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Mycobacterium tuberculosis (M.tb) infection results in approximately 1.3 million human deaths each year. M.tb resides primarily inside macrophages, and maintains persistent infection. In response to infection and inflammation, platelet activating factor C-16 (PAF C16), a phospholipid compound, is released by various cells including neutophils and monocytes. We have recently shown that PAF C-16 can directly inhibit the growth of two representative non-pathogenic mycobacteria, Mycobacterium bovis BCG and Mycobacterium smegmatis (M. smegmatis), by damaging the bacterial cell membrane. Here, we have examined the effect of PAF C-16 on M. smegmatis residing within macrophages, and identified mechanisms involved in their growth inhibitory function. Our results demonstrated that exogenous PAF C-16 inhibited the growth of M. smegmatis inside phagocytic cells of monocytic cell line, THP1; this effect was partially blocked by PAF receptor antagonists, suggesting the involvement of PAF receptor-mediated signalling pathways. Arachidonic acid, a downstream metabolite of PAF C-16 signalling pathway, directly inhibited the growth of M. smegmatis in vitro. Moreover, the inhibition of phospholipase C and phospholipase A2 activities, involved in PAF C-16 signalling pathway, increased survival of intracellular M. smegmatis. Interestingly, we also observed that inhibition of inducible nitric oxide synthase (iNOS) enzyme and antibody-mediated neutralization of TNF-α partially mitigated the intracellular growth inhibitory effect of PAF C-16. Use of a number of PAF C-16 structural analogues, including Lyso-PAF, 2-O-methyl PAF, PAF C-18 and Hexanolamino PAF, revealed that the presence of acetyl group (CH3CO) at sn-2 position of the glycerol backbone of PAF is important for the intracellular growth inhibition activity against M. smegmatis. Taken together, these results suggest that exogenous PAF C-16 treatment inhibits intracellular M. smegmatis growth, at least partially, in a nitric oxide and TNF-α dependent manner.

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

Hydrophilic lung surfactant proteins have emerged as key immunomodulators aimed at recognition and clearance of pulmonary pathogens. Surfactant protein A (SP-A) is a surfactant-associated innate immune pattern recognition molecule, which is known to interact with a variety of pathogens, and display anti-microbial effects. SP-A, being carbohydrate pattern recognition molecule, has been suggested to have a wide range of innate immune functions against pathogens. In addition, SP-A can work against respiratory pathogens, including influenza A virus (IAV). Some pandemic pH1N1 strains resist neutralization by SP-A due to differences in the N-glycosylation of viral hemagglutinin (HA). Here, we provide evidence, for the first time, that a recombinant form of human SP-A (rfhSP-A) composed of α-helical neck and carbohydrate recognition domains can actually promote the IAV replication, as observed by an upregulation of M1 expression in lung epithelial cell line, A549, when challenged with pH1N1 and H3N2 IAV subtypes. rfhSP-A (10 μg/ml) bound neuraminidase (NA) (˜60 kDa), matrix protein 1 (M1) (˜25 kDa) and M2 (˜17 kDa) in a calcium dependent manner, as revealed by far western blotting, and direct binding ELISA. However, human full length native SP-A downregulated mRNA expression levels of M1 in A549 cells challenged with IAV subtypes. Furthermore, qPCR analysis showed that transcriptional levels of TNF-α, IL-12, IL-6, IFN-α and RANTES were enhanced following rfhSP-A treatment by both IAV subtypes at 6 h post-IAV infection of A549 lung epithelial cells. In the case of full length SP-A treatment, mRNA expression levels of TNF-α, and IL-6 were downregulated during the mid-to-late stage of IAV infection of A549 cells. Multiplex cytokine/chemokine array revealed enhanced levels of both IL-6 and TNF-α due to rfhSP-A treatment in the case of both IAV subtypes tested, while no significant effect was seen in the case of IL-12. Enhancement of IAV infection of pH1N1 and H3N2 subtypes by truncated rfhSP-A, concomitant with infection inhibition by full-length SP-A, appears to suggest that a complete SP-A molecule is required for protection against IAV. This is in contrast to a recombinant form of trimeric lectin domains of human SP-D (rfhSP-D) that acts as an entry inhibitor of IAV. KACST (14-MED258-20)

