T-cell and serological responses to Erp, an exported Mycobacterium tuberculosis protein, in tuberculosis patients and healthy individuals

<p>Abstract</p> <p>Background</p> <p>The identification of antigens able to differentiate tuberculosis (TB) disease from TB infection would be valuable. Cellular and humoral immune responses to Erp (Exported repetitive protein) – a recently identified <it>M. tuberculosis </it>protein – have not yet been investigated in humans and may contribute to this aim.</p> <p>Methods</p> <p>We analyzed the cellular and humoral immune responses to Erp, ESAT-6, Ag85B and PPD in TB patients, in BCG⁺individuals without infection, BCG⁺individuals with latent TB infection (LTBI) and BCG^-controls. We used lymphoproliferation, ELISpot IFN-γ, cytokine production assays and detection of specific human antibodies against recombinant <it>M. tuberculosis </it>proteins.</p> <p>Results</p> <p>We included 22 TB patients, 9 BCG⁺individuals without TB infection, 7 LTBI and 7 BCG^-controls. Erp-specific T cell counts were higher in LTBI than in the other groups. Erp-specific T cell counts were higher in LTBI subjects than TB patients (median positive frequency of 211 SFC/10⁶PBMC (range 118–2000) for LTBI subjects compared to 80 SFC/10⁶PBMC (range 50–191), p = 0.019); responses to PPD and ESAT-6 antigens did not differ between these groups. IFN-γ secretion after Erp stimulation differed between TB patients and LTBI subjects (p = 0.02). Moreover, LTBI subjects but not TB patients or healthy subjects produced IgG3 against Erp.</p> <p>Conclusion</p> <p>The frequencies of IFN-γ-producing specific T cells, the IFN-γ secretion and the production of IgG3 after Erp stimulation are higher in LTBI subjects than in TB patients, whereas PPD and ESAT-6 are not.</p

Longevity and Composition of Cellular Immune Responses Following Experimental Plasmodium falciparum Malaria Infection in Humans

Cellular responses to Plasmodium falciparum parasites, in particular interferon-gamma (IFNγ) production, play an important role in anti-malarial immunity. However, clinical immunity to malaria develops slowly amongst naturally exposed populations, the dynamics of cellular responses in relation to exposure are difficult to study and data about the persistence of such responses are controversial. Here we assess the longevity and composition of cellular immune responses following experimental malaria infection in human volunteers. We conducted a longitudinal study of cellular immunological responses to sporozoites (PfSpz) and asexual blood-stage (PfRBC) malaria parasites in naïve human volunteers undergoing single (n = 5) or multiple (n = 10) experimental P. falciparum infections under highly controlled conditions. IFNγ and interleukin-2 (IL-2) responses following in vitro re-stimulation were measured by flow-cytometry prior to, during and more than one year post infection. We show that cellular responses to both PfSpz and PfRBC are induced and remain almost undiminished up to 14 months after even a single malaria episode. Remarkably, not only ‘adaptive’ but also ‘innate’ lymphocyte subsets contribute to the increased IFNγ response, including αβT cells, γδT cells and NK cells. Furthermore, results from depletion and autologous recombination experiments of lymphocyte subsets suggest that immunological memory for PfRBC is carried within both the αβT cells and γδT compartments. Indeed, the majority of cytokine producing T lymphocytes express an CD45RO+ CD62L- effector memory (EM) phenotype both early and late post infection. Finally, we demonstrate that malaria infection induces and maintains polyfunctional (IFNγ+IL-2+) EM responses against both PfRBC and PfSpz, previously found to be associated with protection. These data demonstrate that cellular responses can be readily induced and are long-lived following infection with P. falciparum, with a persisting contribution by not only adaptive but also (semi-)innate lymphocyte subsets. The implications hereof are positive for malaria vaccine development, but focus attention on those factors potentially inhibiting such responses in the field

Plasmodium falciparum genetic diversity can be characterised using the polymorphic merozoite surface antigen 2 (MSA-2) gene as a single locus marker

The genetic diversity of Solomon Island Plasmodium falciparum isolates was examined using MSA-2 as a single locus marker. Amplification of MSA-2 gene fragments showed size polymorphism and the presence of mixed infections. Sequence analysis indicated a global representation of MSA-2 alleles with representatives of 3D7/CAMP allelic subfamilies and the FCQ-27 allelic family being identified. A simplified method of characterisation, utilising PCR-RFLPs of MSA-2 gene fragments, was developed. The RFLPs allowed identification of allelic families and further distinction within the 3D7/CAMP family. The amplification of MSA-2 gene fragments from culture derived lines revealed a loss of diversity for a number of Solomon Island isolates. Genomic diversity was confirmed for Solomon Island lines, along with Papua New Guinean and Thai lines, by the generation of 7H8/6 fingerprints. All lines were distinct and band sharing frequencies and Wagner tree construction failed to identify any geographic clustering

Experimental human Plasmodium falciparum infections: Longitudinal analysis of lymphocyte responses with particular reference to gamma delta T cells

The kinetics of the gamma delta T-cell response was analysed in the context of the overall haematological response in subjects experimentally infected with sporozoites of Plasmodium falciparum. Numbers of gamma delta and alpha beta T cells and NK cells declined markedly during infection to reach minimum values 12-13 days postinfection when the patients were ill. This decline commenced from the beginning of the erythrocytic cycle and well before parasites could be detected microscopically and clinical symptoms developed. Platelet numbers also declined. In vivo activation of gamma delta T cells was evident with sequential up-regulation of the activation markers CD69 and HLA-DR, gamma delta T cell numbers were highest after treatment with the majority being CD4(-)CD8(-), HLA-DR(+) and showing reduced CD45RA expression. Contrary to some published observations gamma delta T-cell percentages remained within the normal range. Little evidence of upregulation of activation or memory markers was observed in the alpha beta T-cell population. In vitro proliferative responses to malaria antigen which involve gamma delta T cells were lost as the infection progressed and the lymphocyte count declined but these could be restored with the addition of exogenous IL-2 to cultures. The authors findings are consistent with a protective and/or immunomodulatory role for gamma delta T cells in malaria