Antigen-specific IFN-γ Responses Research Articles

Synthetic immunotherapy induces HIV virus specific Th1 cytotoxic response and death of an HIV-1 infected human cell line through classic complement activation

BackgroundThis manuscript describes the development of a novel synthetic immunotherapy (HIV-v) composed of four multi-epitope polypeptides targeting conserved regions in the Nef, Rev, Vif and Vpr viral proteins. Immunogenicity and cytotoxicity of HIV-v are discussed.MethodsShort conserved T-cell multi-epitope regions were identified in silico in the HIV proteome. The immunogenicity of the identified HIV-v polypeptides was assessed in vivo by immunisation of C57BLK6 mice transgenic for HLA-A*0201. Splenocytes from immunised animals were exposed in vitro to soluble HIV-v polypeptides or to syngeneic (T1) or allogeneic (Jurkat) cells transfected with these polypeptides. Specific T-cell reactivity was assessed by cell-based IFN-γ ELISA. Virus specific CD3 + CD8+ IFN-γ+ recall responses were also determined by flow cytometry following in vitro exposure of splenocytes from immunised mice to syngeneic (T1) and allogeneic (H9) cells infected with HIV-1 strain IIIB. HIV-v specific antibodies were quantified by ELISA whilst antibody mediated anti-viral immunotherapeutic effect on T1 cells infected with a laboratory adapted and a primary isolate of the HIV-1 virus was assessed in a LDH-based complement mediated lysis assay.ResultsHIV-v elicited antigen-specific IgG and IFN−γ responses against the synthetic polypeptides in the formulation. HIV-v specific T cells recognised polypeptides presented either as soluble antigen or complexed to HLA-A*0201 following natural processing and presentation by syngeneic human T1 cells. Moreover, the CD3 + CD8+ component of the response recognised syngeneic T1 cells naturally infected with HIV-1 in a virus-specific and MHC restricted-manner. The HIV-v specific IgG response was also able to recognise human T1 cells naturally infected with HIV-1 and induce cell death through classic activation of complement.ConclusionsHIV-v induces a vaccine-specific type I immune response characterised by activation of effector CD8+ T cell and antibody responses that recognise and kill human cell lines naturally infected with a laboratory adapted and a primary isolate of the HIV-1 virus. The data supports the hypothesis that alternative HIV protein targets can be effectively used to prime both cellular and antibody immune responses of clinical value in the prevention and treatment of HIV infection.

IL-10 Dependent Suppression of Type 1, Type 2 and Type 17 Cytokines in Active Pulmonary Tuberculosis

BackgroundAlthough Type 1 cytokine responses are considered protective in pulmonary tuberculosis (PTB), their role as well as those of Type 2, 17 and immunoregulatory cytokines in tuberculous lymphadenitis (TBL) and latent tuberculosis (LTB) have not been well studied.Aim and MethodsTo identify cytokine responses associated with pulmonary tuberculosis (TB), TB lymphadenitits and latent TB, we examined mycobacterial antigen-specific immune responses of PTB, TBL and LTB individuals. More specifically, we examined ESAT-6 and CFP-10 induced Type 1, Type 2 and Type 17 cytokine production and their regulation using multiplex ELISA.ResultsPTB individuals exhibited a significantly lower baseline as well as antigen-specific production of Type 1 (IFNγ, TNFα and IL-2); Type 2 (IL-4) and Type 17 (IL-17A and IL-17F) cytokines in comparison to both TBL and LTB individuals. TBL individuals exhibited significantly lower antigen-specific IFNγ responses alone in comparison to LTB individuals. Although, IL-10 levels were not significantly higher, neutralization of IL-10 during antigen stimulation resulted in significantly enhanced production of IFNγ, IL-4 and IL-17A in PTB individuals, indicating that IL-10 mediates (at least partially) the suppression of cytokine responses in PTB.ConclusionPulmonary TB is characterized by an IL-10 dependent antigen-specific suppression of Type 1, Type 2 and Type 17 cytokines, reflecting an important association of these cytokines in the pathogenesis of active TB.

