalie Epstein, Nazihah Beevi, Florence Chioh, Nicholas Tan, Neeraja Kulkarni, Noella Mukobo, Srikanthi R, Yovita Purwanti, Evan Newell, Alessandra Nardin, Michael Fehlings, Katja Fink, Daniel MacLeod. Deep Immunomics pipeline for discovery and validation of novel cancer-specific T cell receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 8.

Estrogen Receptor Mutations as Novel Targets for Immunotherapy in Metastatic Estrogen Receptor-positive Breast Cancer.

Estrogen receptor (ESR1) mutations have emerged as a key factor in endocrine therapy resistance. We identified and validated five novel, immunogenic ESR1-derived peptides that could be targeted through vaccine-based immunotherapy.

In VitroExpansion of Antigen-Specific CD8+ T Cells from Human PBMCs Using Artificial T-cell Stimulating Microparticles

Abstract While current T cell-based immunotherapies have shown moderate success, clinical translation of these methods has been hindered by biological variability, limited efficacy, and high expense. Here, we create a novel T cell stimulating platform for antigen-specific CD8+ T cell activation and expansion, eliminating the extensive ex vivoexpansion steps and thus making it accessible for many more cancer patients. The scaffold is formed using thiol-modified hyaluronic acid cross-linked with PEGDA, magnetic dextran nanoparticles, as well as DTT-reduced MHC-Ig dimer HLA-A*02 (A2) and anti-CD28. The gel is passed through a mesh device to form microparticles (MPs), which are then passively loaded with peptides (here, CMV and MART1) for ease of multiple expansions. They were plated with human PBMCs thawed overnight in culture media supplemented with human AB serum and a cocktail of cytokines (IL-1b, IL-2, IL-4, IL-6, and IFNγ). On days 7 and 14, cells were harvested and analyzed for proliferation of antigen-specific cells. After fourteen days of culturing PBMCs with A2 MPs, we could detect a significant expansion of antigen-specific T cells via flow cytometry. When stimulated with peptide-pulsed target cells for an intracellular cytokine assay, they expressed one or more of IFNγ, TNFα, and IL-2, demonstrating their polyfunctionality. We also observed a greater population of memory T cells, which indicates that these cells may persist longer in the body. We have developed a biomaterial-based T cell activation platform that provides critical signals, mimicking the lymph nodes. These MPs are effective in expanding antigen-specific T cells from human PBMCs, and these cells have robust killing of target splenocytes.

Loss of an Immunodominant HLA-Α *01:01 Restricted Epitope for CD8+ Cytotoxic T Lymphocytes (CTLs) in the Delta Variant of COVID-19: An Example of Immunologic Escape and Implications for Immunologic Treatment

