Epitope-specific Cytotoxic T Lymphocytes Research Articles

Redirector of Vaccine-induced Effector Responses (RoVER) for specific killing of cellular targets

In individuals with malignancy or HIV-1 infection, antigen-specific cytotoxic T lymphocytes (CTLs) often display an exhausted phenotype with impaired capacity to eliminate the disease. Existing cell-based immunotherapy strategies are often limited by the requirement for adoptive transfer of CTLs. We have developed an immunotherapy technology in which potent CTL responses are generated invivo by vaccination and redirected to eliminate target cells using a bispecific Redirector of Vaccine-induced Effector Responses (RoVER). Following Yellow fever (YF) 17D vaccination of 51 healthy volunteers (NCT04083430), single-epitope YF-specific CTL responses were quantified by tetramer staining and multi-parameter flow cytometry. RoVER-mediated redirection of YF-specific CTLs to kill antigen-expressing Raji-Env cells, autologous CD19+ B cells or CD4+ T cells infected invitro with a full-length HIV-1-eGFP was assessed in cell killing assays. Moreover, secreted IFN-γ, granzyme B, and TNF-α were analyzed by mesoscale multiplex assays. YF-17D vaccination induced strong epitope-specific CTL responses in the study participants. In cell killing assays, RoVER-mediated redirection of YF-specific CTLs to autologous CD19+ B cells or HIV-1-infected CD4+ cells resulted in 58% and 53% killing at effector to target ratio 1:1, respectively. We have developed an immunotherapy technology in which epitope-specific CTLs induced by vaccination can be redirected to kill antigen-expressing target cells by RoVER linking. The RoVER technology is highly specific and can be adapted to recognize various cell surface antigens. Importantly, this technology obviates the need for adoptive transfer of CTLs. This work was funded by the Novo Nordisk Foundation (Hallas Møller NNF10OC0054577).

Development of a novel immunoproteasome digestion assay for synthetic long peptide vaccine design.

Recently, many autologous tumor antigens have been examined for their potential use in cancer immunotherapy. However, the success of cancer vaccines in clinical trials has been limited, partly because of the limitations of using single, short peptides in most attempts. With this in mind, we aimed to develop multivalent synthetic long peptide (SLP) vaccines containing multiple cytotoxic T-lymphocyte (CTL) epitopes. However, to confirm whether a multivalent vaccine can induce an individual epitope-specific CTL, the only viable screening strategies currently available are interferon-gamma (IFN-μ enzyme-linked immunospot (ELISPOT) assays using human peripheral blood mononuclear cells, or expensive human leukocyte antigen (HLA)-expressing mice. In this report, we evaluated the use of our developed murine-20S immunoproteasome (i20S) digestion assay, and found that it could predict the results of IFN-μ ELISPOT assays. Importantly, the murine-i20S digestion assay not only predicted CTL induction, but also antitumor activity in an HLA-expressing mouse model. We conclude that the murine-i20S digestion assay is an extremely useful tool for the development of “all functional” multivalent SLP vaccines.

9-Phenanthrol enhances the generation of an CD8+ T cell response following transcutaneous immunization with imiquimod in mice

BackgroundTranscutaneous immunization (TCI) is a non-invasive vaccination strategy targeting the skin-associated lymphoid tissue. Topical application of the TLR7 agonist imiquimod as adjuvant in combination with peptide antigens activates the innate immune system and mounts cytotoxic T lymphocyte (CTL) responses. ObjectiveBased on the commercial 5% imiquimod-containing drug Aldara we aimed to develop an improved formulation with superior vaccination efficiencies. The primary target was the enhancement of mast cell activation as important key for the function of the innate immune system. MethodsWe investigated the effects of 9-phenanthrol (9-phe) on the activation of mast cells in vitro and in vivo. For TCI, we applied 0.2% 9-phe in Aldara or Aldara alone as adjuvants in combination with the MHC class I – restricted peptide SIINFEKL. To monitor vaccination, mast cell degranulation, migration of DC and frequencies of epitope-specific CTL was assessed. In a transgenic tumor model, the efficiencies of prophylactic immunization against a tumor antigen were also monitored. Results9-phe induced degranulation of mast cells in vitro and upon topical application in vivo. A mixture of 0.2% 9-phe in Aldara showed superior results regarding the migration of DC and the expansion of antigen-specific CTL. Consequently, prophylactic immunization with 0.2% 9-phe in Aldara caused enhanced protection against tumor inoculation. ConclusionOur data demonstrate that a simple modification of an adjuvant formulation can yield superior results in experimental vaccination protocols by boosting critical steps leading to the generation of an efficient CTL response.