Surfactant protein D (SP-D), a C-type collagen containing lectin (collectin), is expressed in the mucosal secretion of the lung and contribute to the innate host defence against a variety of pathogens, including influenza A virus (IAV). SP-D has been shown to inhibit haemagglutination activity and infectivity of IAV, in addition to reducing neuraminidase (NA) activity. SP-D exhibits a strong anti-IAV activity by virtue of its carbohydrate recognition domain (CRD) binding to carbohydrate pattern (N-linked mannosylated) on NA and hemagglutinin (HA) of the IAV. Here, we demonstrate that a recombinant fragment of human SP-D (rfhSP-D), containing homotrimeric neck and CRD regions, acts as an entry inhibitor of IAV and down-regulates M1 expression considerably in A549 cells infected with pH1N1 as well as H3N2 IAV strains at 2h treatment. In addition, rfhSP-D down-regulated mRNA levels of TNF-α, IFN-a, IFN-b, IL-6 and RANTES production, as judged by qPCR, particularly during the initial stage of IAV infection of A549 cell line. rfhSP-D also interfered with IAV infection of Madin-Darby canine kidney (MDCK) cells through HA1 binding, as confirmed by luciferase reporter assay and far western blotting. Furthermore, rfhSP-D was found to reduce luciferase reporter activity of MDCK cells transduced with H1+N1 pseudotyped lentiviral particles in a dose-dependent manner, where 50% of reduction was observed with 10 μg/ml rhfSP-D. Thus, binding of rfhSP-D to HA1 and reduction in luciferase reporter activity are suggestive of a critical role of rfhSP-D in mediating the inhibition of IAV infectivity and that of pesudotyped lentivirus as an entry inhibitor. Multiplex cytokine array revealed that rfhSP-D treatment of IAV challenged A549 cells led to a dramatic suppression of some of the key pro-inflammatory cytokines and chemokines in the virus challenged A549 cells. In the case of pH1N1, soluble factors such as TNF-a, IFN- a, IL-10, IL-12 (p40), VEGF, GM-CSF and eotaxin were considerably suppressed by rfhSP51 D treatment at 24h. However, these suppressive effects of IL-10, VEGF, eotaxin and IL-12 (p40) were not so evident in the case of H3N2 strain at the secreted protein level, with the exception of TNF-a, IFN-a, and GM-CSF. These data seem to suggest that the extent of immunomodulatory effect of SP-D on host cells can vary considerably in a strain-specific manner. Thus, rfhSP-D treatment can downregulate pro-inflammatory milieu encouraged by IAV via aberrant inflammatory cell recruitment leading to cell death and other possible long term immune defects and lung damage.

Mycobacterium tuberculosis can proficiently enter phagocytes and diminish complement 23 activation on its cell surface. Within phagocytes, the mycobacterium can suppress 24 macrophage apoptosis and survive the intracellular environment. Complement regulatory 25 proteins such as factor H may facilitate pathogen-macrophage interactions during 26 tuberculosis infection. In this study, we show that M. bovis BCG binds properdin, an up27 regulator of the complement alternative pathway. TSR4+5, a recombinant form of 28 thrombospondin repeats 4 and 5 of human properdin expressed in tandem, which is an 29 inhibitor of the alternative pathway, was also able to bind M. bovis BCG. Properdin and 30 TSR4+5 were found to inhibit uptake of M. bovis BCG by THP-1 macrophage cells in a 31 dose-dependent manner. Quantitative real-time PCR assays using transcripts from the THP-1 32 cells revealed elevated pro-inflammatory responses (TNF-α, IL-1β and IL-6) in the presence 33 of properdin and TSR4+5, which gradually decreased over 6 hours. Correspondingly, anti34 inflammatory responses (IL-10, TGF-β and IL-12) showed suppressed levels of expression in 35 the presence of properdin, which gradually increased over 6 hours. Multiplex cytokine array 36 analysis further revealed that properdin and TSR4+5 significantly enhanced the pro37 inflammatory response (TNF-α, IL-1β and IL-1α) at 24 hours, which declined at 48 hours, 38 whereas the anti-inflammatory response (IL-10 and IL-12) was suppressed. Our results 39 suggest that properdin may interfere with mycobacterial entry into macrophages involving 40 TSR4 and TSR5, particularly during the initial stages of infection, thus, affecting the 41 extracellular survival of the pathogen. This study offers novel insights into non-complement 42 related functions of properdin, which may be independent of other complement proteins 43 during host-pathogen interactions in tuberculosis. Thus, human properdin modulates 44 macrophage-M. bovis BCG interaction via TSR4+5. Properdin residing in the granules of 45 neutrophils is secreted upon stimulation and may also be produced by other cell types such as 3 monocytes, bone marrow progenitor cell lines and T cells. The local production 46 of properdin 47 may be crucial for recruitment at sites of infection and in the control of M. tuberculosis 48 infection.