SARS-COV-2 (COVID-19) has resulted in over 4 million deaths worldwide. While vaccination has decreased mortality, there remains a need for curative therapies for active infections. Uncertainties regarding the duration of post-vaccination immunity and the rapidity of mutational evolution by this virus suggest that it is unwise to rely on preventative measures alone.Humoral and cellular immunity provide selective pressure for the emergence of variant strains which have eliminated target epitopes. Elimination of immunodominant epitopes provides the strongest advantage to newly emerging strains and, consequently, immunodominant epitopes would be expected to be preferentially eliminated compared to subdominant epitopes in emerging variants. Immunologic treatments for SARS-COV-2 need to be continuously reassessed as new sequence information becomes available.TVGN-489 is a clinical grade product consisting of highly enriched, highly potent CD8+ CTLs recognizing peptides derived from COVID-19 gene/ORF products in an HLA restricted manner. CTLs are generated from apheresis products from individuals who have recovered from COVID-19 infections. Lymphocytes are serially primed and selected using APCs from these donors pulsed with small numbers of peptides encoded by the COVID-19 genome predicted or demonstrated to bind to specific HLA class I alleles. The resulting products are typically >95% CD3+/CD8+, >60% positive by tetramer staining and demonstrate strong cytolytic activity with >60% lysis of peptide pulsed targets typically at an effector to target ratio of 3:1 (See Figure).Given the immunologic pressure to lose dominant target epitopes, we assessed whether the peptides derived from genomic sequences from early SARS-COV-2 strains (and successfully used to generate CTLs from donors infected with these early strains) were still present in the more recently evolved Delta variant.Seven peptides were used to generate CTL products restricted by HLA-A*02:01, the most common allele worldwide. These peptides are derived from the spike (S) and nucleocapsid (N) proteins as well as ORF3a and ORF1ab. The contributions of these peptides to the overall cytotoxicity and tetramer staining range from 2% to 18% without clear immunodominance by one of these peptides. Though identified in early viral strains, these sequences persist in 97.5%-100% of the more than 120 Delta variant sequences present in the NIH database.For HLA-A*01:01, eight peptides derived from the matrix (M) protein as well as ORF1ab and ORF3a were utilized to generate CTLs. Seven of the eight peptides showed binding similar to what was seen with the HLA-A*02:01 peptides (1% to 18%). However, in contrast to HLA-A*02:01, an immunodominant peptide (TTDPSFLGRY, ORF1ab 1637-1646) was noted which was responsible for over half of the observed tetramer binding. This region of ORF1ab was mutated in the Delta variant resulting in loss of this immunodominant epitope from nearly 93% of the Delta genomic sequences in the NIH database. The remaining subdominant peptides were all preserved in 100% of the sequences. Given the growing number of Delta cases, it will be essential to remove this peptide from the HLA-A*01:01 peptide pool used to stimulate SARS-COV-2-specific CD8+ CTLs to avoid encouraging the expansion of cells which would recognize early strains of the virus, but not Delta variants. The remaining CTLs, generated in the absence of TTDPSFLGRY, should be capable of eradicating Delta as well as the earlier prototypic strains of COVID-19.The loss of immunodominant epitopes is not surprising in a virus such as SARS-COV-2, with a high frequency of mutation. This provides an example of immunologic escape similar to what has been described for the Delta variant in the case of HLA-A24. These data are consistent with the hypothesis that immunodominant epitopes will be preferentially eliminated as the virus continues to evolve. They further illustrate the need to monitor viral sequences and to tune the production of CTLs in order to ensure that they can continue to recognize and effectively treat newly emerging variants of COVID-19. [Display omitted] DisclosuresNo relevant conflicts of interest to declare. OffLabel Disclosure:The drug is Cytotoxic T lymphocytes that are specific to COVID-19. Preclinical data.

Design and Immunological Validation of Macaca fascicularis Papillomavirus Type 3 Based Vaccine Candidates in Outbred Mice: Basis for Future Testing of a Therapeutic Papillomavirus Vaccine in NHPs.

Persistent human papillomavirus (HPV) infections are causative for cervical neoplasia and carcinomas. Despite the availability of prophylactic vaccines, morbidity and mortality induced by HPV are still too high. Thus, an efficient therapy, such as a therapeutic vaccine, is urgently required. Herein, we describe the development and validation of Macaca fascicularis papillomavirus type 3 (MfPV3) antigens delivered via nucleic-acid and adenoviral vectors in outbred mouse models. Ten artificially fused polypeptides comprising early viral regulatory proteins were designed and optionally linked to the T cell adjuvant MHC-II-associated invariant chain. Transfected HEK293 cells and A549 cells transduced with recombinant adenoviruses expressing the same panel of artificial antigens proved proper and comparable expression, respectively. Immunization of outbred CD1 and OF1 mice led to CD8+ and CD4+ T cell responses against MfPV3 antigens after DNA- and adenoviral vector delivery. Moreover, in vivo cytotoxicity of vaccine-induced CD8+ T cells was demonstrated in BALB/c mice by quantifying specific killing of transferred peptide-pulsed syngeneic target cells. The use of the invariant chain as T cell adjuvant enhanced the T cell responses regarding cytotoxicity and in vitro analysis suggested an accelerated turnover of the antigens as causative. Notably, the fusion-polypeptide elicited the same level of T-cell responses as administration of the antigens individually, suggesting no loss of immunogenicity by fusing multiple proteins in one vaccine construct. These data support further development of the vaccine candidates in a follow up efficacy study in persistently infected Macaca fascicularis monkeys to assess their potential to eliminate pre-malignant papillomavirus infections, eventually instructing the design of an analogous therapeutic HPV vaccine.

