A Candidate HIV/AIDS Vaccine (MVA-B) Lacking Vaccinia Virus Gene C6L Enhances Memory HIV-1-Specific T-Cell Responses.

The vaccinia virus (VACV) C6 protein has sequence similarities with the poxvirus family Pox_A46, involved in regulation of host immune responses, but its role is unknown. Here, we have characterized the C6 protein and its effects in virus replication, innate immune sensing and immunogenicity in vivo. C6 is a 18.2 kDa protein, which is expressed early during virus infection and localizes to the cytoplasm of infected cells. Deletion of the C6L gene from the poxvirus vector MVA-B expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (MVA-B ΔC6L) had no effect on virus growth kinetics; therefore C6 protein is not essential for virus replication. The innate immune signals elicited by MVA-B ΔC6L in human macrophages and monocyte-derived dendritic cells (moDCs) are characterized by the up-regulation of the expression of IFN-β and IFN-α/β-inducible genes. In a DNA prime/MVA boost immunization protocol in mice, flow cytometry analysis revealed that MVA-B ΔC6L enhanced the magnitude and polyfunctionality of the HIV-1-specific CD4(+) and CD8(+) T-cell memory immune responses, with most of the HIV-1 responses mediated by the CD8(+) T-cell compartment with an effector phenotype. Significantly, while MVA-B induced preferentially Env- and Gag-specific CD8(+) T-cell responses, MVA-B ΔC6L induced more Gag-Pol-Nef-specific CD8(+) T-cell responses. Furthermore, MVA-B ΔC6L enhanced the levels of antibodies against Env in comparison with MVA-B. These findings revealed that C6 can be considered as an immunomodulator and that deleting C6L gene in MVA-B confers an immunological benefit by enhancing IFN-β-dependent responses and increasing the magnitude and quality of the T-cell memory immune responses to HIV-1 antigens. Our observations are relevant for the improvement of MVA vectors as HIV-1 vaccines.

RTS,S/AS02A malaria vaccine does not induce parasite CSP T cell epitope selection and reduces multiplicity of infection

Objective: The candidate malaria vaccine RTS,S/AS02A is a recombinant protein containing part of the circumsporozoite protein (CSP) sequence of Plasmodium falciparum, linked to the hepatitis B surface antigen and formulated in the proprietary adjuvant system AS02A. In a recent trial conducted in children younger than age five in southern Mozambique, the vaccinedemonstrated significant and sustained efficacy against both infection and clinical disease. In a follow-up study to the main trial, breakthrough infections identified in the trial were examined to determine whether the distribution of csp sequences was affected by the vaccine and to measure the multiplicity of infecting parasite genotypes. Design: P. falciparum DNA from isolates collected during the trial was used for genotype studies. Setting: The main trial was carried out in the Manhiça district, Maputo province, Mozambique, between April 2003 and May 2004. Participants: Children from the two cohorts of the main trial provided parasite isolates as follows: children from Cohort 1 who were admitted to hospital with clinical malaria; children from Cohort 1 who were parasite-positive in a cross-sectional survey at study month 8.5; children from Cohort 2 identified as parasite-positive during follow-up by active detection of infection. Outcome: Divergence of DNA sequence encoding the CSP T cell-epitope region sequence from that of the vaccine sequence was measured in 521 isolates. The number of distinct P. falciparum genotypes was also determined. Results: We found no evidence that parasite genotypes from children in the RTS,S/AS02A arm were more divergent than those receiving control vaccines. For Cohort 1 (survey at studymonth 8.5) and Cohort 2, infections in the vaccine group contained significantly fewer genotypes than those in the control group, (p 1/4 0.035, p 1/4 0.006), respectively, for the two cohorts. This was not the case for children in Cohort 1 who were admitted to hospital (p 1/4 0.478). Conclusions: RTS,S/AS02A did not select for genotypes encoding divergent T cell epitopes in the C-terminal region of CSP in this trial. In both cohorts, there was a modest reduction in the mean number of parasite genotypes harboured by vaccinated children compared with controls, but only among those with asymptomatic infections.