Rationale: rfhSP-D has been shown to suppress house dust mite and Aspergillus 74 fumigatus-induced allergic inflammation in murine models. 75 Objectives: We sought to elucidate the effect of rfhSP-D on FcεRI and CD23- 76 mediated grass pollen induced allergic inflammatory responses. 77 Methods: rfhSP-D, containing homotrimeric neck and lectin domains, was 78 expressed in Escherichia coli BL21 (λDE3) pLysS. PBMCs and sera were obtained 79 from grass pollen allergic individuals (n=27). The effect of rfhSP-D on basophil 80 activation and histamine release was measured by flow cytometry. IgE-facilitated 81 allergen binding and presentation was assessed by flow cytometry. Th2 cytokines 82 were measured in cell culture supernatants. The effect of rfhSP-D on IgE production 83 by B cells when stimulated with CD40L, IL-4 and IL-21 was also determined. 84 Results: rfhSP-D bound to Phleum pratense in a dose- and calcium-dependent 85 manner. Allergen-induced basophil responsiveness and histamine release was 86 inhibited in the presence of rfhSP-D, as measured by CD63, CD203c 87 (P=0.0086,P=0.04205), and intracellular-labelled DAO (P=0.0003,P=0.0148). The 88 binding of allergen-IgE complexes to B cells was reduced by 51%(P=0.002) in the 89 presence of rfhSP-D. This decrease was concomitant with reduction in CD23 90 expression on B cells (P

Mycobacterium tuberculosis can proficiently enter macrophages and diminish complement activation on its cell surface. Within macrophages, the mycobacterium can suppress macrophage apoptosis and survive within the intracellular environment. Previously, we have shown that complement regulatory proteins such as factor H may interfere with pathogen–macrophage interactions during tuberculosis infection. In this study, we show that Mycobacterium bovis BCG binds properdin, an upregulator of the complement alternative pathway. TSR4+5, a recombinant form of thrombospondin repeats 4 and 5 of human properdin expressed in tandem, which is an inhibitor of the alternative pathway, was also able to bind to M. bovis BCG. Properdin and TSR4+5 were found to inhibit uptake of M. bovis BCG by THP-1 macrophage cells in a dose-dependent manner. Quantitative real-time PCR revealed elevated pro-inflammatory responses (TNF-α, IL-1β, and IL-6) in the presence of properdin or TSR4+5, which gradually decreased over 6 h. Correspondingly, anti-inflammatory responses (IL-10 and TGF-β) showed suppressed levels of expression in the presence of properdin, which gradually increased over 6 h. Multiplex cytokine array analysis also revealed that properdin and TSR4+5 significantly enhanced the pro-inflammatory response (TNF-α, IL-1β, and IL-1α) at 24 h, which declined at 48 h, whereas the anti-inflammatory response (IL-10) was suppressed. Our results suggest that properdin may interfere with mycobacterial entry into macrophages via TSR4 and TSR5, particularly during the initial stages of infection, thus affecting the extracellular survival of the pathogen. This study offers novel insights into the non-complement related functions of properdin during host–pathogen interactions in tuberculosis.

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.

Due to their shape anisotropy, high aspect ratio magnetic nanoparticles offer many advantages in biomedical applications. For biocompatibility, it is essential to have full control over the dimensions and surface chemistry of the particles. The aim of this study was to synthesize biocompatible nanowires with tuneable dimensions. This was achieved by electrodeposition of Ni in polycarbonate membranes. To ensure biocompatibility, a continuous gold coating was deposited onto the Ni wires by a newly developed electroless deposition method. The coating was analysed using electron microscopy and X-ray diffraction. Magnetic properties, anisotropy and Au film thickness were studied using vibrating sample magnetometry. After biofunctionalization, no significant cytotoxic effects were found in studies involving a diverse range of primary and tumour cells exposed to increasing concentrations of nanowires for up to 7 days. These nanowires may thus be used for in vivo applications such as magnetic drug delivery.

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.