Peptides Derived From S and N Proteins of Severe Acute Respiratory Syndrome Coronavirus 2 Induce T Cell Responses: A Proof of Concept for T Cell Vaccines.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that escape vaccine-induced neutralizing antibodies has indicated the importance of T cell responses against this virus. In this study, we highlight the SARS-CoV-2 epitopes that induce potent T cell responses and discuss whether T cell responses alone are adequate to confer protection against SARS-CoV-2 and describe the administration of 20 peptides with an RNA adjuvant in mice. The peptides have been synthesized based on SARS-CoV-2 spike and nucleocapsid protein sequences. Our study demonstrates that immunization with these peptides significantly increases the proportion of effector memory T cell population and interferon-γ (IFN-γ)-, interleukin-4 (IL-4)-, tumor necrosis factor-α (TNF-α)-, and granzyme B-producing T cells. Of these 20 peptides, four induce the generation of IFN-γ-producing T cells, elicit CD8+ T cell (CTL) responses in a dose-dependent manner, and induce cytotoxic T lymphocytes that eliminate peptide-pulsed target cells in vivo. Although it is not statistically significant, these peptide vaccines reduce viral titers in infected hamsters and alleviate pulmonary pathology in SARS-CoV-2-infected human ACE2 transgenic mice. These findings may aid the design of effective SARS-CoV-2 peptide vaccines, while providing insights into the role of T cells in SARS-CoV-2 infection.

TVGN-489: A novel approach for the production of SARS-CoV-2-specific cytotoxic T lymphocytes to treat COVID-19

Abstract SARS-COV-2 (COVID-19) has resulted in over 2 million deaths. While vaccination is expected to decrease mortality, there remains a need for curative therapies for active infections. Uncertainties regarding the duration of post-vaccination immunity and the rapidity of mutational evolution by this virus suggest that it is unwise to rely on preventative measures alone. Dysregulation of endogenous cellular immunity has been implicated in the failure to control COVID-19 infections suggesting that allogeneic T cell therapy using nature’s curative approach to viral infections could successfully be applied to vulnerable patients. Allogeneic virus-specific cytotoxic T lymphocytes (CTLs) have a long track record of safety and efficacy in treating viral infections occurring after allogeneic stem cell transplantation. We hypothesize that this approach can be applied to the treatment of Covid-19. Covid-19 specific CTLs were produced by priming and enriching T cells from convalescent patients using COVID-19 peptides predicted or demonstrated to bind to specific HLA alleles. Utilizing the globally common HLA-A*02:01 allele as the restriction element, 7 peptides from 4 COVID-19 gene/ORF products were identified and used as a pool for serial restimulation and enrichment on a peptide pulsed, HLA-A*02:01 antigen presenting cell monolayer. This preclinical effort produced CTLs of high purity (>95% CD3+/CD8+, >60% positive by tetramer staining) and potency (60% lysis of peptide pulsed targets at an effector to target ratio of 3:1) in sufficient quantities for clinical application. Trials using these TVGN-489 CTLs and those produced with other SARS-COV-2 peptides and HLA restriction elements will hopefully be launched shortly.

Downregulation of HLA-I by the molluscum contagiosum virus mc080 impacts NK-cell recognition and promotes CD8+ T-cell evasion.

Molluscum contagiosum virus (MCV) is a common cause of benign skin lesions in young children and currently the only endemic human poxvirus. Following the infection of primary keratinocytes in the epidermis, MCV induces the proliferation of infected cells and this results in the production of wart-like growths. Full productive infection is observed only after the infected cells differentiate. During this prolonged replication cycle the virus must avoid elimination by the host immune system. We therefore sought to investigate the function of the two major histocompatibility complex class-I-related genes encoded by the MCV genes mc033 and mc080. Following insertion into a replication-deficient adenovirus vector, codon-optimized versions of mc033 and mc080 were expressed as endoglycosidase-sensitive glycoproteins that localized primarily in the endoplasmic reticulum. MC080, but not MC033, downregulated cell-surface expression of endogenous classical human leucocyte antigen (HLA) class I and non-classical HLA-E by a transporter associated with antigen processing (TAP)-independent mechanism. MC080 exhibited a capacity to inhibit or activate NK cells in autologous assays in a donor-specific manner. MC080 consistently inhibited antigen-specific T cells being activated by peptide-pulsed targets. We therefore propose that MC080 acts to promote evasion of HLA-I-restricted cytotoxic T cells.

CD8+ T Cell Immunity Is Compromised by Anti-CD20 Treatment and Rescued by Interleukin-17A.

