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The complement system is an important humoral immune surveillance mechanism against tumours. However, many malignant tumours are resistant to complement mediated lysis. Here, we report secretion of complement factor H related protein 5 (FHR5) by primary tumour cells derived from Glioblastoma multiforme (GBM) patients. We investigated whether the secreted FHR5 exhibited functional activity similar to factor H, including inhibition of complement mediated lysis, acting as a co-factor for factor I mediated cleavage of C3b, and decay acceleration of C3 convertase. Immunoblotting analysis of primary GBM cells (B30, B31 and B33) supernatant showed the active secretion of FHR5, but not of Factor H. ELISA revealed that the secretion of soluble GBM-FHR5 by cultured GBM cells increased in a time-dependent manner. Primary GBM-FHR5 inhibited complement mediated lysis, possessed co-factor activity for factor I mediated cleavage and displayed decay acceleration of C3 convertase. In summary, we detected the secretion of FHR5 by primary GBM cells B30, B31 and B33. The results demonstrated that GBM-FHR5 shares biological function with FH as a mechanism primary GBM cells potentially use to resist complement mediated lysis. peerReviewed

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 fromMycobacterium tuberculosisis 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 toM. 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-γ)-secreting T cells and are of a low magnitude. Preexisting immune responses to mycobacterial antigens were associated with higher CD4+CD25hiCD39+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.

Copyright © 2009 by the American Thoracic Society. Rationale: 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. Objectives: To evaluate the safety and immunogenicity of a leading new TB vaccine, recombinant Modified Vaccinia Ankara expressing Antigen 85A (MVA85A) in individuals with LTBI. Methods: An open-label, phase I trial of MVA85A was performed in 12 subjects with LTBI recruited from TB contact clinics in Oxford and London or by poster advertisements in Oxford hospitals. Patients were assessed clinically and had blood samples drawn for immunological analysis over a 52-week period after vaccination with MVA85A. Thoracic computed tomography scans were performed at baseline and at 10 weeks after vaccination. Safety of MVA85A was assessed by clinical, radiological, and inflammatory markers. The immunogenicity of MVA85A was assessed by IFNγ and IL-2 ELISpot assays and FACS. Measurements and Main Results: MVA85A was safe in subjects with LTBI, with comparable adverse events to previous trials of MVA85A. There were no clinically significant changes in inflammatory markers or thoracic computed tomography scans after vaccination. MVA85A induced a strong antigen-specific IFN-γ and IL-2 response that was durable for 52 weeks. The magnitude of IFN-γ response was comparable to previous trials of MVA85A in bacillus Calmette-Guérin–vaccinated individuals. Antigen 85A–specific polyfunctional CD4+ T cells were detectable prior to vaccination with statistically significant increases in cell numbers after vaccination. Conclusions: MVA85A is safe and highly immunogenic in individuals with LTBI. These results will facilitate further trials in TB-endemic areas. Oxford Biomedical Research Centre, Wellcome Trust, and AFTBVAC.

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-α-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 (AFP137–145 and AFP249–258) and showed that the CD4+ T cells recognizing these epitopes produce Th1 (IFN-γ and TNF-α) but not Th2 (IL-5)-type cytokines. AFP137–145-, AFP249–258-, and AFP364–373-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.

INTRODUCTION: 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. AIMS: We aimed to establish if individual-level covariates were associated with cell-mediated immune response (interferon gamma (IFN-γ)) at vaccine trial enrolment (baseline) in a long-term retrospective analysis (LTR) and after BCG vaccination in a short-term prospective analysis (STP). METHODS: Four covariates were analysed: gender, country, BCG vaccination history and monocyte/lymphocyte cell count ratio. Univariable and multivariable linear regression were conducted on IFN-γ response at baseline for LTR, and area under the curve (AUC), 24 week and peak IFN-γ response for STP. RESULTS: Previous BCG vaccination was strongly associated with higher IFN-γ response at baseline (LTR analysis) (p-values < 0.05). Being male showed a weak association with higher baseline response (p-value = 0.1). BCG revaccination was strongly associated with a larger response increase than primary-vaccination (AUC & peak p-values < 0.01), but did not differ at 24 weeks (STP analysis). All other covariates were non-significant (p-values > 0.1). CONCLUSION: This analysis suggests that previous BCG vaccination and gender are associated with durable IFN-γ responses. Vaccine trials may need to stratify by BCG vaccination history and gender.

