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Ansar Pathan - Top 30 Publications

Association of Marek's Disease induced immunosuppression with activation of a novel regulatory T cells in chickens.

Marek's Disease Virus (MDV) is an alphaherpesvirus that infects chickens, transforms CD4+ T cells and causes deadly lymphomas. In addition, MDV induces immunosuppression early during infection by inducing cell death of the infected lymphocytes, and potentially due to activation of regulatory T (Treg)-cells. Furthermore, immunosuppression also occurs during the transformation phase of the disease; however, it is still unknown how the disease can suppress immune response prior or after lymphoma formation. Here, we demonstrated that chicken TGF-beta+ Treg cells are found in different lymphoid tissues, with the highest levels found in the gut-associated lymphoid tissue (cecal tonsil: CT), fostering an immune-privileged microenvironment exerted by TGF-beta. Surprisingly, significantly higher frequencies of TGF-beta+ Treg cells are found in the spleens of MDV-susceptible chicken lines compared to the resistant line, suggesting an association between TGF-beta+ Treg cells and host susceptibility to lymphoma formation. Experimental infection with a virulent MDV elevated the levels of TGF-beta+ Treg cells in the lungs as early as 4 days post infection, and during the transformation phase of the disease in the spleens. In contrast to TGF-beta+ Treg cells, the levels of CD4+CD25+ T cells remained unchanged during the infection and transformation phase of the disease. Furthermore, our results demonstrate that the induction of TGF-beta+ Treg cells is associated with pathogenesis of the disease, as the vaccine strain of MDV did not induce TGF-beta+ Treg cells. Similar to human haematopoietic malignant cells, MDV-induced lymphoma cells expressed high levels of TGF-beta but very low levels of TGF-beta receptor I and II genes. The results confirm that COX-2/ PGE2 pathway is involved in immunosuppression induced by MDV-lymphoma cells. Taken together, our results revealed a novel TGF-beta+ Treg subset in chickens that is activated during MDV infection and tumour formation.

A Recombinant Fragment of Human Surfactant Protein D Suppresses Basophil Activation and T-Helper Type 2 and B-Cell Responses in Grass Pollen-induced Allergic Inflammation.

Recombinant fragment of human surfactant protein D (rfhSP-D) has been shown to suppress house dust mite- and Aspergillus fumigatus-induced allergic inflammation in murine models.

Using Data from Macaques To Predict Gamma Interferon Responses after Mycobacterium bovis BCG Vaccination in Humans: a Proof-of-Concept Study of Immunostimulation/Immunodynamic Modeling Methods.

Macaques play a central role in the development of human tuberculosis (TB) vaccines. Immune and challenge responses differ across macaque and human subpopulations. We used novel immunostimulation/immunodynamic modeling methods in a proof-of-concept study to determine which macaque subpopulations best predicted immune responses in different human subpopulations. Data on gamma interferon (IFN-γ)-secreting CD4+ T cells over time after recent Mycobacterium bovis BCG vaccination were available for 55 humans and 81 macaques. Human population covariates were baseline BCG vaccination status, time since BCG vaccination, gender, and the monocyte/lymphocyte cell count ratio. The macaque population covariate was the colony of origin. A two-compartment mathematical model describing the dynamics of the IFN-γ T cell response after BCG vaccination was calibrated to these data using nonlinear mixed-effects methods. The model was calibrated to macaque and human data separately. The association between subpopulations and the BCG immune response in each species was assessed. The macaque subpopulations that best predicted immune responses in different human subpopulations were identified using Bayesian information criteria. We found that the macaque colony and the human baseline BCG status were significantly (P < 0.05) associated with the BCG-induced immune response. For humans who were BCG naïve at baseline, Indonesian cynomolgus macaques and Indian rhesus macaques best predicted the immune response. For humans who had already been BCG vaccinated at baseline, Mauritian cynomolgus macaques best predicted the immune response. This work suggests that the immune responses of different human populations may be best modeled by different macaque colonies, and it demonstrates the potential utility of immunostimulation/immunodynamic modeling to accelerate TB vaccine development.

Human C1q Induces Apoptosis in an Ovarian Cancer Cell Line via Tumor Necrosis Factor Pathway.