Immunity Against Heterosubtypic Influenza Virus Induced By Adenovirus And MVA Expressing Nucleoprotein And Matrix Protein-1

Alternate prime/boost vaccination regimens employing recombinant replication-deficient adenovirus or MVA, expressing Influenza A virus nucleoprotein and matrix protein 1, induced antigen-specific T cell responses in intradermally (ID) vaccinated mice; with the strongest responses resulting from Ad/MVA immunization. In BALB/C mice the immunodominant response was shifted from the previously identified immunodominant epitope to a novel epitope when the antigen was derived from A/Panama/2007/1999 rather than A/PR/8. Alternate immunization routes did not affect the magnitude of antigen-specific systemic IFN-γ response, but higher CD8+ T-cell IFN-γ immune responses were seen in the bronchoalveolar lavage following intransal (IN) boosting after intramuscular (IM) priming, whilst higher splenic antigen-specific CD8+ T cell IFN-γ was seen following IM boosting. Partial protection against heterologous influenza virus challenge was achieved following either IM/IM or IM/IN but not ID/ID immunization. These data may be of relevance for the design of optimal immunization regimens for human influenza vaccines, especially for influenza-naïve infants.

Oral administration of an adenovirus vector encoding both an avian influenza A hemagglutinin and a TLR3 ligand induces antigen specific granzyme B and IFN-γ T cell responses in humans

PurposeTo test the safety and immunogenicity of an orally delivered avian influenza vaccine. The vaccine has a non-replicating adenovirus type 5 vector backbone which expresses hemagglutinin from avian influenza and a TLR3 ligand as an adjuvant. MethodsForty-two subjects were randomized into 3 groups dosed with either 1×1010, 1×109, or 1×108IU of the vaccine administered in capsules. Twelve subjects were vaccinated with identical capsules containing placebo. A portion of the 1×109 dose group were immunized a second time 4 weeks after the first immunization. The safety of the vaccine was assessed by measuring the frequency and severity of adverse events in placebo versus vaccine treated subjects. IFN-γ and granzyme B ELISpot assays were used to assess immunogenicity. ResultsThe vaccine had a positive safety profile with no treatment emergent adverse events reported above grade 1, and with an adverse event frequency in the treated groups no greater than placebo. Antigen specific cytotoxic and IFN-γ responses were induced in a dose dependent manner and cytotoxic responses were boosted after a second vaccination. ConclusionThis first in man clinical trial demonstrates that an orally delivered adenovirus vectored vaccine can induce immune responses to antigen with a favorable safety profile. Clinical Trial Registration number: NCT01335347.

Mucosal Immunization with Live Attenuated Francisella novicida U112ΔiglB Protects against Pulmonary F. tularensis SCHU S4 in the Fischer 344 Rat Model

The need for an efficacious vaccine against Francisella tularensis is a consequence of its low infectious dose and high mortality rate if left untreated. This study sought to characterize a live attenuated subspecies novicida-based vaccine strain (U112ΔiglB) in an established second rodent model of pulmonary tularemia, namely the Fischer 344 rat using two distinct routes of vaccination (intratracheal [i.t.] and oral). Attenuation was verified by comparing replication of U112ΔiglB with wild type parental strain U112 in F344 primary alveolar macrophages. U112ΔiglB exhibited an LD50>107 CFU compared to the wild type (LD50 = 5×106 CFU i.t.). Immunization with 107 CFU U112ΔiglB by i.t. and oral routes induced antigen-specific IFN-γ and potent humoral responses both systemically (IgG2a>IgG1 in serum) and at the site of mucosal vaccination (respiratory/intestinal compartment). Importantly, vaccination with U112ΔiglB by either i.t. or oral routes provided equivalent levels of protection (50% survival) in F344 rats against a subsequent pulmonary challenge with ∼25 LD50 (1.25×104 CFU) of the highly human virulent strain SCHU S4. Collectively, these results provide further evidence on the utility of a mucosal vaccination platform with a defined subsp. novicida U112ΔiglB vaccine strain in conferring protective immunity against pulmonary tularemia.