MHC class I-restricted CD8 cytotoxic T lymphocytes (CTL) are essential for protective immunity to Mycobacterium tuberculosis in animal models but their role in humans remains unclear. We therefore studied subjects who had successfully contained M. tuberculosis infection in vivo, i.e. exposed healthy household contacts and individuals with inactive self-healed pulmonary tuberculosis. Using the ELISPOT assay for IFN-gamma, we screened peptides from ESAT-6, a secreted antigen that is highly specific for M. tuberculosis. We identified a novel nonamer epitope: unstimulated peripheral blood-derived CD8 T cells displayed peptide-specific IFN-gamma release ex vivo while CD8 T cell lines and clones exhibited HLA-A68.02-restricted cytolytic activity and recognized endogenously processed antigen. The frequency of CD8 CTL specific for this single M. tuberculosis epitope, 1/2500 peripheral blood lymphocytes, was equivalent to the combined frequency of all IFN-gamma-secreting purified protein derivative-reactive T cells ex vivo. This highly focused CTL response was maintained in an asymptomatic contact over 2 years and is the most potent antigen-specific antimycobacterial CD8 CTL response hitherto described. Thus, human M. tuberculosis-specific CD8 CTL are not necessarily associated with active disease per se. Rather, our results are consistent with a protective role for these ESAT-6-specific CD8 T cells in the long-term control of M. tuberculosis in vivo in humans.

Protective immunity against Mycobacterium tuberculosis depends on the generation of a T(H)1-type cellular immune response, characterized by the secretion of interferon-gamma (IFN-gamma) from antigen-specific T cells. The induction of potent cellular immune responses by vaccination in humans has proven difficult. Recombinant viral vectors, especially poxviruses and adenoviruses, are particularly effective at boosting previously primed CD4(+) and CD8(+) T-cell responses against a number of intracellular pathogens in animal studies. In the first phase 1 study of any candidate subunit vaccine against tuberculosis, recombinant modified vaccinia virus Ankara (MVA) expressing antigen 85A (MVA85A) was found to induce high levels of antigen-specific IFN-gamma-secreting T cells when used alone in bacille Calmette-Guerin (BCG)-naive healthy volunteers. In volunteers who had been vaccinated 0.5-38 years previously with BCG, substantially higher levels of antigen-specific IFN-gamma-secreting T cells were induced, and at 24 weeks after vaccination these levels were 5-30 times greater than in vaccinees administered a single BCG vaccination. Boosting vaccinations with MVA85A could offer a practical and efficient strategy for enhancing and prolonging antimycobacterial immunity in tuberculosis-endemic areas.

The RTS,S/SBAS2 vaccine confers sterile protection against Plasmodium falciparum sporozoite challenge. The mechanisms underlying this are of great interest, yet little is known about the immune effector mechanisms induced by this vaccine. The immune responses induced by RTS,S/SBAS2 were characterized in 10 malaria-naive volunteers. Several epitopes in the circumsporozoite protein (CSP) were identified as targets of cultured interferon (IFN)-gamma-secreting CD4+ T cells. RTS,S-specific IFN-gamma-secreting effector T cells were induced in 8 subjects; this ex vivo response mapped to a single peptide in Th2R. CSP-specific CD8+ cytotoxic T lymphocytes were not detected. RTS, S-specific IFN-gamma production was universal, whereas interleukin-4 and -5 production was rare. RTS,S-specific lymphoproliferative responses and antibodies to CSP were strongly induced in all volunteers. Responses waned with time but were boostable. Thus, RTS, S/SBAS2 is a potent inducer of Th1-type cellular and humoral immunity. These results highlight possible immune mechanisms of protection and have important implications for vaccine design in general.

OBJECTIVES: 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. DESIGN: Phase I open label observational trial, in the UK. Healthy, HIV-negative, BCG vaccinated adults were recruited and vaccinated with BCG. The primary outcome was safety; the secondary outcome was cellular immune responses to antigen 85, overlapping peptides of antigen 85A and tuberculin purified protein derivative (PPD) detected by ex vivo interferon-gamma (IFN-gamma) ELISpot assay and flow cytometry. RESULTS AND CONCLUSIONS: BCG revaccination (BCG-BCG) was well tolerated, and boosting of pre-existing PPD-specific T cell responses was observed. However, when these results were compared with data from a previous clinical trial, where BCG was boosted with MVA85A (BCG-MVA85A), MVA85A induced significantly higher levels (>2-fold) of antigen 85-specific CD4+ T cells (both antigen and peptide pool responses) than boosting with BCG, up to 52 weeks post-vaccination (p = 0.009). To identify antigen 85A-specific CD8+ T cells that were not detectable by ex vivo ELISpot and flow cytometry, dendritic cells (DC) were used to amplify CD8+ T cells from PBMC samples. We observed low, but detectable levels of antigen 85A-specific CD8+ T cells producing IFNgamma (1.5% of total CD8 population) in the BCG primed subjects after BCG boosting in 1 (20%) of 5 subjects. In contrast, in BCG-MVA85A vaccinated subjects, high levels of antigen 85A-specific CD8+ T cells (up to 14% total CD8 population) were observed after boosting with MVA85A, in 4 (50%) of 8 subjects evaluated. In conclusion, revaccination with BCG resulted in modest boosting of pre-existing immune responses to PPD and antigen 85, but vaccination with BCG-MVA85A induced a significantly higher response to antigen 85 and generated a higher frequency of antigen 85A-specific CD8+ T cells. TRIAL REGISTRATION: ClinicalTrials.gov NCT00654316 NCT00427830.