Complement protein C1q is the first recognition subcomponent of the complement classical pathway that plays a vital role in the clearance of immune complexes, pathogens, and apoptotic cells. C1q also has a homeostatic role involving immune and non-immune cells; these functions not necessarily involve complement activation. Recently, C1q has been shown to be expressed locally in the microenvironment of a range of human malignant tumors, where it can promote cancer cell adhesion, migration, and proliferation, without involving complement activation. C1q has been shown to be present in the ascitic fluid formed during ovarian cancers. In this study, we have examined the effects of human C1q and its globular domain on an ovarian cancer cell line, SKOV3. We show that C1q and the recombinant globular head modules induce apoptosis in SKOV3 cells in a time-dependent manner. C1q expression was not detectable in the SKOV3 cells. Exogenous treatment with C1q and globular head modules at the concentration of 10 µg/ml induced apoptosis in approximately 55% cells, as revealed by immunofluorescence microscopy and FACS. The qPCR and caspase analysis suggested that C1q and globular head modules activated tumor necrosis factor (TNF)-α and upregulated Fas. The genes of mammalian target of rapamycin (mTOR), RICTOR, and RAPTOR survival pathways, which are often overexpressed in majority of the cancers, were significantly downregulated within few hours of the treatment of SKOV3 cells with C1q and globular head modules. In conclusion, C1q, via its globular domain, induced apoptosis in an ovarian cancer cell line SKOV3 via TNF-α induced apoptosis pathway involving upregulation of Bax and Fas. This study highlights a potentially protective role of C1q in certain cancers.

Analysis of the Interaction between Globular Head Modules of Human C1q and Its Candidate Receptor gC1qR.

The heterotrimeric globular head (gC1q) domain of human C1q is made up of the C-terminal ends of the three individual chains, ghA, ghB, and ghC. A candidate receptor for the gC1q domain is a multi-functional pattern recognition protein, gC1qR. Since understanding of gC1qR and gC1q interaction could provide an insight into the pleiotropic functions of gC1qR, this study was undertaken to identify the gC1qR-binding site on the gC1q domain, using the recombinant ghA, ghB, and ghC modules and their substitution mutants. Our results show that ghA, ghB, and ghC modules can interact with gC1qR independently, thus reinforcing the notion of modularity within the gC1q domain of human C1q. Mutational analysis revealed that while Arg162 in the ghA module is central to interaction between gC1qR and C1q, a single amino acid substitution (arginine to glutamate) in residue 114 of the ghB module resulted in enhanced binding. Expression of gC1qR and C1q in adherent monocytes with or without pro-inflammatory stimuli was also analyzed by qPCR; it showed an autocrine/paracrine basis of C1q and gC1qR interaction. Microscopic studies revealed that C1q and gC1qR are colocalized on PBMCs. Cell proliferation assays indicated that ghA, ghB, and ghC modules were able to attenuate phytohemagglutinin-stimulated proliferation of PBMCs. Addition of gC1qR had an additive effect on the anti-proliferative effect of globular head modules. In summary, our results identify residues involved in C1q-gC1qR interaction and explain, to a certain level, their involvement on the immune cell surface, which is relevant for C1q-induced functions including inflammation, infection, and immunity.

Complement Deposition on Nanoparticles Can Modulate Immune Responses by Macrophage, B and T Cells.

Nanoparticles are attractive drug delivery vehicles for targeted organ-specific as well as systemic therapy. However, their interaction with the immune system offers an intriguing challenge to the success of nanotherapeutics in vivo. Recently, we showed that pristine and derivatised carbon nanotubes (CNT) can activate complement mainly via the classical pathway leading to enhanced uptake by phagocytic cells, and transcriptional down-regulation of pro-inflammatory cytokines. Here, we report the interaction of complement-activating CC-CNT and RNA-CNT, and non-complement-activating gold-nickel (Au-Ni) nanowires with cell lines representing macrophage, B and T cells. Complement deposition considerably enhanced uptake of CNTs by immune cells known to overexpress complement receptors. Real-Time qPCR and multiplex array analyses showed complement-dependent down-regulation of TNF-α and IL-1β and up-regulation of IL-12 by CMC- and RNA-CNTs, in addition to revealing IL-10 as a crucial regulator during nanoparticle-immune cell interaction. It appears that complement system can recognize molecular patterns differentially displayed by nanoparticles and thus, modulate subsequent processing of nanoparticles by antigen capturing and antigen presenting cells, which can shape innate and adaptive immune axes.

Individual-level factors associated with variation in mycobacterial-specific immune response: Gender and previous BCG vaccination status.