CD133 epitope vaccine with gp96 as adjuvant elicits an antitumor T cell response against leukemia

Cancer stem cells are currently under intensive investigation due to their capabilities for tumor initiation, self-renewal, and resistance to chemotherapy. CD133 is implicated in stemness and the malignancy of tumor cells. Here, we explored heat shock protein gp96 adjuvanted CD133 epitope vaccine against leukemia. We screened and identified three H2-Kd-restricted cytotoxic T lymphocyte (CTL) epitopes derived from CD133, CD133₄₁₉₋₄₂₈, CD133₇₀₂₋₇₁₀ and CD133₇₆₀₋₇₆₉. The immunogenicity and antitumor activity of the epitope vaccine using heat shock protein gp96 as adjuvant were further determined in CD133⁺ leukemia xenograft mice. Finally, we demonstrate that adoptive transfer of epitope-specific CTLs led to suppression of leukemia growth. Our data therefore provide the basis for designing a CD133 epitope vaccine to activate specific CTLs against CD133⁺ leukemia and other cancers.

Differential Potency and Breadth of the Same Epitope-Specific CTLs to Inhibit HIV-1 Replication based on TCR Usage

Increasing evidence suggests that the immune control of viral replication by HIV-1-specific cytotoxic T lymphocytes (CTLs) is relevant to the selection of human leukocyte antigen (HLA) allele-restricted antigen-specific CTL repertoire. But the underlying factors accounting for CTL functional difference from T cell receptor (TCR) clonal diversity are currently unclear. Here we found that CTL repertoire specific for HLA-B*27-restricted HIV-1 Gag p24 epitope KK10 (KRWIILGLNK, residues 263-272) selected multiple TCR clonotypes. By dissection of the bulk KK10-specific CTL compartment, differential potency and breadth of KK10-specific CTL clones to inhibit HIV-1 replication was defined due to the distinct TCR usage. Superior control of viral replication of wild-type HIV-1 isolates was observed by the TCR-clonotypic CTLs characteristic of higher ability to produce MIP-1β. A unique TCR-equipped KK10- specific CTLs efficiently controlled wide-type HIV-1 isolates and broadly cross-recognized viral variants. These data suggest that clonally diverse CTL responses to a viral epitope increase the likelihood that multiple divergent strains of viruses may be recognized by cross-reactive CTLs through immunization with only a single viral epitope sequence.

Delivery of foreign cytotoxic T lymphocyte epitopes to tumor tissues for effective antitumor immunotherapy against pre-established solid tumors in mice.

Cytotoxic T lymphocyte (CTL) can have remarkable abilities to kill tumor cells. However, the establishment of successful CTL-based anticancer therapy has met with many challenges. Within tumor cells, there exist subpopulations with low or no expression of the targeted antigen (termed as antigen-loss variants). In addition, tumor cells can downregulate the levels of major histocompatibility complex class I (MHC-I) molecules on cell surface due to immune pressure. As a result, some tumor cells can escape the immune pressure bestowed by CTLs, resulting in treatment failure. To address these difficulties, a new approach is developed to deliver foreign high-affinity CTL epitopes to tumor tissues utilizing pH-responsive "smart" microparticles (MPs). These MPs could encapsulate CTL peptide epitope, release the peptide under acidic condition encountered in tumor tissues and enhance CTL activation. Mice bearing pre-established tumor as "antigen-loss variant" solid tumor models were administered intratumorally with MPs containing the CTL peptide, which showed 100% survival following the treatment. In contrast, all control mice died from tumor. Significant protection from tumor-induced death was also observed with systemic administration of CTL peptide-MPs. The therapeutic efficacy can be attributed to enhanced delivery of the epitope to tumor tissues, presentation of the epitope by tumor cells as well as tumor stromal cells and/or generation of epitope-specific CTLs by the peptide-containing MPs. These findings offer a promising new direction for treating established solid tumor using CTL therapy.

DSP-7888, a Novel Cocktail Design of WT1 Peptide Vaccine, and Its Combinational Immunotherapy with Immune Checkpoint-Blocking Antibody Against PD-1