Correlation of antigen-specific IFN-γ responses of fresh blood samples from Mycobacterium avium subsp. paratuberculosis infected heifers with responses of day-old samples co-cultured with IL-12 or anti-IL-10 antibodies

Paratuberculosis is a chronic infection of the intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Early stage MAP infection can be detected by measuring cell-mediated immune responses using the interferon gamma (IFN-γ) assay. Whole blood samples are cultured overnight with specific MAP antigens followed by quantification of IFN-γ by ELISA. It is recommended that the time interval from sampling to culture does not exceed eight hours but addition of the co-stimulating cytokine interleukin 12 (IL-12) or anti-IL-10 antibodies to culture have been demonstrated to enhance IFN-γ responses of cultures stimulated with Johnin purified protein derivative (PPDj). Here we examined the correlation of IFN-γ production in response to PPDj and 15 recombinant antigens in day-old blood samples from heifers 10–21 months of age from a MAP infected herd with addition of either recombinant bovine IL-12 or anti-bovine IL-10 antibody with IFN-γ production in sample day samples. IFN-γ responses of sample day samples showed high correlation with responses to some antigens in day-old samples with addition of IL-12 or anti-IL-10 antibodies to cultures, indicating that day-old protocols can be applied as an alternative to the conventional IFN-γ protocol. Immunogenicity of the novel antigens was generally low for day-old samples. The most promising antigen using the day-old protocol with addition of IL-12 was latency protein LATP-2 as correlations, immunogenicity and diagnostic specificity collectively was high. The latency protein LATP-1 was the most promising antigen in the day-old protocol with addition of anti-IL-10 antibodies.

Immunogenicity of eight Mycobacterium avium subsp. paratuberculosis specific antigens in DNA vaccinated and Map infected mice

Mycobacterium avium subsp. paratuberculosis (Map), the etiological agent of chronic enteritis of the small intestine in domestic and wild ruminants, causes substantial losses to livestock industry. Control of this disease is seriously hampered by the lack of adequate diagnostic tools and vaccines. Here we report on the immunogenicity of eight Map specific antigens, i.e. MAP1693c, Ag3, MAP2677c (identified by post-genomic and immunoproteomic analysis of Map secretome) and Ag5, Ag6, MAP1637c, MAP0388 and MAP3743 (identified by bioinformatic in silico screening of the Map genome). Strong, antigen-specific IFN-γ responses were induced in mice vaccinated with plasmid DNA encoding MAP1693c, MAP1637c, MAP0388 and MAP3743. In contrast, T cell responses in Map infected mice were directed preferentially against Ag5 and to a lesser extent against MAP3743. None of the tested DNA vaccines conferred protection against subsequent challenge with Map.

Regulation of Mycobacterium-Specific Mononuclear Cell Responses by 25-Hydroxyvitamin D3

The active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), has been shown to be an important regulator of innate and adaptive immune function. In addition, synthesis of 1,25(OH)2D3 from 25-hydroxyvitamin D3 (25(OH)D3) by the enzyme 1α-hydroxylase in monocytes upon activation by TLR signaling has been found to regulate innate immune responses of monocytes in an intracrine fashion. In this study we wanted to determine what cells expressed 1α-hydroxylase in stimulated peripheral blood mononuclear cell (PBMC) cultures and if conversion of 25(OH)D3 to 1,25(OH)2D3 in PBMC cultures regulated antigen-specific immune responses. Initially, we found that stimulation of PBMCs from animals vaccinated with Mycobacterium bovis (M. bovis) BCG with purified protein derivative of M. bovis (M. bovis PPD) induced 1α-hydroxylase gene expression and that treatment with a physiological concentration of 25(OH)D3 down-regulated IFN-γ and IL-17F gene expression. Next, we stimulated PBMCs from M. bovis BCG-vaccinated and non-vaccinated cattle with M. bovis PPD and sorted them by FACS according to surface markers for monocytes/macrophages (CD14), B cells (IgM), and T cells (CD3). Sorting the PBMCs revealed that 1α-hydroxylase expression was induced in the monocytes and B cells, but not in the T cells. Furthermore, treatment of stimulated PBMCs with 25(OH)D3 down-regulated antigen-specific IFN-γ and IL-17F responses in the T cells, even though 1α-hydroxylase expression was not induced in the T cells. Based on evidence of no T cell 1α-hydroxylase we hypothesize that activated monocytes and B cells synthesize 1,25(OH)2D3 and that 1,25(OH)2D3 down-regulates antigen-specific expression of IFN-γ and IL-17F in T cells in a paracrine fashion.