A more effective tuberculosis (TB) vaccine is needed to eliminate TB disease. Many new vaccine candidates enhance the immunogenicity of the existing vaccine, Bacillus Calmette-Guérin (BCG). Understanding BCG induced immune variation is key to developing a new vaccine.

Serum indoleamine 2,3-dioxygenase activity is associated with reduced immunogenicity following vaccination with MVA85A.

There is an urgent need for improved vaccines to protect against tuberculosis. The currently available vaccine Bacille Calmette-Guerin (BCG) has varying immunogenicity and efficacy across different populations for reasons not clearly understood. MVA85A is a modified vaccinia virus expressing antigen 85A from Mycobacterium tuberculosis which has been in clinical development since 2002 as a candidate vaccine to boost BCG-induced protection. A recent efficacy trial in South African infants failed to demonstrate enhancement of protection over BCG alone. The immunogenicity was lower than that seen in UK trials. The enzyme Indoleamine 2,3-dioxygenase (IDO) catalyses the first and rate-limiting step in the breakdown of the essential amino acid tryptophan. T cells are dependent on tryptophan and IDO activity suppresses T-cell proliferation and function.

Roles for Treg expansion and HMGB1 signaling through the TLR1-2-6 axis in determining the magnitude of the antigen-specific immune response to MVA85A.

A better understanding of the relationships between vaccine, immunogenicity and protection from disease would greatly facilitate vaccine development. Modified vaccinia virus Ankara expressing antigen 85A (MVA85A) is a novel tuberculosis vaccine candidate designed to enhance responses induced by BCG. Antigen-specific interferon-γ (IFN-γ) production is greatly enhanced by MVA85A, however the variability between healthy individuals is extensive. In this study we have sought to characterize the early changes in gene expression in humans following vaccination with MVA85A and relate these to long-term immunogenicity. Two days post-vaccination, MVA85A induces a strong interferon and inflammatory response. Separating volunteers into high and low responders on the basis of T cell responses to 85A peptides measured during the trial, an expansion of circulating CD4+ CD25+ Foxp3+ cells is seen in low but not high responders. Additionally, high levels of Toll-like Receptor (TLR) 1 on day of vaccination are associated with an increased response to antigen 85A. In a classification model, combined expression levels of TLR1, TICAM2 and CD14 on day of vaccination and CTLA4 and IL2Rα two days post-vaccination can classify high and low responders with over 80% accuracy. Furthermore, administering MVA85A in mice with anti-TLR2 antibodies may abrogate high responses, and neutralising antibodies to TLRs 1, 2 or 6 or HMGB1 decrease CXCL2 production during in vitro stimulation with MVA85A. HMGB1 is released into the supernatant following atimulation with MVA85A and we propose this signal may be the trigger activating the TLR pathway. This study suggests an important role for an endogenous ligand in innate sensing of MVA and demonstrates the importance of pattern recognition receptors and regulatory T cell responses in determining the magnitude of the antigen specific immune response to vaccination with MVA85A in humans.

Safety and immunogenicity of an FP9-vectored candidate tuberculosis vaccine (FP85A), alone and with candidate vaccine MVA85A in BCG-vaccinated healthy adults: a phase I clinical trial.

The safety and immunogenicity of a new candidate tuberculosis (TB) vaccine, FP85A was evaluated alone and in heterologous prime-boost regimes with another candidate TB vaccine, MVA85A. This was an open label, non-controlled, non-randomized Phase I clinical trial. Healthy previously BCG-vaccinated adult subjects were enrolled sequentially into three groups and vaccinated with FP85A alone, or both FP85A and MVA85A, with a four week interval between vaccinations. Passive and active data on adverse events were collected. Immunogenicity was evaluated by Enzyme Linked Immunospot (ELISpot), flow cytometry and Enzyme Linked Immunosorbent assay (ELISA). Most adverse events were mild and there were no vaccine-related serious adverse events. FP85A vaccination did not enhance antigen 85A-specific cellular immunity. When MVA85A vaccination was preceded by FP85A vaccination, cellular immune responses were lower compared with when MVA85A vaccination was the first immunisation. MVA85A vaccination, but not FP85A vaccination, induced anti-MVA IgG antibodies. Both MVA85A and FP85A vaccinations induced anti-FP9 IgG antibodies. In conclusion, FP85A vaccination was well tolerated but did not induce antigen-specific cellular immune responses. We hypothesize that FP85A induced anti-FP9 IgG antibodies with cross-reactivity for MVA85A, which may have mediated inhibition of the immune response to subsequent MVA85A. ClinicalTrials.gov identification number: NCT00653770.