Background:Wilms' tumor gene 1 (WT1), broadly expressed in hematological malignancies and solid tumors, is a promising candidate for development as a cancer vaccine. WT4869, one of our WT1 vaccine candidates demonstrated early signs of clinical activity in Azacitidine (AZA)-resistant higher-risk MDS patients with a median survival (OS) of 13.0 months in a phase 1/2 study (Suzuki T, Ueda Y, Ogura M, et al. A Phase 1/2 Study of WT1 Peptide Cancer Vaccine WT4869 in Patients with Myelodysplastic Syndromes (MDS). Blood. 2015;126:2868). In this study, we report the characterization of DSP-7888 and the potential synergic effect with anti-PD-1 antibody. DSP-7888, which is a novel cocktail peptide vaccine designed to induce cytotoxic T lymphocytes (CTLs) that recognize WT1 antigen in an HLA-A*02:01, HLA-A*02:06 or HLA-A*24:02 restricted manner, has entered early clinical trials in patients with MDS and pediatric brain cancer in Japan and solid tumors in the U.S.Methods:WT1-specific T cells were measured by WT1 tetramer staining following stimulation of human peripheral blood mononuclear cells (PBMCs) with DSP-7888. In vivo CTL inducing activity of DSP-7888 was evaluated by ELISPOT assay and 51Cr-release assay after immunization of HLA-A2.1/DR1 transgenic (Tg) mice or HLA-A24.2 Tg mice. The response of induced CTLs against the WT1 epitope was measured by IFN-γ ELISA assay following co-culture with irradiated cancer cells. The frequency of PD-1 positive CTLs in spleen and tumor was analyzed by flow cytometry. The anti-tumor effect of DSP-7888 with or without anti-PD-1 antibody was evaluated using HLA-A24.2 Tg mice bearing mouse EL4 cells expressing both HLA-A24.2 and WT1.Results:HLA-A*02:01- or HLA-A*24:02-restricted and WT1-specific CTLs were induced in HLA-A2.1/DR1 Tg mice, HLA-A24.2 Tg mice and human PBMCs. Two groups of DSP-7888-induced CTLs recognized WT1 in an HLA-A*02:01-restricted manner and one in an HLA-A*24:02-restricted manner. Compared to its component vaccine without helper peptide, DSP-7888 induced a large number of WT1-specific CTLs in HLA-A2.1/DR1 Tg mice. The activity of DSP-7888-induced CTLs was relatively maintained in the environment including cancer cells. In addition, it was further activated by treatment with anti-PD-1 antibody. On the other hand, the activity of the component vaccine-induced CTLs was very low even when treated with anti-PD-1 antibody. In DSP-7888-administered tumor-bearing mice, half of WT1-specific CTLs in spleen expressed PD-1; however, in tumor more than 90% of the CTLs expressed this protein. Combination therapy of DSP-7888 and anti-PD-1 antibody showed a higher anti-tumor effect than each monotherapy alone in this model.Conclusions:In this study, DSP-7888 induced CTLs that recognize multiple WT1 epitopes. The helper peptide included in DSP-7888 enhanced the response of the CTL induction, and contributed to the lasting cytotoxic activities even in the tumor immunosuppressive environment, suggesting potential of DSP-7888 as a cancer vaccine. In addition, the in vitro treatment with anti-PD-1 antibody furthermore enhanced the activity of DSP-7888-induced CTLs. The combination of DSP-7888 and anti-PD-1 antibody may induce multiple WT1 epitope-specific CTLs and maintain the intratumoral cytotoxic activity. Further evaluations of DSP-7888 are warranted. DisclosuresGoto:Sumitomo Dainippon Pharma Co.,Ltd: Employment. Nakamura:Sumitomo Dainippon Pharma Co.,Ltd.: Employment. Suginobe:Sumitomo Dainippon Pharma Co.,Ltd.: Employment. Takasu:Sumitomo Dainippon Pharma Co.,Ltd.: Employment. Takanashi:Sumitomo Dainippon Pharma Co.,Ltd.: Employment. Ban:Sumitomo Dainippon Pharma Co.,Ltd.: Employment. Li:Sumitomo Dainippon Pharma Co.,Ltd.: Employment.

Biphasic CD8+ T-Cell Defense in Simian Immunodeficiency Virus Control by Acute-Phase Passive Neutralizing Antibody Immunization.

Identifying human immunodeficiency virus type 1 (HIV-1) control mechanisms by neutralizing antibodies (NAbs) is critical for anti-HIV-1 strategies. Recent in vivo studies on animals infected with simian immunodeficiency virus (SIV) and related viruses have shown the efficacy of postinfection NAb passive immunization for viremia reduction, and one suggested mechanism is its occurrence through modulation of cellular immune responses. Here, we describe SIV control in macaques showing biphasic CD8(+) cytotoxic T lymphocyte (CTL) responses following acute-phase NAb passive immunization. Analysis of four SIVmac239-infected rhesus macaque pairs matched with major histocompatibility complex class I haplotypes found that counterparts receiving day 7 anti-SIV polyclonal NAb infusion all suppressed viremia for up to 2 years without accumulating viral CTL escape mutations. In the first phase of primary viremia control attainment, CD8(+) cells had high capacities to suppress SIVs carrying CTL escape mutations. Conversely, in the second, sustained phase of SIV control, CTL responses converged on a pattern of immunodominant CTL preservation. During this sustained phase of viral control, SIV epitope-specific CTLs showed retention of phosphorylated extracellular signal-related kinase (ERK)(hi)/phosphorylated AMP-activated protein kinase (AMPK)(lo) subpopulations, implying their correlation with SIV control. The results suggest that virus-specific CTLs functionally boosted by acute-phase NAbs may drive robust AIDS virus control. In early HIV infection, NAb responses are lacking and CTL responses are insufficient, which leads to viral persistence. Hence, it is important to identify immune responses that can successfully control such HIV replication. Here, we show that monkeys receiving NAb passive immunization in early SIV infection strictly control viral replication for years. Passive infusion of NAbs with CTL cross-priming capacity resulted in induction of functionally boosted early CTL responses showing enhanced suppression of CTL escape mutant virus replication. Accordingly, the NAb-infused animals did not show accumulation of viral CTL escape mutations during sustained SIV control, and immunodominant CTL responses were preserved. This early functional augmentation of CTLs by NAbs provides key insights into the design of lasting and viral escape mutation-free protective immunity against HIV-1 infection.