Treatment with anti-CD20, used in many diseases in which B cells play a pathogenic role, has been associated with susceptibility to intracellular infections. Here, we studied the effect of anti-CD20 injection on CD8+ T cell immunity using an experimental model of Trypanosoma cruzi infection, in which CD8+ T cells play a pivotal role. C57BL/6 mice were treated with anti-CD20 for B cell depletion prior to T. cruzi infection. Infected anti-CD20-treated mice exhibited a CD8+ T cell response with a conserved expansion phase followed by an early contraction, resulting in a strong reduction in total and parasite-specific CD8+ T cell numbers at 20 days postinfection. Anti-CD20 injection increased the frequency of apoptotic CD8+ T cells, decreased the number of effector and memory CD8+ T cells, and reduced the frequency of proliferating and cytokine-producing CD8+ T cells. Accordingly, infected anti-CD20-treated mice presented lower cytotoxicity of T. cruzi peptide-pulsed target cells in vivo All of these alterations in CD8+ T cell immunity were associated with increased tissue parasitism. Anti-CD20 injection also dampened the CD8+ T cell response, when this had already been generated, indicating that B cells were involved in the maintenance rather than the induction of CD8+ T cell immunity. Anti-CD20 injection also resulted in a marked reduction in the frequency of interleukin-6 (IL-6)- and IL-17A-producing cells, and recombinant IL-17A (rIL-17A) injection partially restored the CD8+ T cell response in infected anti-CD20-treated mice. Thus, anti-CD20 reduced CD8+ T cell immunity, and IL-17A is a candidate for rescuing deficient responses either directly or indirectly.IMPORTANCE Monoclonal antibody targeting the CD20 antigen on B cells is used to treat the majority of non-Hodgkin lymphoma patients and some autoimmune disorders. This therapy generates adverse effects, notably opportunistic infections and activation of viruses from latency. Here, using the infection murine model with the intracellular parasite Trypanosoma cruzi, we report that anti-CD20 treatment affects not only B cell responses but also CD8+ T cell responses, representing the most important immune effectors involved in control of intracellular pathogens. Anti-CD20 treatment, directly or indirectly, affects cytotoxic T cell number and function, and this deficient response was rescued by the cytokine IL-17A. The identification of IL-17A as the cytokine capable of reversing the poor response of CD8+ T cells provides information about a potential therapeutic treatment aimed at enhancing defective immunity induced by B cell depletion.

Naturally processed HLA-DR3-restricted HHV-6B peptides are recognized broadly with polyfunctional and cytotoxic CD4 T-cell responses.

Human herpes virus 6B (HHV-6B) is a widespread virus that infects most people early in infancy and establishes a chronic life-long infection with periodic reactivation. CD4 Tcells have been implicated in control of HHV-6B, but antigenic targets and functional characteristics of the CD4 T-cell response are poorly understood. We identified 25 naturally processed MHC-II peptides, derived from six different HHV-6B proteins, and showed that they were recognized by CD4 T-cell responses in HLA-matched donors. The peptides were identified by mass spectrometry after elution from HLA-DR molecules isolated from HHV-6B-infected Tcells. The peptides showed strong binding to matched HLA alleles and elicited recall T-cell responses in vitro. T-cell lines expanded in vitro were used for functional characterization of the response. Responding cells were mainly CD3+ CD4+ , produced IFN-γ, TNF-α, and low levels of IL-2, alone or in combination, highlighting the presence of polyfunctional Tcells in the overall response. Many of the responding cells mobilized CD107a, stored granzyme B, and mediated specific killing of peptide-pulsed target cells. These results highlight a potential role for polyfunctional cytotoxic CD4 Tcells in the long-term control of HHV-6B infection.

Introduction of Genetically Modified CD3ζ Improves Proliferation and Persistence of Antigen-Specific CTLs.

The clinical efficacy of T-cell therapies based on T cells transduced with genes encoding tumor-specific T-cell receptors (TCR-T) is related to the in vivo persistence of the T cells. To improve persistence without modifying TCR affinity, we instead modified intracellular signaling, using artificial T cell-activating adapter molecules (ATAM), generated by inserting the intracellular domain (ICD) of activating T-cell signaling moieties into CD3ζ. ATAMs with the ICD of either CD28 or 4-1BB were generated, assembled into the TCR complex as a part of CD3ζ, and enhanced downstream signaling from the supramolecular activation cluster. ATAMs were retrovirally introduced into human CMV-specific or NY-ESO-1-specific TCR-transduced CD8+ T lymphocytes, and downstream functionality was then examined. ATAM-transduced NY-ESO-1 TCR-T cells were also investigated using the U266-xenograft mouse model. ATAMs were successfully transduced and localized to the cell membrane. ATAM-transduced CMV-specific T cells retained their cytotoxic activity and cytokine production against peptide-pulsed target cells without altering antigen-specificity and showed resistance to activation-induced cell death. Upon both single and repeated stimulation, CD3ζ/4-1BB-transduced T cells had superior proliferation to the CD3ζ-transduced T cells in both the CMV-specific and the NY-ESO-1 TCR-T models and significantly improved antitumor activity compared with untransduced T cells both in vitro and in a mouse xenograft model. ATAM-transduced TCR-T cells demonstrated improved proliferation and persistence in vitro and in vivo This strategy to control the intracellular signaling of TCR-T cells by ATAM transduction in combination with various tumor-specific TCRs may improve the efficacy of TCR-T therapy. Cancer Immunol Res; 6(6); 733-44. ©2018 AACR.