Effect of vaccine dose on the safety and immunogenicity of a candidate TB vaccine, MVA85A, in BCG vaccinated UK adults.

A non-randomised, open-label, Phase I safety and immunogenicity dose-finding study to assess the safety and immunogenicity of the candidate TB vaccine Modified Vaccinia virus Ankara expressing Antigen 85A (MVA85A) from Mycobacterium tuberculosis (MTB) in healthy adult volunteers previously vaccinated with BCG.

Induction of CD152 (CTLA-4) and LAP (TGF-β1) in human Foxp3- CD4+ CD25- T cells modulates TLR-4 induced TNF-α production.

CD152 (CTLA-4) is a co-stimulatory molecule that is expressed by T cells and negatively regulates immune responses. Here, we report the identification of a novel ligand, GPC(81-95), with the ability to induce both CD152 and LAP (TGF-β1) on human Foxp3(-) CD25(-) CD4(+) T cells. The results demonstrate that GPC(81-95) peptide-induced cell surface CD152 is endocytosed back into the cell during stimulation. The protein export and exocytosis of CD152 is also induced by this ligand. The inhibitory effects of GPC(81-95) on LPS-induced TNF-α production was shown to be closely associated with its ability to induce both LAP (TGF-β1) and CD152. Taken together, we have shown that a novel peptide ligand stimulates LAP (TGF-β1) and CD152 expression on resting CD4 T cells and have demonstrated that GPC(81-95) is a useful tool to study the functional properties of LAP (TGF-β1)(+) CD152(+) CD4(+) T cells.

A Phase I study evaluating the safety and immunogenicity of MVA85A, a candidate TB vaccine, in HIV-infected adults.

Objectives Control of the tuberculosis (TB) epidemic is a global health priority and one that is likely to be achieved only through vaccination. The critical overlap with the HIV epidemic requires any effective TB vaccine regimen to be safe in individuals who are infected with HIV. The objectives of this clinical trial were to evaluate the safety and immunogenicity of a leading candidate TB vaccine, MVA85A, in healthy, HIV-infected adults. Design This was an open-label Phase I trial, performed in 20 healthy HIV-infected, antiretroviral-naïve subjects. Two different doses of MVA85A were each evaluated as a single immunisation in 10 subjects, with 24 weeks of follow-up. The safety of MVA85A was assessed by clinical and laboratory markers, including regular CD4 counts and HIV RNA load measurements. Vaccine immunogenicity was assessed by ex vivo interferon γ (IFN-γ) ELISpot assays and flow-cytometric analysis. Results MVA85A was safe in subjects with HIV infection, with an adverse-event profile comparable with historical data from previous trials in HIV-uninfected subjects. There were no clinically significant vaccine-related changes in CD4 count or HIV RNA load in any subjects, and no evidence from qPCR analyses to indicate that MVA85A vaccination leads to widespread preferential infection of vaccine-induced CD4 T cell populations. Both doses of MVA85A induced an antigen-specific IFN-γ response that was durable for 24 weeks, although of a lesser magnitude compared with historical data from HIV-uninfected subjects. The functional quality of the vaccine-induced T cell response in HIV-infected subjects was remarkably comparable with that observed in healthy HIV-uninfected controls, but less durable. Conclusion MVA85A is safe and immunogenic in healthy adults infected with HIV. Further safety and efficacy evaluation of this candidate vaccine in TB- and HIV-endemic areas is merited.

Th1/Th17 cell induction and corresponding reduction in ATP consumption following vaccination with the novel Mycobacterium tuberculosis vaccine MVA85A.