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.

There is limited information on the influence of tumor growth on the expansion of tumor-specific TGF-β-producing CD4+ T cells in humans. α-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 (AFP46–55) and develop into TGF-β-producing CD4+ T cells. In a peptide-specific and dose-dependent manner, AFP46–55 CD4+ T cells produce TGF-β, 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 AFP46–55 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-β-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.

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 CMC-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.

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.

Animal models of tuberculosis point to a protective role for MHC class I-restricted CD8(+) T cells, yet it is unclear how these cells protect or whether such findings extend to humans. Here we report that macrophages infected with Mycobacterium tuberculosis, rapidly process and present an early secreted antigenic target (ESAT-6)-specific HLA class I-restricted CD8(+) T cell epitope. When cocultured with CD8(+) T cells restricted through classical HLA class I molecules the growth of bacilli within macrophages is significantly impaired after 7 days. This slow antimycobacterial activity did not correlate with macrophage lysis but required cell contact. We also found that inhibitors of apoptosis either had no effect or augmented the CD8-mediated suppressive activity, suggesting that an activation signal might be involved. Indeed we show that CD8(+) T cells were able to activate macrophages through receptors that include CD95 (Fas). Consistent with these findings the CD8-mediated suppression of mycobacterial growth was partially reversed by Fas blockade. These data identify a previously unrecognized CD8(+) T cell-mediated mechanism used to control an intracellular infection of macrophages.

In animals, effective immune responses against malignancies and against several infectious pathogens, including malaria, are mediated by T cells. Here we show that a heterologous prime-boost vaccination regime of DNA either intramuscularly or epidermally, followed by intradermal recombinant modified vaccinia virus Ankara (MVA), induces high frequencies of interferon (IFN)-gamma-secreting, antigen-specific T-cell responses in humans to a pre-erythrocytic malaria antigen, thrombospondin-related adhesion protein (TRAP). These responses are five- to tenfold higher than the T-cell responses induced by the DNA vaccine or recombinant MVA vaccine alone, and produce partial protection manifest as delayed parasitemia after sporozoite challenge with a different strain of Plasmodium falciparum. Such heterologous prime-boost immunization approaches may provide a basis for preventative and therapeutic vaccination in humans.

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.

Background 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. Methods Using samples collected during phase I trials with MVA85A across the UK and South Africa we have investigated the relationship between vaccine immunogenicity and IDO using IFN-γ ELISPOT, qPCR and liquid chromatography mass spectrometry. Results We demonstrate an IFN-γ dependent increase in IDO mRNA expression in peripheral blood mononuclear cells (PBMC) following MVA85A vaccination in UK subjects. IDO mRNA correlates positively with the IFN-γ ELISPOT response indicating that vaccine specific induction of IDO in PBMC is unlikely to limit the development of vaccine specific immunity. IDO activity in the serum of volunteers from the UK and South Africa was also assessed. There was no change in serum IDO activity following MVA85A vaccination. However, we observed higher baseline IDO activity in South African volunteers when compared to UK volunteers. In both UK and South African serum samples, baseline IDO activity negatively correlated with vaccine-specific IFN-γ responses, suggesting that IDO activity may impair the generation of a CD4+ T cell memory response. Conclusions Baseline IDO activity was higher in South African volunteers when compared to UK volunteers, which may represent a potential mechanism for the observed variation in vaccine immunogenicity in South African and UK populations and may have important implications for future vaccination strategies. Trial registration Trials are registered at ClinicalTrials.gov; UK cohort NCT00427830, UK LTBI cohort NCT00456183, South African cohort NCT00460590, South African LTBI cohort NCT00480558. Electronic supplementary material The online version of this article (doi:10.1186/s12879-014-0660-7) contains supplementary material, which is available to authorized users.

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.

© 2013 Matsumiya et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited 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. This work was funded by the Wellcome Trust. MM has a Wellcome Trust PhD studentship and HM is a Wellcome Trust Senior Fellow

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.

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.

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., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published