Novel MVA-based vaccine expressing influenza NP and M1 activates cross-reactive T cell responses in human nasopharynx-associated lymphoid tissue (NALT)

Abstract Influenza virus infection continues to cause widespread morbidity and mortality, resulting in major challenges for healthcare systems. Current influenza vaccines are of limited efficacy against mismatched or emerging new virus strains. Recent efforts are focused on the development of novel vaccines capable of conferring broad protection against various influenza virus subtypes. We have studied the potential of Modified Vaccinia Ankara (MVA)-vectored vaccines expressing nucleoprotein and matrix protein 1 (MVA-NP+M1) to induce cross-reactive mucosal immunity using human adenotonsillar mononuclear cell (MNC) culture system with flow cytometry and ELISPOT analysis. Influenza NP and M1 antigens were abundantly expressed following vaccine stimulation in MNC particularly in B cells and dendritic cells. M1-specific T cell response was analyzed in detail, and MVA-NP+M1 was shown to activate IFN-γ-producing CD8+ T cells against conserved M1 epitopes. M158–66-specific CD8+ T cells in HLA-matched individuals were markedly increased in an age-dependent manner. These M1-specific T cells coexpressed cytotoxic molecules such as perforin and granzyme A and B. Upon the recognition of M158–66 peptide, these cells displayed marked increase in surface CD107a expression (implicating degranulation), together with the production of pro-inflammatory cytokines including IFN-γ and TNF-α, that correlated to their ability to kill the peptide-pulsed target cells. In summary, we showed MVA-NP+M1 activates conserved influenza antigen-specific cytotoxic T cell response in human NALT. The results suggest MVA-NP+M1 vaccine has the potential to induce cross-reactive mucosal T cell immunity against different types of influenza viruses.

Induction of Genotype Cross-Reactive, Hepatitis C Virus-Specific, Cell-Mediated Immunity in DNA-Vaccinated Mice.

A universal hepatitis C virus (HCV) vaccine should elicit multiantigenic, multigenotypic responses, which are more likely to protect against challenge with the range of genotypes and subtypes circulating in the community. A vaccine cocktail and vaccines encoding consensus HCV sequences are attractive approaches to achieve this goal. Consequently, in a series of mouse vaccination studies, we compared the immunogenicity of a DNA vaccine encoding a consensus HCV nonstructural 5B (NS5B) protein to that of a cocktail of DNA plasmids encoding the genotype 1b (Gt1b) and Gt3a NS5B proteins. To complement this study, we assessed responses to a multiantigenic cocktail regimen by comparing a DNA vaccine cocktail encoding Gt1b and Gt3a NS3, NS4, and NS5B proteins to a single-genotype NS3/4/5B DNA vaccine. To thoroughly evaluate in vivo cytotoxic T lymphocyte (CTL) and T helper (Th) cell responses against Gt1b and Gt3a HCV peptide-pulsed target cells, we exploited a novel fluorescent-target array (FTA). FTA and enzyme-linked immunosorbent spot (ELISpot) analyses collectively indicated that the cocktail regimens elicited higher responses to Gt1b and Gt3a NS5B proteins than those with the consensus vaccine, while the multiantigenic DNA cocktail significantly increased the responses to NS3 and NS5B compared to those elicited by the single-genotype vaccines. Thus, a DNA cocktail vaccination regimen is more effective than a consensus vaccine or a monovalent vaccine at increasing the breadth of multigenotypic T cell responses, which has implications for the development of vaccines for communities where multiple HCV genotypes circulate.IMPORTANCE Despite the development of highly effective direct-acting antivirals (DAA), infections with hepatitis C virus (HCV) continue, particularly in countries where the supply of DAA is limited. Furthermore, patients who eliminate the virus as a result of DAA therapy can still be reinfected. Thus, a vaccine for HCV is urgently required, but the heterogeneity of HCV strains makes the development of a universal vaccine difficult. To address this, we developed a novel cytolytic DNA vaccine which elicits robust cell-mediated immunity (CMI) to the nonstructural (NS) proteins in vaccinated animals. We compared the immune responses against genotypes 1 and 3 that were elicited by a consensus DNA vaccine or a DNA vaccine cocktail and showed that the cocktail induced higher levels of CMI to the NS proteins of both genotypes. This study suggests that a universal HCV vaccine can most readily be achieved by use of a DNA vaccine cocktail.