Vaccination with Bacille Calmette-Guérin (BCG) has traditionally been used for protection against disease caused by the bacterium Mycobacterium tuberculosis (M.tb). The efficacy of BCG, especially against pulmonary tuberculosis (TB) is variable. The best protection is conferred in temperate climates and there is close to zero protection in many tropical areas with a high prevalence of both tuberculous and non-tuberculous mycobacterial species. Although interferon (IFN)-γ is known to be important in protection against TB disease, data is emerging on a possible role for interleukin (IL)-17 as a key cytokine in both murine and bovine TB vaccine studies, as well as in humans. Modified Vaccinia virus Ankara expressing Antigen 85A (MVA85A) is a novel TB vaccine designed to enhance responses induced by BCG. Antigen-specific IFN-γ production has already been shown to peak one week post-MVA85A vaccination, and an inverse relationship between IL-17-producing cells and regulatory T cells expressing the ectonucleosidease CD39, which metabolises pro-inflammatory extracellular ATP has previously been described. This paper explores this relationship and finds that consumption of extracellular ATP by peripheral blood mononuclear cells from MVA85A-vaccinated subjects drops two weeks post-vaccination, corresponding to a drop in the percentage of a regulatory T cell subset expressing the ectonucleosidase CD39. Also at this time point, we report a peak in co-production of IL-17 and IFN-γ by CD4(+) T cells. These results suggest a relationship between extracellular ATP and effector responses and unveil a possible pathway that could be targeted during vaccine design.

Investigating the induction of vaccine-induced Th17 and regulatory T cells in healthy, Mycobacterium bovis BCG-immunized adults vaccinated with a new tuberculosis vaccine, MVA85A.

Tuberculosis (TB) remains a threat to global health. While advances in diagnostics and treatment are crucial to the containment of the epidemic, it is likely that elimination of the disease can only be achieved through vaccination. Vaccine-induced protection from Mycobacterium tuberculosis is dependent, at least in part, on a robust Th1 response, yet little is known of the ability of TB vaccines to induce other T-cell subsets which may influence vaccine efficacy. Interleukin-17A (IL-17A) is a proinflammatory cytokine produced by Th17 cells which has been associated with both immune pathology and protection against infectious disease. Following vaccination with MVA85A, a viral vector vaccine aimed at enhancing immune responses to M. tuberculosis, antigen-specific IL-17A-producing T cells were induced in the peripheral blood of healthy volunteers. These T cells are detected later than gamma interferon (IFN-gamma)-secreting T cells and are of a low magnitude. Preexisting immune responses to mycobacterial antigens were associated with higher CD4(+) CD25(hi) CD39(+) T-cell levels in the periphery and a reduced capacity to produce IL-17A following immunization. These data highlight the intricate balance of effector and regulatory immune responses induced by vaccination and that preexisting immunity to mycobacterial antigens may affect the composition of vaccine-induced T-cell subsets.

Identification of major factors influencing ELISpot-based monitoring of cellular responses to antigens from Mycobacterium tuberculosis.

A number of different interferon-gamma ELISpot protocols are in use in laboratories studying antigen-specific immune responses. It is therefore unclear how results from different assays compare, and what factors most significantly influence assay outcome. One such difference is that some laboratories use a short in vitro stimulation period of cells before they are transferred to the ELISpot plate; this is commonly done in the case of frozen cells, in order to enhance assay sensitivity. Other differences that may be significant include antibody coating of plates, the use of media with or without serum, the serum source and the number of cells added to the wells. The aim of this paper was to identify which components of the different ELISpot protocols influenced assay sensitivity and inter-laboratory variation. Four laboratories provided protocols for quantifying numbers of interferon-gamma spot forming cells in human peripheral blood mononuclear cells stimulated with Mycobacterium tuberculosis derived antigens. The differences in the protocols were compared directly. We found that several sources of variation in assay protocols can be eliminated, for example by avoiding serum supplementation and using AIM-V serum free medium. In addition, the number of cells added to ELISpot wells should also be standardised. Importantly, delays in peripheral blood mononuclear cell processing before stimulation had a marked effect on the number of detectable spot forming cells; processing delay thus should be minimised as well as standardised. Finally, a pre-stimulation culture period improved the sensitivity of the assay, however this effect may be both antigen and donor dependent. In conclusion, small differences in ELISpot protocols in routine use can affect the results obtained and care should be given to conditions selected for use in a given study. A pre-stimulation step may improve the sensitivity of the assay, particularly when cells have been previously frozen.

The relationship between human effector and memory T cells measured by ex vivo and cultured ELISPOT following recent and distal priming.