Immunogenicity of dominant epitopes of complex antigen rhoptry protein 2-sunface protein 1 derived from Toxoplasma gondii

Objective To analyze the immunogenicity of dominant epitope of complex antigen of rhoptry protein 2 (ROP2) and major surface protein 1 (SAG1) derived from Toxoplasma gondii (T. gondii). Methods Dominant epitope of ROP2-SAG1 containing both dominant T- and B-cell epitopes was predicted and selected from T. gondii with bioinformatics methods. The gene fragment cloned into pET32a expression vector was transformed into the competent cell (Escherichia coli strain Rosetta) and expressed under the induction. The protein purified by nitrilotriacetic acid (Ni-NTA) agarose resin were finally identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Japanese rabbits were immunized subcutaneously with purified epitope protein in contrast with control group immunized with pET32a protein or phosphate buffevred saline (PBS). The sera from immunized rabbits were collected at week 0, week 2 and week 4 for determination of epitope-specific antibody IgG using enzyme-linked immunosorbent assay, and immunodot assay was used to further confirm the specificity of antibody. After BALB/c mice were immunized with purified epitope proteins, the capacity of production of interferon-γ(IFN-γ) in splenocytes was detected by enzyme-linked immunospot assay. Results Relative molecular weight 30 000 of dominant epitope was derived from prokaryotic system. Then the rabbits immunized with purified dominant epitope could produce corresponding epitope-specific antibody IgG. And with the increased frequency of immunization, the level of antibody gradually increased. At week 2 and 4, higher antibody response were observed in group of rabbit immunized with dominant epitope than those of control group (1.454±0.098 vs 0.616±0.084, F=0.000, P<0.05; 2.299±0.224 vs 1.580±0.192, F=0.112, P<0.05). The antibody titer at week 4 was as high as 1∶40 960. Immunodot assay further confirmed the antibody specificity against the dominant epitope. The level of IFN-γ in splenocytes from mice immunized with dominant epitope after stimulation with three epitope specific CTL peptides (epitope peptides 1 [19.333±1.528]/100 000 cells, epitope peptides 2 ([40.333±1.528]/100 000 cells), epitope peptides 3 ([70.667±1.890]/100 000 cells) was significantly higher than that of PBS control group (epitope peptides 1 [1.033±0.150]/100 000 cells, epitope peptides 2 [1.045±0.110]/100 000 cells, epitope peptides 3 [1.041±0.120]/100 000 cells, F=0.284, 0.000 and 0.284, respectively; all P<0.05). The level of IFN-γ from splenocytes stimulated with combined peptide with cytotoxic T lymphocyte (CTL) epitope peptide and helper T cell epitope peptide (epitope peptides 3) was significantly higher than that with single CTL epitope peptides (epitope peptides 1 and epitope peptides 2, F=5.796 and 0.000, respectively; both P<0.05). Conclusion Screened dominant epitopes of ROP2-SAG1 from T. gondii derived from prokaryotic expression system exhibit remarkable immunogenicity. Key words: Toxoplasma; ROP2-SAG1; Epitopes; Immunogenicity

Differential Recognition of Influenza A Viruses by M158-66 Epitope-Specific CD8+ T Cells Is Determined by Extraepitopic Amino Acid Residues.

Natural influenza A virus infections elicit both virus-specific antibody and CD4(+) and CD8(+) T cell responses. Influenza A virus-specific CD8(+) cytotoxic T lymphocytes (CTLs) contribute to clearance of influenza virus infections. Viral CTL epitopes can display variation, allowing influenza A viruses to evade recognition by epitope-specific CTLs. Due to functional constraints, some epitopes, like the immunodominant HLA-A*0201-restricted matrix protein 1 (M158-66) epitope, are highly conserved between influenza A viruses regardless of their subtype or host species of origin. We hypothesized that human influenza A viruses evade recognition of this epitope by impairing antigen processing and presentation by extraepitopic amino acid substitutions. Activation of specific T cells was used as an indication of antigen presentation. Here, we show that the M158-66 epitope in the M1 protein derived from human influenza A virus was poorly recognized compared to the M1 protein derived from avian influenza A virus. Furthermore, we demonstrate that naturally occurring variations at extraepitopic amino acid residues affect CD8(+) T cell recognition of the M158-66 epitope. These data indicate that human influenza A viruses can impair recognition by M158-66-specific CTLs while retaining the conserved amino acid sequence of the epitope, which may represent a yet-unknown immune evasion strategy for influenza A viruses. This difference in recognition may have implications for the viral replication kinetics in HLA-A*0201 individuals and spread of influenza A viruses in the human population. The findings may aid the rational design of universal influenza vaccines that aim at the induction of cross-reactive virus-specific CTL responses. Influenza viruses are an important cause of acute respiratory tract infections. Natural influenza A virus infections elicit both humoral and cellular immunity. CD8(+) cytotoxic T lymphocytes (CTLs) are directed predominantly against conserved internal proteins and confer cross-protection, even against influenza A viruses of various subtypes. In some CTL epitopes, mutations occur that allow influenza A viruses to evade recognition by CTLs. However, the immunodominant HLA-A*0201-restricted M158-66 epitope does not tolerate mutations without loss of viral fitness. Here, we describe naturally occurring variations in amino acid residues outside the M158-66 epitope that influence the recognition of the epitope. These results provide novel insights into the epidemiology of influenza A viruses and their pathogenicity and may aid rational design of vaccines that aim at the induction of CTL responses.