Immunothérapie: une solution dans les carcinomes épidermoïdes de la sphère ORL [Immunotherapy: a new alternative treatment for head and neck squamous cell carcinoma]

In the past decades, prognosis of head and neck squamous cell carcinoma (HNSCC) has not improved even though treatment has made substantial progress. Since the description of immune response against some cancers, antitumoral immunotherapy has been studied to be used as adjunctive treatment in various cancer types. This article review contributions made in the field of immunotherapy on HNSCC in the past few years. It appears that this approach may play an important role in the treatment of head and neck squamous cell carcinoma. Among various TAAs, cancer testis antigens family may be promising candidates for specific immune therapy in HNSCC. Ongoing studies will confirm whether expression CTAs generate an immune response in clinical vaccine trials.

Comparison of human and rhesus macaque T-cell responses elicited by boosting with NYVAC encoding human immunodeficiency virus type 1 clade C immunogens.

Rhesus macaques (Macaca mulatta) have played a valuable role in the development of human immunodeficiency virus (HIV) vaccine candidates prior to human clinical trials. However, changes and/or improvements in immunogen quality in the good manufacturing practice (GMP) process or changes in adjuvants, schedule, route, dose, or readouts have compromised the direct comparison of T-cell responses between species. Here we report a comparative study in which T-cell responses from humans and macaques to HIV type 1 antigens (Gag, Pol, Nef, and Env) were induced by the same vaccine batches prepared under GMP and administered according to the same schedules in the absence and presence of priming. Priming with DNA (humans and macaques) or alphavirus (macaques) and boosting with NYVAC induced robust and broad antigen-specific responses, with highly similar Env-specific gamma interferon (IFN-gamma) enzyme-linked immunospot assay responses in rhesus monkeys and human volunteers. Persistent cytokine responses of antigen-specific CD4(+) and CD8(+) T cells of the central memory as well as the effector memory phenotype, capable of simultaneously eliciting multiple cytokines (IFN-gamma, interleukin 2, and tumor necrosis factor alpha), were induced. Responses were highly similar in humans and primates, confirming earlier data indicating that priming is essential for inducing robust NYVAC-boosted IFN-gamma T-cell responses. While significant similarities were observed in Env-specific responses in both species, differences were also observed with respect to responses to other HIV antigens. Future studies with other vaccines using identical lots, immunization schedules, and readouts will establish a broader data set of species similarities and differences with which increased confidence in predicting human responses may be achieved.

Ionizing radiation enhances immunogenicity of cells expressing a tumor-specific T-cell epitope.

BACKGROUND: p53 point mutations represent potential tumor-specific cytolytic T lymphocyte (CTL) epitopes. Whether ionizing radiation (IR) alters the immunological properties of cells expressing mutant p53 in respect of the CTL epitope generated by a defined point mutation has not been evaluated. METHODS: Mutant p53-expressing syngeneic, nontumor forming BALB/c 3T3 fibroblasts, tumor forming ras-transfected BALB/c 3T3 sarcomas, and DBA/2-derived P815 mastocytoma cells, which differ at the level of minor histocompatibility antigens, were used as cellular vaccines. Cells were either injected with or without prior IR into naive BALB/c mice. Cellular cytotoxicity was assessed after secondary restimulation of effector spleen cells in vitro. RESULTS: Injection of P815 mastocytoma cells expressing the mutant p53 induced mutation-specific CTL in BALB/c mice irrespective of prior irradiation. However, syngeneic fibroblasts or fibrosarcomas endogenously expressing mutant p53 were able to induce significant mutation-specific CTL only when irradiated prior to injection into BALB/c mice. IR of fibroblasts did not detectably alter the expression of cell surface molecules involved in immune response induction, nor did it alter the short-term in vitro viability of the fibroblasts. Interestingly, radioactively-labeled fibroblasts injected into mice after irradiation showed altered organ distribution, suggesting that the in vivo fate of these cells may play a crucial role in their immunogenicity. CONCLUSIONS: These findings indicate that IR can alter the immunogenicity of syngeneic normal as well as tumor forming fibroblasts in vivo, and support the view that ionizing radiation enhances immunogenicity of cellular tumor vaccines.

Immunization with HIV Gag targeted to dendritic cells followed by recombinant New York vaccinia virus induces robust T-cell immunity in nonhuman primates.