Maintenance of T-cell responses is an essential feature in protection from many infectious diseases that must be harnessed in vaccination. The relationship between effector T-cell responses and more durable and highly proliferative T-cell memory, particularly in humans, is not well understood. In this study, effector T-cell responses were measured by overnight ex vivo interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot-forming cell assay (ELISPOT), whereas memory T cells were measured by 10-day culture followed by IFN-gamma ELISPOT (cultured ELISPOT). We observed a significant correlation between IFN-gamma responses to CD4-stimulatory, but not to CD8-stimulatory, recall antigens measured by these assays, suggesting a divergence in regulation. In vaccine trial participants who received a prime-boost vaccination regimen comprising malaria antigens delivered by poxviruses, there was a correlation between ex vivo and cultured responses on day 7, but not 3 months post-vaccination, with the ratio of cultured : ex vivo response increasing over time. To compare responses revealed by cultured ELISPOT in more detail, tetramers comprising viral recall antigens were used to ascribe effector-memory and central-memory T-cell phenotypes through CCR7 and CD62L costaining. For CD8(+) responses the effector phenotype decreased during the initial culture period and memory populations remained high within the resulting 20-fold to 50-fold increased IFN-gamma-secreting or tetramer(+) population. This was less marked for CD4(+) responses, which had higher starting memory phenotype. Depletion of these central-memory T-cell populations generally ablated responses in cultured ELISPOT and reduced ex vivo responses. This study highlights differences between CD4(+) and CD8(+) effector and memory T cells, and the more complex phenotype of CD4(+) T cells.

Safety and immunogenicity of boosting BCG vaccinated subjects with BCG: comparison with boosting with a new TB vaccine, MVA85A.

To investigate the safety and immunogenicity of a booster BCG vaccination delivered intradermally in healthy, BCG vaccinated subjects and to compare with a previous clinical trial where BCG vaccinated subjects were boosted with a new TB vaccine, MVA85A.

Safety and immunogenicity of a new tuberculosis vaccine, MVA85A, in Mycobacterium tuberculosis-infected individuals.

An effective new tuberculosis (TB) vaccine regimen must be safe in individuals with latent TB infection (LTBI) and is a priority for global health care.

A comparison of IFNgamma detection methods used in tuberculosis vaccine trials.

Interferon gamma (IFNgamma) is a critical component of the pro-inflammatory immune response that provides protection against Mycobacterium tuberculosis. In the absence of an immunological correlate of protection, antigen-specific production of IFNgamma is a commonly used marker of a protective immune response. To facilitate the evaluation of tuberculosis candidate vaccines three different IFNgamma detection methods were compared. The cultured whole blood ELISA, ex vivo IFNgamma ELISpot and whole blood ex vivo intracellular cytokine staining (ICS) assays were performed head-to-head during a Phase I clinical trial using the candidate vaccine MVA85A. Whilst all three assays detected significant increases in IFNgamma production immediately following vaccination, distinctions between the assays were apparent. Higher baseline IFNgamma responses were detected using the cultured whole blood ELISA, whereas the ex vivo ELISpot assay was the most sensitive in detecting long-term (52 weeks) post-vaccination responses. The whole blood ex vivo ICS assay provided novel information by dissecting the IFNgamma response into responding CD4, CD8 and gamma/delta T cell subsets. Future tuberculosis vaccine trials and immunology studies should ideally include a combination of ex vivo and cultured assays to ensure a thorough and multifaceted evaluation of the immune response is achieved.

Boosting BCG vaccination with MVA85A down-regulates the immunoregulatory cytokine TGF-beta1.

In clinical trials recombinant-modified vaccinia virus Ankara expressing the Mycobacterium tuberculosis antigen 85A (MVA85A) induces approximately 10 times more effector T cells than any other recombinant MVA vaccine. We have found that in BCG primed subjects MVA85A vaccination reduces transforming growth factor beta 1 (TGF-beta1) mRNA in peripheral blood lymphocytes and reduces TGF-beta1 protein in the serum, but increases IFN-gamma ELISPOT responses to the recall antigen SK/SD. TGF-beta1 is essential for the generation of regulatory T cells and we see a correlation across vaccinees between CD4+CD25hiFoxP3+ cells and TGF-beta1 serum levels. This apparent ability to counteract regulatory T cell effects suggests a potential use of MVA85A as an adjuvant for less immunogenic vaccines.

Human CD4(+) T cells recognize an epitope within alpha-fetoprotein sequence and develop into TGF-beta-producing CD4(+) T cells.