Presentation of available CTL epitopes that induction of cell-mediated immune response against HIV-1 Koran clade B strain using computational technology.

Theoretical predicting cytotoxic T lymphocyte (CTL) epitopes are an important tool in vaccine design and CTL therapy for enhancing our understanding of the cellular immune system. We would like to identify available CTL epitopes against HIV-1 Korean clade B. CTL activity was assessed in freshly isolated peripheral blood mononuclear cells from Korean HIV patients in order to assess whether these CTL epitopes induce a cell-mediated immune response (CMI). NetCTLpan1.1 software, which is the most popular prediction computer software package, and full atom-based simulation (FABS), which is a 3D modelling system for binding activity between epitopes and human leucocyte antigen (HLA) molecules, were used to predict the peptide-spanning Env region binding to HLA-A*24:02, HLA-A*02:01 and HLA-B*15:01, which are frequently found in the Korean population. Granzyme B and interferon-γ ELISPOT assays were used to determine whether identified CTL epitopes induce CMI. Three HIV-1 Korean clade B-specific Env CTL epitopes were identified: Gp41-RYL and Gp41-RQG are localized within gp41, and Gp120-LLQ within gp120. In in vitro assays using granzyme B ELISPOT, Gp120-LLQ and Gp41-RQG induced epitope-specific CTL responses in HLA-restricted cells. In ex vivo assay using IFN-γ ELISPOT, cell-mediated immune responses to Gp41-RYL were present in 50% of HLA-matched patients, and responses to Gp120-LLQ and Gp41-RQG were found in 33% of HLA-matched patients. In this study, we found that a prediction pipeline for CTL epitopes might be based on the most popular computer prediction software and FABS methods. Our results suggest that these CTL epitopes may provide useful tools and information for the development of a therapeutic vaccine against HIV-1 Korean clade B.

Naturally Selected Rilpivirine-Resistant HIV-1 Variants by Host Cellular Immunity

Rilpivirine is listed as an alternative key drug in current antiretroviral therapy (ART) guidelines. E138G/A/K in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) are rilpivirine resistance-associated mutations and can be identified in a few ART-naive patients, although at low frequency. The 138th position in HIV-1 RT is located in one of the putative epitopes of human leukocyte antigen (HLA)-B*18-restricted cytotoxic T lymphocytes (CTLs). CTL-mediated immune pressure selects escape mutations within the CTL epitope. Here we tested whether E138G/A/K could be selected by HLA-B*18-restricted CTLs. The amino acid variation at the 138th position was compared between ART-naive HIV-1-infected patients with and without HLA-B*18. The optimal epitope containing the 138th position was determined and the impact of E138G/A/K on CTL response was analyzed by epitope-specific CTLs. The effect of E138G/A/K on drug susceptibility was determined by constructing recombinant HIV-1 variants. The prevalence of E138G/A/K was 21% and 0.37% in 19 and 1088 patients with and without HLA-B*18, respectively (odds ratio, 72.3; P = 4.9 × 10(-25)). The CTL response was completely abolished by the substitution of E138G/A/K in the epitope peptide. E138G/A/K conferred 5.1-, 7.1-, and 2.7-fold resistance to rilpivirine, respectively. E138G/A/K can be selected by HLA-B*18-restricted CTLs and confer significant rilpivirine resistance. We recommend drug resistance testing before the introduction of rilpivirine-based ART in HLA-B*18-positive patients.

Polymorphisms and tissue expression of the Feline Leukocyte Antigen class I loci FLA I-E, -H and -K (P6086)

Abstract The domestic cat is a popular model of human retroviral diseases, but surprisingly, knowledge of the MHC class I complex (FLA I) in this species is limited. Only a few full-length coding sequences with uncertain locus assignments are known, and allelic variability, genotypic frequency and expression patterns remain undetermined. Of 19 class I genes, 3 loci - FLA I-E, -H and -K - are predicted to encode classical molecules. Our objective was to confirm their presumptive class Ia status by investigating polymorphisms and tissue expression of these genes. To accomplish this task, we developed a PCR-based, locus-specific, genotyping protocol, and then amplified 33 different FLA I-E, -H, and -K alleles from the gDNA of 12 cats of various breeds. We identified, for the first time, allele genotypes across all three loci in a feline species. We also amplified alleles from cDNAs from eight different tissues using an FLA I multi-loci-permissive primer set; only FLA I-E, -H, and -K transcripts were detected, and all three were widely expressed. The putative proteins encoded by our FLA I-E, -H, and -K alleles are highly variable and fit the class Ia model of homologous domains. Previously reported full-length FLA I sequences were confirmed, as were the predictions of classical status from the feline genome; FLA I-E, -H, and -K meet two major criteria of class Ia genes. These data lay the important groundwork for studying epitope-specific cytotoxic T-lymphocyte responses in the cat.