Protein vaccines, if rendered immunogenic, would facilitate vaccine development against HIV and other pathogens. We compared in nonhuman primates (NHPs) immune responses to HIV Gag p24 within 3G9 antibody to DEC205 ("DEC-HIV Gag p24"), an uptake receptor on dendritic cells, to nontargeted protein, with or without poly ICLC, a synthetic double stranded RNA, as adjuvant. Priming s.c. with 60 μg of both HIV Gag p24 vaccines elicited potent CD4(+) T cells secreting IL-2, IFN-γ, and TNF-α, which also proliferated. The responses increased with each of three immunizations and recognized multiple Gag peptides. DEC-HIV Gag p24 showed better cross-priming for CD8(+) T cells, whereas the avidity of anti-Gag antibodies was ∼10-fold higher with nontargeted Gag 24 protein. For both protein vaccines, poly ICLC was essential for T- and B-cell immunity. To determine whether adaptive responses could be further enhanced, animals were boosted with New York vaccinia virus (NYVAC)-HIV Gag/Pol/Nef. Gag-specific CD4(+) and CD8(+) T-cell responses increased markedly after priming with both protein vaccines and poly ICLC. These data reveal qualitative differences in antibody and T-cell responses to DEC-HIV Gag p24 and Gag p24 protein and show that prime boost with protein and adjuvant followed by NYVAC elicits potent cellular immunity.

PD-1 and Tim-3 regulate the expansion of tumor antigen-specific CD8⁺ T cells induced by melanoma vaccines.

Although melanoma vaccines stimulate tumor antigen-specific CD8(+) T cells, objective clinical responses are rarely observed. To investigate this discrepancy, we evaluated the character of vaccine-induced CD8(+) T cells with regard to the inhibitory T-cell coreceptors PD-1 and Tim-3 in patients with metastatic melanoma who were administered tumor vaccines. The vaccines included incomplete Freund's adjuvant, CpG oligodeoxynucleotide (CpG), and the HLA-A2-restricted analog peptide NY-ESO-1 157-165V, either by itself or in combination with the pan-DR epitope NY-ESO-1 119-143. Both vaccines stimulated rapid tumor antigen-specific CD8(+) T-cell responses detected ex vivo, however, tumor antigen-specific CD8(+) T cells produced more IFN-γ and exhibited higher lytic function upon immunization with MHC class I and class II epitopes. Notably, the vast majority of vaccine-induced CD8(+) T cells upregulated PD-1 and a minority also upregulated Tim-3. Levels of PD-1 and Tim-3 expression by vaccine-induced CD8(+) T cells at the time of vaccine administration correlated inversely with their expansion in vivo. Dual blockade of PD-1 and Tim-3 enhanced the expansion and cytokine production of vaccine-induced CD8(+) T cells in vitro. Collectively, our findings support the use of PD-1 and Tim-3 blockades with cancer vaccines to stimulate potent antitumor T-cell responses and increase the likelihood of clinical responses in patients with advanced melanoma.

Induction of human papillomavirus oncogene-specific CD8 T-cell effector responses in the genital mucosa of vaccinated mice.

Cervical cancer, the second leading cause of cancer mortality in women worldwide, results from infection with a subset of human papillomaviruses (HPV), HPV-16 being the most prevalent type. The available prophylactic vaccines are an effective strategy to prevent this cancer in the long term. However, they only target 70-80% of all cervical cancers and cannot control existing HPV infections and associated lesions. Therapeutic vaccines are thus necessary for women who cannot benefit from prophylactic vaccination. Induction of protective immune responses in the genital mucosa (GM) may be crucial for efficacy of HPV therapeutic vaccines. We report here that mice that received a single subcutaneous (s.c.) vaccination of an adjuvanted long synthetic HPV16 E7(1-98) polypeptide showed induction of 100% tumor protection against s.c. TC-1 tumors and that tumor regression was mainly provided by CD8 T cells. In vivo cytotoxic assay revealed high E7-specific cytolytic T lymphocytes activity in spleen and in genital draining lymph nodes (LN), and E7-specific CD8 T cells could be detected in GM by tetramer staining. More importantly, high-avidity E7-specific INF-gamma secreting CD8 T cells were induced not only in blood, spleen and LN but also in GM of vaccinated mice, thus providing evidence that a parenteral vaccination may be sufficient to provide regression of genital tumors. In addition, there was no correlation between the responses measured in blood with those measured in GM, highlighting the necessity and relevance to determine the immune responses in the mucosa where HPV-tumors reside.

Current state of vaccine therapies in non-small-cell lung cancer.