There is limited information on the influence of tumor growth on the expansion of tumor-specific TGF-beta-producing CD4(+) T cells in humans. alpha-Fetoprotein (AFP) is an oncofetal Ag and has intrinsic immunoregulatory properties. In this study, we report the identification and characterization of subsets of CD4(+) T cells that recognize an epitope within the AFP sequence (AFP(46-55)) and develop into TGF-beta-producing CD4(+) T cells. In a peptide-specific and dose-dependent manner, AFP(46-55) CD4(+) T cells produce TGF-beta, GM-CSF, and IL-2 but not Th1-, Th2-, Th17-, or Tr1-type cytokines. These cells express CTLA-4 and glucocorticoid-induced TNR receptor and inhibit T cell proliferation in a contact-dependent manner. In this study, we show that the frequency of AFP(46-55) CD4(+) T cells is significantly higher (p = 001) in patients with hepatocellular carcinoma than in healthy donors, suggesting that these cells are expanded in response to tumor Ag. In contrast, tumor necrosis-inducing treatments that are shown to improve survival rate can shift the Th1/TGF-beta-producing CD4(+) T cell balance in favor of Th1 responses. Our data demonstrate that tumor Ags may contain epitopes which activate the expansion of inducible regulatory T cells, leading to evasion of tumor control.

Boosting BCG with recombinant modified vaccinia ankara expressing antigen 85A: different boosting intervals and implications for efficacy trials.

To investigate the safety and immunogenicity of boosting BCG with modified vaccinia Ankara expressing antigen 85A (MVA85A), shortly after BCG vaccination, and to compare this first with the immunogenicity of BCG vaccination alone and second with a previous clinical trial where MVA85A was administered more than 10 years after BCG vaccination.

Immunisation with BCG and recombinant MVA85A induces long-lasting, polyfunctional Mycobacterium tuberculosis-specific CD4+ memory T lymphocyte populations.

In the search for effective vaccines against intracellular pathogens such as HIV, tuberculosis and malaria, recombinant viral vectors are increasingly being used to boost previously primed T cell responses. Published data have shown prime-boost vaccination with BCG-MVA85A (modified vaccinia virus Ankara expressing antigen 85A) to be highly immunogenic in humans as measured by ex vivo IFN-gamma ELISPOT. Here, we used polychromatic flow cytometry to investigate the phenotypic and functional profile of these vaccine-induced Mycobacterium tuberculosis (M.tb) antigen 85A-specific responses in greater detail. Promisingly, antigen 85A-specific CD4(+) T cells were found to be highly polyfunctional, producing IFN-gamma, TNF-alpha, IL-2 and MIP-1beta. Surface staining showed the responding CD4(+) T cells to be relatively immature (CD45RO(+) CD27(int)CD57(-)); this observation was supported by the robust proliferative responses observed following antigenic stimulation. Furthermore, these phenotypic and functional properties were independent of clonotypic composition and epitope specificity, which was maintained through the different phases of the vaccine-induced immune response. Overall, these data strongly support the use of MVA85A in humans as a boosting agent to expand polyfunctional M.tb-specific CD4(+) T cells capable of significant secondary responses.

Unmasking of alpha-fetoprotein-specific CD4(+) T cell responses in hepatocellular carcinoma patients undergoing embolization.

Necrosis of tumor cells can activate both innate and adaptive antitumor immunity. However, there is little information on the effects of necrosis-inducing cancer treatments on tumor-specific T cell immune responses in humans. We studied the effects of a necrosis-inducing treatment (embolization) on anti-alpha-fetoprotein (AFP)-specific CD4(+) T cell responses in hepatocellular carcinoma (HCC) patients and controls using an array of AFP-derived peptides. In this study, we show that AFP-specific CD4(+) T cell responses to three immunodominant epitopes in HCC patients were significantly expanded during (p < 0.0001) and after embolization (p < 0.002). The development of higher frequencies of AFP-specific CD4(+) T cells after treatment were significantly associated with the induction of >50% necrosis of tumor and an improved clinical outcome (p < 0.007). In addition, we identified two novel HLA-DR-restricted AFP-derived CD4(+) T cell epitopes (AFP(137-145) and AFP(249-258)) and showed that the CD4(+) T cells recognizing these epitopes produce Th1 (IFN-gamma and TNF-alpha) but not Th2 (IL-5)-type cytokines. AFP(137-145)-, AFP(249-258)-, and AFP(364-373)-specific CD4(+) T cells were detected in HCC patients but not in patients with chronic liver diseases or healthy donors. In conclusion; our study shows that induction of tumor necrosis by a conventional cancer treatment can unmask tumor rejection Ag cell-mediated immunity and provides a rationale for combining embolization with immunotherapy in HCC patients.