Improvement of cytomegalovirus pp65 DNA vaccine efficacy by co-administration of siRNAs targeting BAK and BAX.

The efficacy of DNA vaccines may be improved by small interfering (si)RNA adjuvants targeting pro-apoptotic genes. The aim of the present study was to investigate the capacity of siRNAs targeting B-cell lymphoma 2 homologous antagonist killer (BAK) and B-cell lymphoma 2-associated X protein (BAX) to improve the efficacy of a cytomegalovirus (CMV) vaccine. BALB/c mice were divided into four groups (n=18 in each): unimmunized and immunized with pcDNA 3.1-pp65 expressing CMV 65 kDa matrix phosphoprotein and BAK + BAX siRNAs, pcDNA 3.1-pp65 and control siRNA, or control pcDNA 3.1 and BAK + BAX siRNAs. Immunizations were performed twice with an interval of 3 weeks. CMV-specific mouse splenocyte interferon (IFN)-γ secretion was assessed by ELISPOT; furthermore, an in vivo cytotoxic T lymphocyte assay was performed 2 weeks after the last immunization. After lethal CMV challenge of the mice, body weight, virus titers in the spleens and salivary glands as well as survival were recorded. The amount of splenocytes secreting IFN-γ in response to CMV pp65 peptides and specific lysis of peptide-pulsed target cells were significantly higher in mice administered pcDNA3.1-pp65 and BAK + BAX siRNAs than those in mice administered pcDNA3.1-pp65 and control siRNA (P<0.05 for each). After the virus challenge, the virus titers in the spleens and salivary glands of mice given pcDNA3.1-pp65 and BAK + BAX siRNAs were significantly lower than those in mice immunized with pcDNA3.1-pp65 and control siRNA (P<0.05 for each). Furthermore, mice immunized with pcDNA 3.1-pp65 and control siRNA or BAK + BAX siRNAs survived for longer, and at 21 days after lethal CMV challenge, 66 and 100% of these mice survived, respectively. These mice also experienced less weight loss compared with mice immunized with pcDNA3.1-pp65 and control siRNA (P<0.05). In conclusion, intradermal administration of siRNAs targeting BAK and BAX improved the efficacy of CMV pp65 DNA vaccine.

Hypoxia-inducing factor (HIF)-1α-derived peptide capable of inducing cancer-reactive cytotoxic T lymphocytes from HLA-A24+ patients with renal cell carcinoma

Hypoxic tumor microenvironment makes cancer cells to be therapy-resistant and hypoxia-inducing factors (HIFs) play a central role in hypoxic adaptation. Especially, renal cell carcinoma (RCC) is often associated with von Hippel-Lindau (VHL) gene mutations, leading to up-regulation of HIFs. However, from a different point of view, this suggests the possibility that HIFs could be promising targets in anti-cancer therapy. In this study, we searched for HIF-1α-derived peptides that are able to induce RCC-reactive cytotoxic T lymphocytes (CTLs) from HLA-A24+ RCC patients. Among five peptides derived from HIF-1α, which were prepared based on the binding motif to the HLA-A24 allele, a HIF-1α278–287 peptide induced peptide-specific CTLs from peripheral blood mononuclear cells of HLA-A24+ RCC patients most effectively. In immunoblot assays, the expression of HIF-1α was lowly detected in whole and nuclear lysates of RCC cell lines even under normoxia (20% O2), and their expression in whole lysates was increased under hypoxia (1% O2). Additionally, HIF-1α278–287 peptide-stimulated T cells showed a higher cytotoxicity against HLA-A24+ HIF-1α-expressing RCC cells than against HLA-A24− HIF-1α-expressing RCC cells. The cytotoxicity was inhibited by the addition of HIF-1α278–287 peptide-pulsed cold target cells. Altogether, these results indicate that the HIF-1α278–287 peptide could be a candidate for peptide-based anti-cancer vaccines for HLA-A24+ RCC patients.