Identification of T cell epitopes on soluble liver antigen in Chinese patients with auto-immune hepatitis

To identify soluble liver antigen (SLA)-specific dominant epitopes and analyse the correlation between SLA-specific T cell response and the status of the disease. A cross-sectional analysis of SLA-specific T cell responses to 54 overlapping peptides covering the entire SLA sequence was performed using an interferon (IFN)-γ ELISpot assay in 31 patients with auto-immune hepatitis (AIH)-1, 15 patients with primary biliary cirrhosis, 16 hepatitis B virus, seven hepatitis C virus infection and 10 healthy subjects, in order to assess the correlation between SLA-specific T cell responses and the clinical outcome. Soluble liver antigen-specific IFN-γ responses in AIH were significantly more frequent in AIH patients (58.1%) than those in controls (6.7% in PBC, P=0.001; 4.3% in hepatitis B/C, P<0.001 and 0% in healthy subjects, P=0.0015). Among 31 AIH patients, the frequency of recognition and the magnitude of response to SLA peptides in anti-SLA antibody-positive patients were higher and stronger than those negative for anti-SLA antibodies (P=0.02 and 0.037 respectively). We further analysed T-cell restriction and found that six individual SLA peptides (4, 9, 11, 12, 41 and 44) were recognized by CD4 T cells, and the most frequently recognized peptides were peptides 12 (61.1% of participants), followed by peptide 4 and peptide 44 (55.6 and 38.9% respectively). Moreover, a positive association was found between the breadth of recognition of SLA peptides and the indices of liver damage. T cell response to SLA in Chinese patients with AIH is broad and associated with hepatocyte damage.

Immune Responses and Protection of Aotus Monkeys Immunized with Irradiated Plasmodium vivax Sporozoites

A non-human primate model for the induction of protective immunity against the pre-erythrocytic stages of Plasmodium vivax malaria using radiation-attenuated P. vivax sporozoites may help to characterize protective immune mechanisms and identify novel malaria vaccine candidates. Immune responses and protective efficacy induced by vaccination with irradiated P. vivax sporozoites were evaluated in malaria-naive Aotus monkeys. Three groups of six monkeys received two, five, or ten intravenous inoculations, respectively, of 100,000 irradiated P. vivax sporozoites; control groups received either 10 doses of uninfected salivary gland extract or no inoculations. Immunization resulted in the production low levels of antibodies that specifically recognized P. vivax sporozoites and the circumsporozoite protein. Additionally, immunization induced low levels of antigen-specific IFN-γ responses. Intravenous challenge with viable sporozoites resulted in partial protection in a dose-dependent manner. These findings suggest that the Aotus monkey model may be able to play a role in preclinical development of P. vivax pre-erythrocytic stage vaccines.

Early Immune Markers Associated withMycobacterium aviumsubsp.paratuberculosisInfection in a Neonatal Calf Model

The objective of this study was to observe early markers of cell-mediated immunity in naïve calves infected with Mycobacterium avium subsp. paratuberculosis and how expression of these markers evolved over the 12-month period of infection. Groups for experimental infection included control (noninfected), oral (infected orally with M. avium subsp. paratuberculosis strain K-10), oral/DXM (pretreatment with dexamethasone before oral inoculation), intraperitoneal (i.p.) inoculation, and oral/M (oral inoculation with mucosal scrapings from a cow with clinical disease) groups. One of the earliest markers to emerge was antigen-specific gamma interferon (IFN-γ). Only i.p. inoculated calves had detectable antigen-specific IFN-γ responses at 7 days, with responses of the other infection groups becoming detectable at 90 and 120 days. All infection groups maintained robust IFN-γ responses for the remainder of the study. At 1 month, calves in the oral and oral/M groups had higher antigen-stimulated interleukin-10 (IL-10) levels than calves in the other treatment groups, but IL-10 secretion declined by 12 months for all calves. T-cell activation markers such as CD25, CD26, CD45RO, and CD5 were significantly upregulated in infected calves compared to noninfected controls. Oral inoculation of calves resulted in significantly increased antigen-specific lymphocyte proliferation at 9 and 12 months, as well as inducible nitric oxide synthase (iNOS) secretion at 6 and 12 months. These results demonstrate that infection of naïve calves with M. avium subsp. paratuberculosis invoked early immunologic responses characterized by robust antigen-specific IFN-γ responses and induction of CD25 and CD45RO expression on T-cell subsets. These were followed by antigen-specific lymphocyte proliferation, iNOS secretion, and expression of CD26 and CD5(bright) markers in the latter part of the 12-month study.