Purpose: α-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. Experimental Design: We analyzed the ability of CD4+ T cells to recognize an HLA-DR-restricted AFP-derived epitope in 41 hepatocellular carcinoma patients and 24 non-hepatocellular carcinoma control patients using intracellular cytokine assays for IFN-γ. Results: Here, for the first time, we report the identification of an AFP-derived CD4+ T-cell epitope that is recognized by circulating lymphocytes from hepatocellular carcinoma patients in association with HLA-DR. The absence of detectable responses in healthy donors and patients with chronic liver disease suggests that AFP-specific CD4+ T cells in the responder patients had been previously expanded in vivo in response to the tumor. The anti-AFP CD4+ T-cell response was only detected in hepatocellular carcinoma patients with normal or mildly elevated serum AFP levels who were in the early stage of disease. Conclusion: Our data will be instrumental in the development of cancer vaccine using AFP-derived immunogens.

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.

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.We assessed the usefulness of a T-cell-based rapid blood test for Mycobacterium tuberculosis infection, the enzyme-linked immunospot assay (ELISPOT), in routine clinical practice. We did a prospective blinded study of 293 African children with suspected tuberculosis in kwaZulu-Natal, a region with high HIV prevalence. Children had full clinical assessment, ELISPOT, and a tuberculin skin test. Test results were compared with final clinical and microbiological diagnoses.In children with tuberculosis, sensitivity of ELISPOT was 83% (95% CI 75-89, n=133), significantly higher (p0.001) than the 63% (54-72) sensitivity of tuberculin skin test (n=116). Sensitivity of tuberculin skin test fell significantly in children younger than 3 years (to 51%), with HIV co-infection (36%), or with malnutrition (44%). Sensitivity of ELISPOT, which was not significantly adversely affected by these factors, was 85%, 73%, and 78%, respectively in these subgroups. In 116 children with both test results available, sensitivity of the two tests combined was 91% (85-95).Diagnostic sensitivity of ELISPOT is higher than that of the skin test and is less affected by factors frequently associated with childhood tuberculosis in developing countries. Used together with the skin test, ELISPOT provides a clinically useful diagnostic sensitivity in African children with suspected tuberculosis.

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., Author summary Treg cells are crucial for the maintenance of tolerance and control of immune responses, especially during viral infection and tumour formation. Marek’s Disease Virus (MDV) infection causes immunosuppression and induces transformation of CD4+ T cells in chicken. Here we demonstrate that a population of chicken CD4+ T cells express inhibitory molecules including TGF-beta and have immune-regulatory properties. TGF-beta+ Treg cells are detected in different chicken lymphoid tissues, the highest being detected in cecal tonsils. Chicken lines susceptible to MDV-induced lymphoma formation have higher frequencies of TGF-beta+ Treg cells compared to the MDV resistant chicken line. Infection of chicken with a virulent MDV increased the numbers of TGF-beta+ Treg cells, which was not changed after infection with the MDV vaccine strain. MDV-transformed CD4+ T cells produce high levels of TGF-beta, while they express very low levels of TGF-beta receptors compared to non-transformed CD4+ T cells. In addition, MDV-induced lymphoma cells express soluble suppressive factors that can inhibit T cell function; however these soluble factors cannot suppress proliferation of the lymphoma cells. For the first time, we identified chicken TGF-beta+ Treg cells and demonstrate that these cells are involved in pathogenesis and immunosuppression of MDV infection.

The wide spectrum of clinical outcomes following infection with Mycobacterium tuberculosis is largely determined by the host immune response; therefore, we studied several clinically defined groups of individuals (n = 120) that differ in their ability to contain the bacillus. To quantitate M. tuberculosis-specific T cells directly ex vivo, we enumerated IFN-γ-secreting CD4 T cells specific for ESAT-6, a secreted Ag that is highly specific for M. tuberculosis, and a target of protective immune responses in animal models. We found that frequencies of circulating ESAT-6 peptide-specific IFN-γ-secreting CD4 T cells were higher in latently infected healthy contacts and subjects with minimal disease and low bacterial burdens than in patients with culture-positive active pulmonary tuberculosis (p = 0.009 and p = 0.002, respectively). Importantly, the frequency of these Ag-specific CD4 T cells fell progressively in all groups with treatment (p = 0.005), suggesting that the lower responses in patients with more extensive disease were not due to tuberculosis-induced immune suppression. This population of M. tuberculosis Ag-specific Th1-type CD4 T cells appears to correlate with clinical phenotype and declines during successful therapy; these features are consistent with a role for these T cells in the containment of M. tuberculosis in vivo. Such findings may assist in the design and evaluation of novel tuberculosis vaccine candidates.