A large variety of cancer vaccines have undergone extensive testing in early-phase clinical trials. A limited number have also been tested in randomized phase II clinical trials. Encouraging trends toward increased survival in the vaccine arms have been recently observed for 2 vaccine candidates in patients with non-small-cell lung cancer. These have provided the impetus for the initiation of phase III trials in large groups of patients with lung cancer. These vaccines target 2 antigens widely expressed in lung carcinomas: melanoma-associated antigen 3, a cancer testis antigen; and mucin 1, an antigen overexpressed in a largely deglycosylated form in advanced tumors. Therapeutic cancer vaccines aim at inducing strong CD8 and CD4 T-cell responses. The majority of vaccines recently tested in phase I clinical trials show efficacy in terms of induction of specific tumor antigen immunity. However, clinical efficacy remains to be determined but appears limited. Efforts are thus aimed at understanding the basis for this apparent lack of effect on tumors. Two major factors are involved. On one hand, current vaccines are suboptimal. Strong adjuvant agents and appropriate tumor antigens are needed. Moreover, dose, route, and schedule also need optimization. On the other hand, it is now clear that large tumors often present a tolerogenic microenvironment that hampers effective antitumor immunity. The partial understanding of the molecular pathways leading to functional inactivation of T cells at tumor sites has provided new targets for intervention. In this regard, blockade of cytotoxic T-lymphocyte antigen-4 and programmed death-1 with humanized monoclonal antibodies has reached the clinical testing stage. In the future, more potent cancer vaccines will benefit from intense research in antigen discovery and adjuvant agents. Furthermore, it is likely that vaccines need to be combined with compounds that reverse major tolerogenic pathways that are constitutively active at the tumor site. Developing these combined approaches to vaccination in cancer promises new, exciting findings and, at the same time, poses important challenges to academic research institutions and the pharmaceutical industry.

Single cell analysis reveals similar functional competence of dominant and non-dominant CD8 T-cell clonotypes

The properties of CD8 T-cells requiredfor protection from infectiousdisease and cancer are only partiallycharacterized, and only limited data isavailable regarding T-cell clonotypes.It has been proposed that dominantT-cell clonotypes may have higherprotective potential than their nondominantcounterparts. Our objectiveswere to assess memory andeffector functions, stage of differentiationand clonotype selection of tumor-reactive T lymphocytes followingpeptide vaccination in melanomapatients.We also characterized dominantversus non-dominant clonotypesto further understand the in vivo functionof these T-cells based on theirprevalence. Using a novel single-cellapproach for simultaneous ex vivomolecular and functional analysis, wereport the preferential selection andexpansion of several tumor-specificco-dominant clonotypes of intermediateto high frequencies, irrespectiveof whether native or analog peptidewas used for vaccination. Theseclonotypes made up 40 - 95% of thedifferentiated "effector-like" T-cells,but only 25% of the less-differentiated"effector-memory" cells. Bothsubsets also contained non-dominantT-cell clonotypes, but these were significantlymore frequent in the lessdifferentiatedcells. Thus, cell differentiationwas clonotype-dependent.Surprisingly however, the acquisitionof memory and effector T-cell propertieswas clonotype independent, as wefound similar functional profiles indominant and low/ non-dominantT-cell clonotypes. In contrast to analogpeptide vaccination, native peptidevaccination induced T-cell functionsthat were more comprehensive,with more pronounced effector functionscombined with memory cellproperties. In summary, this study revealsthat T-cell functions are determinedprimarily by the antigen andthe stage of T-cell differentiation, butare similar in dominant and non-dominantclonotypes participating in aCD8 T-cell response. The identifiedclonotypic basis of T-cell responsescontributes to the rational developmentof vaccines.

Critical role for the chemokine receptor CXCR6 in NK cell-mediated antigen-specific memory of haptens and viruses.

Hepatic natural killer (NK) cells mediate antigen-specific contact hypersensitivity (CHS) in mice deficient in T cells and B cells. We report here that hepatic NK cells, but not splenic or naive NK cells, also developed specific memory of vaccines containing antigens from influenza, vesicular stomatitis virus (VSV) or human immunodeficiency virus type 1 (HIV-1). Adoptive transfer of virus-sensitized NK cells into naive recipient mice enhanced the survival of the mice after lethal challenge with the sensitizing virus but not after lethal challenge with a different virus. NK cell memory of haptens and viruses depended on CXCR6, a chemokine receptor on hepatic NK cells that was required for the persistence of memory NK cells but not for antigen recognition. Thus, hepatic NK cells can develop adaptive immunity to structurally diverse antigens, an activity that requires NK cell-expressed CXCR6.