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

ObjectivesControl 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...

Co-incubation with IL-18 potentiates antigen-specific IFN-γ response in a whole-blood stimulation assay for measurement of cell-mediated immune responses in pigs experimentally infected with Lawsonia intracellularis

The whole-blood interferon-gamma (IFN-γ) assay is a quantitative in vitro assay for a direct read-out of Ag-specific cell-mediated immune (CMI) responses to infectious diseases. The IFN-γ assay is robust in severe intracellular infections like Brucella or mycobacteria, but more difficult to evaluate for less severe or immunocompromising infections. Here we investigated the performance of the assay when recombinant co-stimulatory cytokines IL-12 and/or IL-18 were added along with Ag or PBS to cultures of whole-blood from pigs infected with Lawsonia intracellularis. In pigs recovering from a natural infection, addition of rIL-12 or rIL-18 alone increased the Ag-specific IFN-γ release while addition of both cytokines resulted in increased IFN-γ release also in PBS cultures. In analyses after experimental infections with L. intracellularis, significant increased levels of Ag-specific IFN-γ production were measured in Ag + rIL-18 cultures from infected pigs compared to the background response in PBS + rIL-18 control samples ( p < 0.01) or to Ag + rIL-18 cultures from non-inoculated control pigs ( p < 0.05). Flow cytometry identified two lymphocyte subsets as the Ag-specific IFN-γ producers. The highest IFN-γ production was by CD4 +CD8 + cells while a more numerous population of CD4 −CD8 + cells produced lower amounts of IFN-γ in response to rIL-18 and L. intracellularis Ag.

Immunogenicity and protective efficacy of an elastase-dependent live attenuated swine influenza virus vaccine administered intranasally in pigs

Influenza A virus is an important respiratory pathogen of swine that causes significant morbidity and economic impact on the swine industry. Vaccination is the first choice for prevention and control of influenza infections. Live attenuated influenza vaccines (LAIV) are approved for use in humans and horses and their application provides broad protective immunity, however no LAIV against swine influenza virus (SIV) exists in the market. Previously we reported that an elastase-dependant mutant SIV A/Sw/Sk-R345V (R345V) derived from A/Sw/Saskatchewan/18789/02 (H1N1) (SIV/Sk02) is highly attenuated in pigs. Two intratracheal administrations of R345V induced strong cell-mediated and humoral immune responses and provided a high degree of protection to antigenically different SIV infection in pigs. Here we evaluated the immunogenicity and the protective efficacy of R345V against SIV infection by intranasal administration, the more practical route for vaccination of pigs in the field. Our data showed that intranasally administered R345V live vaccine is capable of inducing strong antigen-specific IFN-γ response from local tracheo-bronchial lymphocytes and antibody responses in serum and respiratory mucosa after two applications. Intranasal vaccination of R345V provided pigs with complete protection not only from parental wild type virus infection, but also from homologous antigenic variant A/Sw/Indiana/1726/88 (H1N1) infection. Moreover, intranasal administration of R345V conferred partial protection from heterologous subtypic H3N2 SIV infection in pigs. Thus, R345V elastase-dependent mutant SIV can serve as a live vaccine against antigenically different swine influenza viruses in pigs.