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.

Highlights ► We compared 3 doses of a the candidate TB vaccine MVA85A. ► All doses of the vaccine were safe and induced a Th1 type immune response. ► The strongest and most sustained response was seen with the highest dose of MVA85A. ► A high dose of 1 × 108 PFU of MVA85A is safe and induces sustained immunity., Purpose 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. Methods Healthy BCG-vaccinated volunteers were vaccinated with either 1 × 107 or 1 × 108 PFU of MVA85A. All adverse events were documented and antigen specific T cell responses were measured using an ex vivo IFN-γ ELISPOT assay. Safety and immunogenicity were compared between the 2 dose groups and with a previous trial in which a dose of 5 × 107 PFU MVA85A had been administered. Results There were no serious adverse events recorded following administration of either 1 × 107 or 1 × 108 PFU of MVA85A. Systemic adverse events were more frequently reported following administration of 1 × 108 PFU of MVA85A when compared to either 5 × 107 or 1 × 107 PFU of MVA85A but were mild or moderate in severity and resolved completely within 7 days of immunisation. Antigen specific T cell responses as measured by the IFN-γ ELISPOT were significantly higher following immunisation in adults receiving 1 × 108 PFU compared to the 5 × 107 and 1 × 107 doses. Additionally, a broader range of Ag85A epitopes are detected following 1 × 108 PFU of MVA85A. Conclusion A higher dose of 1 × 108 PFU of MVA85A is well-tolerated, increases the frequency of IFN-γ secreting T cells detected following immunisation and broadens the range of Ag85A epitopes detected.

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., Article summary Article focus HIV infection increases susceptibility to TB, and globally, TB is the cause of death in up to half of AIDS deaths. There is an urgent need for a safe and effective TB vaccine in HIV-infected people. Key messages MVA85A, a leading candidate TB vaccine, is safe and well tolerated in HIV-infected people and does not induce changes in either CD4 count or HIV RNA load. MVA85A is immunogenic in HIV-infected people, and induces a similar immune profile to that seen in HIV-uninfected people, but the immunogenicity is less durable in HIV-infected people. Strengths and limitations of this study This is a Phase I study with 20 subjects, and further studies are needed in TB endemic countries in this important target population.

© 2011 Griffiths et al. Vaccination with Bacille Calmette-Gue´ rin (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)-c 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-c 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-c 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. KLG’s Wellcome Trust studentship

Copyright @ 2010 Cancer Research UK. This work is licensed under the Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/. Background: α-Fetoprotein (AFP) is a tumour-associated antigen in hepatocellular carcinoma (HCC) and is a target for immunotherapy. However, there is little information on the pattern of CD4 (Th1) and CD8 (Tc1) T-cell response to AFP in patients with HCC and their association with the clinical characteristics of patients. Methods: We therefore analysed CD4 and CD8 T-cell responses to a panel of AFP-derived peptides in a total of 31 HCC patients and 14 controls, using an intracellular cytokine assay for IFN-γ. Results: Anti-AFP Tc1 responses were detected in 28.5% of controls, as well as in 25% of HCC patients with Okuda I (early tumour stage) and in 31.6% of HCC patients with stage II or III (late tumour stages). An anti-AFP Th1 response was detected only in HCC patients (58.3% with Okuda stage I tumours and 15.8% with Okuda stage II or III tumours). Anti-AFP Th1 response was mainly detected in HCC patients who had normal or mildly elevated serum AFP concentrations (P=0.00188), whereas there was no significant difference between serum AFP concentrations in these patients and the presence of an anti-AFP Tc1 response. A Th1 response was detected in 44% of HCC patients with a Child–Pugh A score (early stage of cirrhosis), whereas this was detected in only 15% with a B or C score (late-stage cirrhosis). In contrast, a Tc1 response was detected in 17% of HCC patients with a Child–Pugh A score and in 46% with a B or C score. Conclusion: These results suggest that anti-AFP Th1 responses are more likely to be present in patients who are in an early stage of disease (for both tumour stage and liver cirrhosis), whereas anti-AFP Tc1 responses are more likely to be present in patients with late-stage liver cirrhosis. Therefore, these data provide valuable information for the design of vaccination strategies against HCC. Association for International Cancer Research and Polkemmet Fund, London Clinic.