MAVS Expressed by Hematopoietic Cells Is Critical for Control of West Nile Virus Infection and Pathogenesis.

West Nile virus (WNV) is the most important cause of epidemic encephalitis in North America. Innate immune responses, which are critical for control of WNV infection, are initiated by signaling through pathogen recognition receptors, RIG-I and MDA5, and their downstream adaptor molecule, MAVS. Here, we show that a deficiency of MAVS in hematopoietic cells resulted in increased mortality and delayed WNV clearance from the brain. In Mavs(-/-) mice, a dysregulated immune response was detected, characterized by a massive influx of macrophages and virus-specific T cells into the infected brain. These T cells were polyfunctional and lysed peptide-pulsed target cells in vitro However, virus-specific T cells in the brains of infected Mavs(-/-) mice exhibited lower functional avidity than those in wild-type animals, and even virus-specific memory T cells generated by prior immunization could not protect Mavs(-/-) mice from WNV-induced lethal disease. Concomitant with ineffective virus clearance, macrophage numbers were increased in the Mavs(-/-) brain, and both macrophages and microglia exhibited an activated phenotype. Microarray analyses of leukocytes in the infected Mavs(-/-) brain showed a preferential expression of genes associated with activation and inflammation. Together, these results demonstrate a critical role for MAVS in hematopoietic cells in augmenting the kinetics of WNV clearance and thereby preventing a dysregulated and pathogenic immune response. West Nile virus (WNV) is the most important cause of mosquito-transmitted encephalitis in the United States. The innate immune response is known to be critical for protection in infected mice. Here, we show that expression of MAVS, a key adaptor molecule in the RIG-I-like receptor RNA-sensing pathway, in hematopoietic cells is critical for protection from lethal WNV infection. In the absence of MAVS, there is a massive infiltration of myeloid cells and virus-specific T cells into the brain and overexuberant production of proinflammatory cytokines. These results demonstrate the important role that MAVS expression in hematopoietic cells has in regulating the inflammatory response in the WNV-infected brain.

Control of HIV-1 replication in vitro by vaccine-induced human CD8+ T cells through conserved subdominant Pol epitopes

ObjectiveThe specificity of CD8+ T cells is critical for early control of founder/transmitted and reactivated HIV-1. To tackle HIV-1 variability and escape, we designed vaccine immunogen HIVconsv assembled from 14 highly conserved regions of mainly Gag and Pol proteins. When administered to HIV-1-negative human volunteers in trial HIV-CORE 002, HIVconsv vaccines elicited CD8+ effector T cells which inhibited replication of up to 8 HIV-1 isolates in autologous CD4+ cells. This inhibition correlated with interferon-γ production in response to Gag and Pol peptide pools, but direct evidence of the inhibitory specificity was missing. Here, we aimed to define through recognition of which epitopes these effectors inhibit HIV-1 replication. DesignCD8+ T-cells from the 3 broadest HIV-1 inhibitors out of 23 vaccine recipients were expanded in culture by Gag or Pol peptide restimulation and tested in viral inhibition assay (VIA) using HIV-1 clade B and A isolates. MethodsFrozen PBMCs were expanded first using peptide pools from Gag or Pol conserved regions and tested on HIV-1-infected cells in VIA or by individual peptides for their effector functions. Single peptide specificities responsible for inhibition of HIV-1 replication were then confirmed by single-peptide expanded effectors tested on HIV-1-infected cells. ResultsWe formally demonstrated that the vaccine-elicited inhibitory human CD8+ T cells recognized conserved epitopes of both Pol and Gag proteins. We defined 7 minimum epitopes, of which 3 were novel, presumably naturally subdominant. The effectors were oligofunctional producing several cytokines and chemokines and killing peptide-pulsed target cells. ConclusionsThese results implicate the use of functionally conserved regions of Pol in addition to the widely used Gag for T-cell vaccine design. Proportion of volunteers developing these effectors and their frequency in circulating PBMC are separate issues, which can be addressed, if needed, by more efficient vector and regimen delivery of conserved immunogens.