Influenza, but not HIV‐specific CTL epitopes, elicits delayed‐type hypersensitivity (DTH) reactions in HIV‐infected patients

The induction of cytotoxic T lymphocytes (CTLs) is believed to be an important defense mechanism against viral infections. The availability of simple, sensitive, specific and physiologically informative in vivo tests, applicable to humans, would greatly elucidate the nature of protective immune responses and facilitate immune monitoring in large vaccine trials. Here we studied the possibility of using defined HLA-A*02:01-restricted CTL epitopes from influenza matrix protein (GL9, GILGFVFTL) and HIV Gag p17 (SL9, SLYNTVATL) to elicit a cutaneous delayed-type hypersensitivity (DTH) reaction. Our results show that the GL9 but not the SL9 epitope was able to induce a DTH reaction. HIV infection status, HIV RNA level and CD4(+) T-cell counts were not predictive of the extent of DTH reactions. However, a markedly reduced expression of skin homing markers CD103 and cutaneous lymphocyte associated Ag (CLA) on epitope-specific CTL populations was associated with a lack of SL9 DTH reactivity. These data demonstrate that DTH reactions can be elicited by optimally defined CTL epitopes per se and point towards specific homing markers that are required for such reactions. These data may offer new insights into the immune pathogenesis of HIV infection and provide the basis of novel immune monitoring approaches for large-scale HIV vaccine trials.

Therapeutic Vaccination Expands and Improves the Function of the HIV-Specific Memory T-Cell Repertoire

The licensing of herpes zoster vaccine has demonstrated that therapeutic vaccination can help control chronic viral infection. Unfortunately, human trials of immunodeficiency virus (HIV) vaccine have shown only marginal efficacy. In this double-blind study, 17 HIV-infected individuals with viral loads of <50 copies/mL and CD4(+) T-cell counts of >350 cells/µL were randomly assigned to the vaccine or placebo arm. Vaccine recipients received 3 intramuscular injections of HIV DNA (4 mg) coding for clade B Gag, Pol, and Nef and clade A, B, and C Env, followed by a replication-deficient adenovirus type 5 boost (10(10) particle units) encoding all DNA vaccine antigens except Nef. Humoral, total T-cell, and CD8(+) cytotoxic T-lymphocyte (CTL) responses were studied before and after vaccination. Single-copy viral loads and frequencies of latently infected CD4(+) T cells were determined. Vaccination was safe and well tolerated. Significantly stronger HIV-specific T-cell responses against Gag, Pol, and Env, with increased polyfunctionality and a broadened epitope-specific CTL repertoire, were observed after vaccination. No changes in single-copy viral load or the frequency of latent infection were observed. Vaccination of individuals with existing HIV-specific immunity improved the magnitude, breadth, and polyfunctionality of HIV-specific memory T-cell responses but did not impact markers of viral control. NCT00270465.

Mathematical modeling of escape of HIV from cytotoxic T lymphocyte responses

Human immunodeficiency virus (HIV-1 or simply HIV) induces a persistent infection, which in the absence of treatment leads to AIDS and death in almost all infected individuals. HIV infection elicits a vigorous immune response starting about 2–3 weeks postinfection that can lower the amount of virus in the body, but which cannot eradicate the virus. How HIV establishes a chronic infection in the face of a strong immune response remains poorly understood. It has been shown that HIV is able to rapidly change its proteins via mutation to evade recognition by virus-specific cytotoxic T lymphocytes (CTLs). Typically, an HIV-infected patient will generate 4–12 CTL responses specific for parts of viral proteins called epitopes. Such CTL responses lead to strong selective pressure to change the viral sequences encoding these epitopes so as to avoid CTL recognition. Indeed, the viral population ‘escapes’ from about half of the CTL responses by mutation in the first year. Here we review experimental data on HIV evolution in response to CTL pressure, mathematical models developed to explain this evolution, and highlight problems associated with the data and previous modeling efforts. We show that estimates of the strength of the epitope-specific CTL response depend on the method used to fit models to experimental data and on the assumptions made regarding how mutants are generated during infection. We illustrate that allowing CTL responses to decay over time may improve the model fit to experimental data and provides higher estimates of the killing efficacy of HIV-specific CTLs. We also propose a novel method for simultaneously estimating the killing efficacy of multiple CTL populations specific for different epitopes of HIV using stochastic simulations. Lastly, we show that current estimates of the efficacy at which HIV-specific CTLs clear virus-infected cells can be improved by more frequent sampling of viral sequences and by combining data on sequence evolution with experimentally measured CTL dynamics.