Analysis of CD4+ T-Cell responses to a novel alpha-fetoprotein-derived epitope in hepatocellular carcinoma patients.

Alpha-fetoprotein (AFP) is a tumor-associated antigen in hepatocellular carcinoma and is a target for the development of cancer vaccine. Four immunodominant AFP-derived HLA-A*0201-restricted peptides have been identified and the administration of these peptides with an adjuvant has stimulated AFP-specific CTL responses in hepatocellular carcinoma patients. However, no AFP-derived CD4 T-cell epitope has yet been reported and the status of AFP-specific CD4(+) T-cell responses in hepatocellular carcinoma patients is not fully understood. The aim of this study was to analyze naturally occurring CD4(+) T-cell responses to AFP.

Synergistic DNA-MVA prime-boost vaccination regimes for malaria and tuberculosis.

T-cell-mediated responses against the liver-stage of Plasmodium falciparum are critical for protection in the human irradiated sporozoite model and several animal models. Heterologous prime-boost approaches, employing plasmid DNA and viral vector delivery of malarial DNA sequences, have proved particularly promising for maximising T-cell-mediated protection in animal models. The T-cell responses induced by this prime-boost regime, in animals and humans, are substantially greater than the sum of the responses induced by DNA or MVA vaccines used alone, leading to the term introduced here of "synergistic" prime-boost immunisation. The insert in our first generation clinical constructs is known as multiple epitope-thrombospondin-related adhesion protein (ME-TRAP). We have performed an extensive series of phase I/II trials evaluating various prime-boost combination regimens for delivery of ME-TRAP in over 500 malaria-naïve and malaria-exposed individuals. The three delivery vectors are DNA, modified vaccinia virus Ankara (MVA) and, more recently, fowlpox strain 9 (FP9). Administration was intra-epidermal and intramuscular for DNA and intradermal for MVA and FP9. Doses of DNA ranged from 4 microg to 2mg. Doses of MVA were up to 1.5 x 10(8) plaque forming units (pfu) and of FP9, up to 1.0 x 10(8)pfu. Further trials employing bacille Calmette-Guérin (BCG) as the priming agent and MVA expressing antigen 85A of Mycobacterium tuberculosis as the boosting agent has extended the scope of synergistic prime-boost vaccination. In this review we summarise the safety, immunogenicity and efficacy results from these malaria and tuberculosis vaccine clinical trials.

Boosting BCG with MVA85A: the first candidate subunit vaccine for tuberculosis in clinical trials.

There is an urgent need for an improved vaccine against tuberculosis. Heterologous prime-boost immunization regimes induce higher levels of cellular immunity than homologous boosting with the same vaccine. Using BCG as the priming immunization in such a regime allows for the retention of the beneficial protective effects of BCG against disseminated disease in childhood. Recombinant poxviruses are powerful boosting agents, for both CD4+ and CD8+ T cells. Here we review the preclinical data from a BCG prime-recombinant modified vaccinia virus Ankara expressing antigen 85A (MVA85A) boost strategy. MVA85A is now in clinical trials in the UK and Africa and the design of these trials, including the ethical and regulatory issues are discussed.

Ex vivo characterization of early secretory antigenic target 6-specific T cells at sites of active disease in pleural tuberculosis.

Presence of early secretory antigenic target-6 (ESAT-6)-specific, interferon- gamma -secreting T cells in blood accurately marks tuberculosis infection. In tuberculous pleural effusions from 10 patients with tuberculosis, these cells were concentrated a mean of 15-fold (standard deviation, +/-6-fold), relative to their level in peripheral blood (P=.014), and displayed rapid effector function. Such cells were absent in 8 control patients with nontuberculous pleural disease. The recruitment of ESAT-6-specific T cells to inflamed tuberculous tissue demonstrates their function in vivo and suggests a novel way to diagnose tuberculous pleuritis.

Diagnosis of tuberculosis in South African children with a T-cell-based assay: a prospective cohort study.

Childhood tuberculosis often presents non-specifically and is a common differential diagnosis in high prevalence areas. Current diagnostic tools have poor sensitivity and cannot reliably exclude tuberculosis, so overdiagnosis is common. HIV co-infection exacerbates this problem and accounts for an increasing proportion of paediatric tuberculosis worldwide.