Boosting with a DNA vaccine expressing ESAT-6 (DNAE6) obliterates the protection imparted by recombinant BCG (rBCGE6) against aerosol Mycobacterium tuberculosis infection in guinea pigs

Owing to its highly immunodominant nature and ability to induce long-lived memory immunity, ESAT-6, a prominent antigen of Mycobacterium tuberculosis, has been employed in several approaches to develop tuberculosis vaccines. Here, for the first time, we combined ESAT-6 based recombinant BCG (rBCG) and DNA vaccine (DNAE6) in a prime boost approach. Interestingly, in spite of inducing an enhanced antigen specific IFN-γ response in mice, a DNAE6 booster completely obliterated the protection imparted by rBCG against tuberculosis in guinea pigs. Analysis of immunopathology and cytokine responses suggests involvement of an exaggerated immunity behind the lack of protection imparted by this regimen.

Rag deletion in peripheral T cells prevents TCR revision (82.10)

Abstract In Vβ5 TCR transgenic (Tg) mice, CD4+Vβ5+ T cells are reactive to a peripherally expressed endogenous superantigen. Most self-reactive CD4+ T cells are deleted in the periphery, but through the process of TCR revision, some lose Vβ5 surface expression, re-express Rag genes, undergo rearrangement of endogenous TCRβ, and express a newly formed TCR. As Vβ5 Tg mice age, TCR revision results in the accumulation of post-revision CD4+TCRβ+Vβ5- cells. We now show that post-revision CD4+ T cells undergo extensive homeostatic proliferation in lymphopenic hosts. MHC drives maximal proliferation. Additionally, post-revision CD4+ T cells in Vβ5 Tg mice generate an antigen-specific IFNγ response to Listeria infection. To further investigate the importance of TCR revision, we devised a strategy for removing the Rag2 gene in peripheral T cells. Mice bearing a floxed Rag2 gene and a Cre transgene driven by the distal lck promoter were crossed to ROSA26 YFP reporter mice, in which YFP reports Cre activity. Adoptive transfer of sorted Vβ5+YFP+ T cells from control Rag+/+ mice results in the generation of post-revision T cells, while Vβ5+YFP+ T cells from Ragfl/fl mice remain Vβ5+. Our results illustrate that TCR revision contributes to the ongoing immunocompetence of Vβ5 Tg mice, and that Rag expression in peripheral T cells is necessary for the generation of Vβ5-CD4+ T cells in Vβ5 Tg mice. This work was funded by NIH RO1 Grant #AG13078.

Evaluation of immune responses and protective efficacy in a goat model following immunization with a coctail of recombinant antigens and a polyprotein of Mycobacterium avium subsp. paratuberculosis

The protective efficacy of four recombinant antigens (85A, 85B, superoxide dismutase [SOD], and a fusion polypeptide [Map74F]) of Mycobacterium avium subsp. paratuberculosis (MAP) along with the adjuvant dimethydioctadecyl ammonium bromide (DDA) was assessed in a goat challenge model. Animals were immunized with the four antigens with adjuvant DDA (Group I, eight goat kids) or without the adjuvant (Group II, eight goat kids) or adjuvant only (Group III, nine goat kids). Animals were boostered 3 weeks after the primary vaccination and challenged 3 weeks after the booster. Significant antigen-specific lymphoproliferation was observed in the immunized animals 3 weeks after the booster immunization. This response increased further at 4 weeks after the booster. Similarly, antigen-specific IFN-γ responses increased in the immunized animals 3 weeks after the booster. The response was significantly higher for 85A and Map74F at 10 weeks after primary vaccination (APV) in Group I animals compared to the other two groups. CD4 + T-cell populations were higher in the vaccinated animals from 6 to 10 weeks APV than those of the control animals. A significant increase in recombinant antigen-specific IFN-γ gene expression was detected in the vaccinated animals. At necropsy (38 weeks APV), our multicomponent subunit vaccine imparted a significant protection in terms of reduction of MAP burden in target organs as compared to sham-immunized goats. This study indicates that our multicomponent subunit vaccine induced a good Th1 response and conferred protection against MAP infection in a goat challenge model.