Evolution of viral life-cycle in response to cytotoxic T lymphocyte-mediated immunity

Viruses in mammals are constantly faced with the problem of elimination by the host immunity. Cytotoxic T lymphocyte (CTL) responses are thought to play a major role in the control and clearance of several viral infections in mice and humans. It is therefore expected that over evolutionary time, viruses would be forced to evolve to avoid recognition by CTLs. Indeed, a number of studies have documented the accumulation of viral variants with escape mutations. These mutations allow viruses to hide from CTL responses common in the host population. CTLs recognize viruses by short protein sequences, named epitopes, derived from viral proteins. The efficiency of viral recognition by epitope-specific CTL responses depends on the expression pattern of the proteins carrying these epitopes, and the total amount of that protein (and thus epitopes) in the cell. When a virus replicates in a cell, some viral genes are expressed early in the life cycle of the virus, while other proteins are expressed late. For example, HIV infected cells first express Rev and Tat proteins, and the Gag proteins are expressed late. Here we propose a dynamical model of the viral life cycle to study how expression level of early vs. late genes may affect viral dynamics within the host and virus transmission over the course of infection. We find that for acute and chronic viral infections lower expression of early genes than that of the late genes is expected to give selective advantage and higher transmission to viruses.

DCs Pulsed with Novel HLA-A2-Restricted CTL Epitopes against Hepatitis C Virus Induced a Broadly Reactive Anti-HCV-Specific T Lymphocyte Response

ObjectiveTo determine the capacity of dendritic cells (DCs) loaded with single or multiple-peptide mixtures of novel hepatitis C virus (HCV) epitopes to stimulate HCV-specific cytotoxic T lymphocyte (CTL) effector functions.MethodsA bioinformatics approach was used to predict HLA-A2-restricted HCV-specific CTL epitopes, and the predicted peptides identified from this screen were synthesized. Subsequent IFN-γ ELISPOT analysis detected the stimulating function of these peptides in peripheral blood mononuclear cells (PBMCs) from both chronic and self-limited HCV infected subjects (subjects exhibiting spontaneous HCV clearance). Mature DCs, derived in vitro from CD14+ monocytes harvested from the study subjects by incubation with appropriate cytokine cocktails, were loaded with novel peptide or epitope peptide mixtures and co-cultured with autologous T lymphocytes. Granzyme B (GrB) and IFN-γ ELISPOT analysis was used to test for epitope-specific CTL responses. T-cell-derived cytokines contained in the co-cultured supernatant were detected by flow cytometry.ResultsWe identified 7 novel HLA-A2-restricted HCV-specific CTL epitopes that increased the frequency of IFN-γ-producing T cells compared to other epitopes, as assayed by measuring spot forming cells (SFCs). Two epitopes had the strongest stimulating capability in the self-limited subjects, one found in the E2 and one in the NS2 region of HCV; five epitopes had a strong stimulating capacity in both chronic and self-limited HCV infection, but were stronger in the self-limited subjects. They were distributed in E2, NS2, NS3, NS4, and NS5 regions of HCV, respectively. We also found that mDCs loaded with novel peptide mixtures could significantly increase GrB and IFN-γ SFCs as compared to single peptides, especially in chronic HCV infection subjects. Additionally, we found that DCs pulsed with multiple epitope peptide mixtures induced a Th1-biased immune response.ConclusionsSeven novel and strongly stimulating HLA-A2-restricted HCV-specific CTL epitopes were identified. Furthermore, DCs loaded with multiple-epitope peptide mixtures induced epitope-specific CTLs responses.

Selection and accumulation of an HIV-1 escape mutant by three types of HIV-1-specific cytotoxic T lymphocytes recognizing wild-type and/or escape mutant epitopes.

It is known that cytotoxic T lymphocytes (CTLs) recognizing HIV-1 escape mutants are elicited in HIV-1-infected individuals, but their role in the control of HIV-1 replication remains unclear. We investigated the antiviral ability of CTLs recognizing the HLA-A*24:02-restricted Gag28 -36 (KYKLKHIVW) epitope and/or its escape mutant (KYRLKHIVW) elicited in the early and chronic phases of the infection. Wild-type (WT)-epitope-specific CTLs, as well as cross-reactive CTLs recognizing both WT and K30R (3R) epitopes, which were predominantly elicited at early and/or chronic phases in HLA-A*24:02(+) individuals infected with the WT virus, suppressed the replication of the WT virus but failed to suppress that of the 3R virus, indicating that the 3R virus was selected by these 2 types of CTLs. On the other hand, cross-reactive and 3R-specific CTLs, which were elicited in those infected with the 3R virus, did not suppress the replication of either WT or 3R virus, indicating that these CTLs did not contribute to the control of 3R virus replication. High accumulation of the 3R mutation was found in a Japanese population recently recruited. The selection and accumulation of this 3R mutation resulted from the antiviral ability of these Gag28-specific CTLs and high prevalence of HLA-A*24:02 in a Japanese population. The present study highlighted the mechanisms for the roles of cross-reactive and mutant-epitope-specific CTLs, as well as high accumulation of escape mutants, in an HIV-1-infected population.