OBJECTIVES: 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. DESIGN: There are two clinical trials reported here: a Phase I observational trial with MVA85A; and a Phase IV observational trial with BCG. These clinical trials were all conducted in the UK in healthy, HIV negative, BCG naïve adults. Subjects were vaccinated with BCG alone; or BCG and then subsequently boosted with MVA85A four weeks later (short interval). The outcome measures, safety and immunogenicity, were monitored for six months. The immunogenicity results from this short interval BCG prime-MVA85A boost trial were compared first with the BCG alone trial and second with a previous clinical trial where MVA85A vaccination was administered many years after vaccination with BCG. RESULTS: MVA85A was safe and highly immunogenic when administered to subjects who had recently received BCG vaccination. When the short interval trial data presented here were compared with the previous long interval trial data, there were no significant differences in the magnitude of immune responses generated when MVA85A was administered shortly after, or many years after BCG vaccination. CONCLUSIONS: The clinical trial data presented here provides further evidence of the ability of MVA85A to boost BCG primed immune responses. This boosting potential is not influenced by the time interval between prior BCG vaccination and boosting with MVA85A. These findings have important implications for the design of efficacy trials with MVA85A. Boosting BCG induced anti-mycobacterial immunity in either infancy or adolescence are both potential applications for this vaccine, given the immunological data presented here. TRIAL REGISTRATION: ClinicalTrials.gov NCT00427453 (short boosting interval), NCT00427830 (long boosting interval), NCT00480714 (BCG alone).

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-naive 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 μg to 2 mg. Doses of MVA were up to 1.5 × 10 8 plaque forming units (pfu) and of FP9, up to 1.0 × 10 8 pfu. Further trials employing bacille Calmette–Guerin (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.

Summary Background Identification of individuals latently infected with Mycobacterium tuberculosis is an important part of tuberculosis control. The current method, the tuberculin skin test (TST), has poor specificity because of the antigenic cross-reactivity of purified protein derivative (PPD) with M bovis BCG vaccine and environmental mycobacteria. ESAT-6 is a secreted antigen that is highly specific for M tuberculosis complex, but is absent from M bovis BCG. With an enzyme-linked immunospot (ELISPOT) assay for interferon gamma, we have identified ESAT-6-specific T cells as an accurate marker of M tuberculosis infection. Methods We did a prospective, masked study of 50 healthy contacts, with varying but well defined degrees of exposure to M tuberculosis , who attended an urban contact-tracing clinic. We assessed and compared the efficacy of our assay and TST for detection of symptomless infected individuals by correlation of test results with the degree of exposure to an infectious index case. Findings The ESAT-6 ELISPOT assay results had a strong positive relation with increasing intensity of exposure (odds ratio=9·0 per unit increase in level of exposure [95% CI 2·6–31·6], p=0·001), whereas TST results had a weaker relation with exposure (1-9 [1·0–3·5], p=0·05). By contrast, ELISPOT results were not correlated with BCG vaccination status (p=0·7), whereas TST results were significantly more likely to be positive in BCG-vaccinated contacts (12·1 [1·3–115·7], p=0·03). Interpretation This new antigen-specific T cell-based assay could allow more accurate identification of symptom-free individuals recently exposed to M tuberculosis , and thereby help to improve tuberculosis control.

There is no reliable means of detecting latent M. tuberculosis infection, and even in patients with active tuberculosis, infection is often unconfirmed. We hypothesized that M. tuberculosis antigen-specific T cells might reliably indicate infection. We enumerated peripheral blood-derived interferon gamma (IFN-gamma)-secreting T cells responding to epitopes from ESAT-6, an antigen that is highly specific for M. tuberculosis complex but absent from BCG, in four groups of individuals. Forty-five of 47 patients with bacteriologically confirmed tuberculosis had ESAT-6-specific IFN-gamma-secreting T cells, compared with four of 47 patients with nontuberculous illnesses, indicating that these T cells are an accurate marker of M. tuberculosis infection. This assay thus has a sensitivity of 96% (95% confidence interval [CI] 92-100) for detecting M. tuberculosis infection in this patient population. By comparison, of the 26 patients with tuberculosis who had a diagnostic tuberculin skin test (TST), only 18 (69%) were positive (p = 0.003). In addition, 22 of 26 (85%) TST-positive exposed household contacts had ESAT-6-specific T cells, whereas zero of 26 unexposed BCG-vaccinated subjects responded. This approach enables rapid detection of M. tuberculosis infection in patients with active tuberculosis and in exposed asymptomatic individuals at high risk of latent infection; it also successfully distinguishes between M. tuberculosis infection and BCG vaccination. This capability may facilitate tuberculosis control in nonendemic regions.