Low TCR avidity and lack of tumor cell recognition in CD8(+) T cells primed with the CEA-analogue CAP1-6D peptide.

The use of "altered peptide ligands" (APL), epitopes designed for exerting increased immunogenicity as compared with native determinants, represents nowadays one of the most utilized strategies for overcoming immune tolerance to self-antigens and boosting anti-tumor T cell-mediated immune responses. However, the actual ability of APL-primed T cells to cross-recognize natural epitopes expressed by tumor cells remains a crucial concern. In the present study, we show that CAP1-6D, a superagonist analogue of a carcinoembriyonic antigen (CEA)-derived HLA-A*0201-restricted epitope widely used in clinical setting, reproducibly promotes the generation of low-affinity CD8(+) T cells lacking the ability to recognized CEA-expressing colorectal carcinoma (CRC) cells. Short-term T cell cultures, obtained by priming peripheral blood mononuclear cells from HLA-A*0201(+) healthy donors or CRC patients with CAP1-6D, were indeed found to heterogeneously cross-react with saturating concentrations of the native peptide CAP1, but to fail constantly lysing or recognizing through IFN- gamma release CEA(+)CRC cells. Characterization of anti-CAP1-6D T cell avidity, gained through peptide titration, CD8-dependency assay, and staining with mutated tetramers (D227K/T228A), revealed that anti-CAP1-6D T cells exerted a differential interaction with the two CEA epitopes, i.e., displaying high affinity/CD8-independency toward the APL and low affinity/CD8-dependency toward the native CAP1 peptide. Our data demonstrate that the efficient detection of self-antigen expressed by tumors could be a feature of high avidity CD8-independent T cells, and underline the need for extensive analysis of tumor cross-recognition prior to any clinical usage of APL as anti-cancer vaccines.

Ex vivo detectable human CD8 T-cell responses to cancer-testis antigens.

Clinical trials have shown that strong tumor antigen-specific CD8 T-cell responses are difficult to induce but can be achieved for T-cells specific for melanoma differentiation antigens, upon repetitive vaccination with stable emulsions prepared with synthetic peptides and incomplete Freund's adjuvant. Here, we show in four melanoma patients that ex vivo detectable T-cells and thus strong T-cell responses can also be induced against the more universal cancer-testis antigens NY-ESO-1 and Mage-A10. Interestingly, all patients had ex vivo detectable T-cell responses against multiple antigens after serial vaccinations with three peptides emulsified in incomplete Freund's adjuvant. Antigen-specific T-cells displayed an activated phenotype and secreted IFNgamma. The robust immune responses provide a solid basis for further development of human T-cell vaccination.

A novel approach to characterize clonality and differentiation of human melanoma-specific T cell responses: spontaneous priming and efficient boosting by vaccination.

Despite major progress in T lymphocyte analysis in melanoma patients, TCR repertoire selection and kinetics in response to tumor Ags remain largely unexplored. In this study, using a novel ex vivo molecular-based approach at the single-cell level, we identified a single, naturally primed T cell clone that dominated the human CD8(+) T cell response to the Melan-A/MART-1 Ag. The dominant clone expressed a high-avidity TCR to cognate tumor Ag, efficiently killed tumor cells, and prevailed in the differentiated effector-memory T lymphocyte compartment. TCR sequencing also revealed that this particular clone arose at least 1 year before vaccination, displayed long-term persistence, and efficient homing to metastases. Remarkably, during concomitant vaccination over 3.5 years, the frequency of the pre-existing clone progressively increased, reaching up to 2.5% of the circulating CD8 pool while its effector functions were enhanced. In parallel, the disease stabilized, but subsequently progressed with loss of Melan-A expression by melanoma cells. Collectively, combined ex vivo analysis of T cell differentiation and clonality revealed for the first time a strong expansion of a tumor Ag-specific human T cell clone, comparable to protective virus-specific T cells. The observed successful boosting by peptide vaccination support further development of immunotherapy by including strategies to overcome immune escape.