A robust and scalable TCR-based reporter cell assay to measure HIV-1 Nef-mediated T cell immune evasion

HIV-1 evades cytotoxic T cell responses through Nef-mediated downregulation of HLA class I molecules from the infected cell surface. Methods to quantify the impact of Nef on T cell recognition typically employ patient-derived T cell clones; however, these assays are limited by the cost and effort required to isolate and maintain primary cell lines. The variable activity of different T cell clones and the limited number of cells generated by re-stimulation can also hinder assay reproducibility and scalability. Here, we describe a heterologous T cell receptor reporter assay and use it to study immune evasion by Nef. Induction of NFAT-driven luciferase following co-culture with peptide-pulsed or virus-infected target cells serves as a rapid, quantitative and antigen-specific measure of T cell recognition of its cognate peptide/HLA complex. We demonstrate that Nef-mediated downregulation of HLA on target cells correlates inversely with T cell receptor-dependent luminescent signal generated by effector cells. This method provides a robust, flexible and scalable platform that is suitable for studies to measure Nef function in the context of different viral peptide/HLA antigens, to assess the function of patient-derived Nef alleles, or to screen small molecule libraries to identify novel Nef inhibitors.

Amplification Ex Vivo and Cytocidal Activity of Leukemia Tumor-Associated Antigen-Specific Cytotoxic T Lymphocytes

To explore the feasibility of amplifying the leukemia tumor associated antigens-specific cytotoxic T lymphocytes (TAA-CTL) ex vivo and to evaluate the cytotoxicity of TAA-CTL. The peripheral blood mononuclear cells were enriched by density gradient centrifugtion; TAA-CTL were generated by stimulation of PBMNC with peptide-pulsed DC at an effector-to-target ratio of 10:1; immunophenotype of TAA-CTL was analyzed by flow cytometry; cytotoxicity assay was used to evaluate the cytotoxic activity of TAA-CTL against peptide-pulsed autologous target cells (PHA-Blasts). TAA-CTL expanded from volunteer showed a mean expansion of 3.81±1.61, the phenotyping of the TAA-CTL was predominantly CD3+ (97.22±0.71)% with varying content of CD4+ (41.47±27.08)% and CD8+ (56.40±11.15)% T cells, it also contained few nature killer cells (0.50±0.31)% and rare residual B cells (0.14±0.20)%; the subpopulations of TAA-CTL and CTL were not statisticaly significantly different in the proportion (P>0.05); the detection of intracellular cytokines after stimulation with peptide showed that the secretion rates of IFN-γ and TNF-α in CD8+ TAA-CTL were (27.67±2.21)% and (34.2±0.71)%, while the secretion rates were (21.6±2.55)% and (9.97±3.44)% in CD4+ TAA-CTL. Compared with the CD8+ TAA-CTL group, the secretion rates of IFN-γ and TNF-α were (1.36±0.04)% and (5.58±0.03)% in CD8+ CTL, the rates of IFN-γ and TNF-α were (0.91±0.06)% and (1.60±0.07)% in CD4+ CTL. The secretions of IFN-γ and TNF-α in CTL were both significantly lower than those in TAA-CTL (P<0.01); the specific killing efficiency of the TAA-CTL against TAA-pulesd target cells were (77.00±1.00)%, (67.40±3.60)%, (60.55±2.45)% and (26.85±5.25)%, when the effecto-target ratios were 40:1, 20:1, 10:1 and 5:1, and there was negligible lysis of TAA-CTL for PHA-blast (P<0.01). TAA-CTL can be successfully induced and generated ex vivo from the healthy volunteer peripheral blood, and the TAA-CTL possess a specific killing activity.

ITOC2 – 021. The melanoma immune-peptidome for T-cell-based anti-tumour immunotherapies

Metastatic malignant melanoma (MMM) is a highly aggressive disease. Immune checkpoint inhibitors have proved immunotherapeutic efficacy; however, not all patients profit and severe side effects may occur. Currently, little is known about the tumour-specific targets and the tumour-reactive effector cells that are involved. Aiming to characterise the melanoma immune-peptidome, we purified HLA-I and HLA-II complexes from MMM patient tumours and accurately identified thousands of unique HLA binding peptides per tissue sample, using state-of-the-art mass spectrometry. We applied algorithms for database search in combination with de-novo sequencing and identified many novel cancer-associated peptides from known melanoma-specific antigens (PRAME, Tyrosinase, MAGEA3, PMEL, CTAG1A, PAX3 etc.). We further extended our search to post-translationally modified peptides that potentially serve as neo-antigens. After in vitro T-cell stimulation assays, we detected elevated levels of peptide-specific T-cells that displayed functional responses against peptide-pulsed target cells as well as tumour reactivity. We established this large-scale resource of the melanoma in vivo presented immune-peptidome that contains many novel targets validated by immune reactivity. These data provide the fundament for further optimisation of treatment modalities for this life-threatening disease.