TCL1: A Shared Tumor-Associated Antigen for Immunotherapy Against B-Cell Lymphomas

Abstract 105Rituximab-based chemotherapy regimens have improved complete response rates, progression-free survival, and overall survival of B-cell non-Hodgkin lymphoma patients. However, most patients relapse and die of their lymphoma. To further improve clinical outcome, novel strategies that eradicate minimal residual disease (MRD) after induction therapy are needed. Therapeutic vaccines may induce antitumor antibody and T-cell responses and may eradicate MRD by complementary mechanisms. A recent randomized phase III clinical trial showed that patient-specific idiotype vaccination improves disease-free survival when administered in the setting of MRD in follicular lymphoma (FL), providing proof of principle that therapeutic vaccines can improve clinical outcome (Schuster et al, J Clin Oncol, 2011). However, generation of a custom-made vaccine formulation for each patient can be expensive and time consuming. To overcome these difficulties, identification of novel shared lymphoma-associated antigens is necessary.The T-cell leukemia/lymphoma 1 (TCL1) oncoprotein encoded by the TCL1 gene is a co-activator of Akt and promotes cell proliferation and survival. Ectopic expression of TCL1 in B cells in transgenic mice results in the development of B-cell malignancies and TCL1 was reported to be aberrantly expressed in multiple human B cell malignancies. Here, we determined whether TCL1 can serve as a novel shared tumor-associated antigen in B cell lymphomas. We analyzed the expression pattern of TCL1 in human B cell lymphomas and normal tissues by real-time PCR, flow cytometry, immunohistochemistry, and Western blotting. TCL1 mRNA transcripts were hyperexpressed in multiple types of primary B cell lymphomas [chronic lymphocytic leukemia (CLL), n=7; mantle cell lymphoma (MCL), n=6; FL, n=12, diffuse large B-cell lymphoma (DLBCL), n=5] as compared with B cells derived from normal donors. TCL1 mRNA was not detected in other normal tissues that included T cells, adipose tissue, esophagus, ovary, spleen, bladder, heart, placenta, brain, kidney, prostate, liver, muscle, thyroid, colon, lung, intestine, trachea, thymus, and cervix. Very low TCL1 mRNA was detected in testis. By flow cytometry, immunohistochemistry, and/or Western blotting we observed that the TCL1 protein is hyperexpressed in CLL, MCL, DLBCL, Burkitts lymphoma, and FL, but not splenic marginal zone lymphoma. To determine whether TCL1 is immunogenic, we synthesized overlapping 15-mer peptides spanning the entire length of the TCL1 protein and stimulated PBMC from HLA-A*0201+ (HLA-A2+) normal donors to generate peptide-specific T cells. We found that TCL165–79 peptide (TQIGPSLLPIMWQLY) consistently induced T cells that secreted significant amount of IFN-γ from 3 normal donors. By intracellular cytokine assay, we determined that the TCL165–79 peptide-specific IFN-g was produced by CD8+ T cells but not CD4+ T cells. Using a panel of peptide-pulsed EBV-transformed B-cell lines that were mismatched at the MHC Class I locus and MHC Class I blocking antibodies, we determined that HLA-A2 is the restriction element of the TCL165–79 peptide-specific T cells. Consistent with this, TCL165–79 peptide-specific CD8+ T cells lysed TCL1 expressing HLA-A2+ MCL cell lines but did not lyse, TCL1 expressing HLA-A2− myeloma cell line. Using a panel of peptides truncated progressively by one amino acid at the N- and C-termini of the TCL165–79peptide, we determined that TCL171–78(LLPIMWQL) is the minimal epitope that bound to HLA-A2. Furthermore, cytotoxic T lymphocytes (CTL) specific to this peptide efficiently killed lymphoma cell lines and primary lymphoma cells from CLL, MCL, DLBCL, and FL in an HLA-A2-restricted manner. Using TCL171–78 peptide-specific HLA-A2 tetramers, we found that TCL1 epitope-specific CTLs were present in the peripheral blood and/or tumor-infiltrating lymphocytes of B-cell lymphoma patients (n=8). More importantly, these TCL1 epitope-specific CTLs could be expanded in vitro and lysed autologous and allogeneic tumor cells in an HLA-A2-restricted manner. In conclusion, our results suggest that TCL1 is naturally processed and presented on the surface of primary lymphoma cells for recognition by CTL and can serve as a novel, shared tumor-associated antigen for therapeutic vaccine development against common B-cell lymphomas including CLL, MCL, DLBCL, and FL. Disclosures:Neelapu:Biovest International, Inc.: Research Funding.