Knowledge of the prevalence of latent Mycobacterium tuberculosis infection is crucial for effective tuberculosis control, but tuberculin skin test surveys have major limitations, including poor specificity because of the broad antigenic cross-reactivity of tuberculin. The M. tuberculosis RD1 genomic segment encodes proteins, such as early secretory antigenic target (ESAT)-6, that are absent from M. bovis bacille Calmette-Guérin (BCG) and most environmental mycobacteria. We recently identified circulating ESAT-6-specific T cells as an accurate marker of M. tuberculosis infection. Here, interferon-gamma-secreting T cells specific for peptides derived from ESAT-6 and a second RD1 gene product, CFP10, were enumerated in 100 prospectively recruited healthy adults in Bombay (Mumbai), India. Eighty percent responded to >/=1 antigen, and many donors had high frequencies of T cells that were specific for certain immunodominant peptides. In contrast, of 40 mostly BCG-vaccinated, United Kingdom-resident healthy adults, none responded to either antigen. This study suggests an 80% prevalence of latent M. tuberculosis infection in urban India.

© 2012 Elsevier Ltd. All rights reserved Background: The development of a new, more effective vaccine against tuberculosis (TB) for use in healthy and HIV-infected adults, children and infants, remains a global health priority. MVA85A is a candidate tuberculosis vaccine designed to enhance immunity to the existing vaccine, Bacillus Calmette-Guerin (BCG). MVA85A entered clinical trials in 2002 and has now progressed to Phase IIb proof-of-concept efficacy trials in infants and HIV-infected adults in Africa. Methods: A detailed analysis was conducted of the cumulative safety data of intradermal delivery of MVA85A in 112 healthy adult subjects in a series of open label, single arm, non-controlled, Phase I safety and immunogenicity clinical trials in the UK. The trials differed with respect to previous mycobacterial exposure, vaccine regime and dose. Objective safety measures (local reaction size and body temperature) were evaluated for correlations with adaptive antigen-specific immune responses. Results: All subjects in the combined mid-dose group developed a local reaction, of which 92% were mild, 8% were moderate and no reactions were severe. Around 90% of subjects in each group reported at least one systemic adverse event, most commonly headache, myalgia, malaise, feeling feverish, fatigue and arthralgia. Of all systemic adverse events in the combined mid-dose group, 96% were mild, 3% were moderate and 1% were severe (but none of these were judged to be vaccine-related). Pre-vaccination mycobacterial exposure did not affect the adverse event profile. The size of local reaction and frequency of systemic adverse events increased with MVA85A vaccine dose. There were no documented fevers in the low-dose group, whilst 3% of subjects in the combined mid-dose group and 21% in the high-dose group had documented fevers. Peak local reactions were larger after a second poxvirus vaccination, but other local and systemic adverse events were comparable to a single MVA85A vaccination. No severe systemic AEs or serious adverse events in any group were judged to be vaccine-related. Local AEs compared favourably to BCG vaccine-induced local AE and systemic AEs after MVA85A vaccination were comparable to those after the live viral Yellow Fever vaccine in similar populations. There were no correlations found between local reaction size or body temperature and adaptive immune responses (measured by ex vivo interferon gamma Enzyme Linked Immunospot). Conclusions: The candidate TB vaccine, MVA85A has been safely administered to over 100 healthy adults in the UK. Intradermal vaccination with MVA85A induced a transient, superficial reaction local to the injection site and mild short-lived viral symptoms. The local and systemic AE profile of MVA85A vaccination was comparable to published data of other intradermal vaccines and live viral vaccines respectively. Local reaction sizes and body temperature measurements did not correlate with the adaptive cellular immune response to MVA85A. Funded by charitable grants from Europe Aid; TBVAC (EU 6th Framework Programme); The Oxford Biomedical Research Centre and the Wellcome Trust

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.