Hepatitis C Virus-Specific T-Cell Gamma Interferon and Proliferative Responses Are More Common in Perihepatic Lymph Nodes than in Peripheral Blood or Liver

The activation state, differentiation state, and functions of liver lymphocytes and perihepatic lymph nodes during chronic hepatitis C virus (HCV) infection are not well understood. Here, we performed phenotypic and functional analyses of freshly prepared lymphocytes isolated from the livers, perihepatic lymph nodes, and peripheral blood compartments of chronic HCV-infected and disease control subjects with end-stage liver disease undergoing liver transplantation. We measured lymphocyte subset frequency and memory T-cell gamma interferon (IFN-gamma) and proliferative responses to HCV peptide and control viral antigens in direct ex vivo assays. We found higher frequencies of CD4 cells in the lymph node compartment than in the other compartments for both HCV-infected and disease control subjects. Lymph node CD4 and CD8 cells less commonly expressed the terminal differentiation marker CD57, a finding consistent with an earlier differentiation state. In HCV-infected subjects, HCV-specific IFN-gamma-producing and proliferative responses were commonly observed in the lymph node fraction, while they were uncommonly observed in the peripheral blood or liver fractions. In contrast, control viral CD4 protein antigen and CD8 peptide antigen-specific IFN-gamma responses were commonly observed in the periphery and uncommonly observed in the lymph nodes of these same subjects. These findings are consistent with a selective defect in HCV-specific T-cell effector function or distribution in patients with advanced chronic HCV infection. The high frequency of HCV-reactive T cells in perihepatic lymph nodes indicates that a failure to generate or sustain T-lymphocyte HCV reactivity is not responsible for the paucity of functional cells even in end-stage liver disease.

The LTK63 adjuvant improves protection conferred by Ag85B DNA-protein prime-boosting vaccination against Mycobacterium tuberculosis infection by dampening IFN-γ response

T helper type-1 response is essential to control Mycobacterium tuberculosis (MTB) infection but excessive antigen-mediated inflammation concurs to pathology. In mice challenged with MTB, the protection elicited by an Ag85B-encoding DNA vaccine, was lost when mice were boosted with Ag85B-protein in the absence of adjuvant. This effect was due to the expansion of a set of IFN-γ secreting-CD4 + T cells highly responsive to Ag85B-protein but which lost the ability to interact with MTB-infected macrophages and control MTB growth. Ag85B-protein co-administration with the adjuvant LTK63 reduced the expansion of Ag85B-protein-responding CD4 + T cells and allowed the survival of those protective Ag85B-specific CD4 + T cells induced by the Ag85B-encoding DNA vaccine. Consequently, the protection against MTB-infection was restored. LTK63 caused also a marked augmentation of Ag85B-specific antibodies, in particular those belonging to the IgG2b isotype. The recovery of protection through a down-modulation of antigen-specific IFN-γ response by an adjuvant is a novel finding which could be of relevance in tuberculosis vaccination.

Adaptation of IFN-gamma ELISA and ELISPOT tests for feline tuberculosis

There are currently no reliable immunodiagnostic tests for feline tuberculosis. Infection of domestic cats in the UK is thought to occur via their contact with the relevant reservoir of infection, e.g. cattle and badgers for Mycobacterium bovis, and rodents for M. microti. In the African National Parks, where M. bovis infection of Bovidae is an increasing problem, transmission to big cats is occurring via their ingestion of infected carcasses. We have adapted feline ELISA and ELISPOT assays to potentially provide the first cell-based diagnostic test for the detection of tuberculosis in cats. We tested peripheral blood mononuclear cell antigen-specific IFN-γ responses of 18 cats suspected of mycobacterial infection for which biopsy material was co-submitted to the Veterinary Laboratories Agency for mycobacterial culture and identification. Seventeen cats were tested by ELISA while seven cats were tested by ELISPOT (six cats were tested by both ELISA and ELISPOT). Six healthy control cats provided baseline data for these tests. Responses to bovine and avian tuberculins (PPDB and PPDA) and a protein cocktail of ESAT6 and CFP10 were measured, together with positive mitogen (PMA and calcium ionophore) and negative (medium) controls. Overall, both ELISPOT and ELISA tests were found to be suitable for generating rapid results (2 and 4 days, respectively), which provided good predictive information for M. bovis and M. microti infections, but were unable to reliably discern M. avium infection.