Malaria seroprevalence in blood bank donors from endemic and non-endemic areas of Venezuela

In Venezuela, a total of 363,466 malaria cases were reported between 1999-2009. Several states are experiencing malaria epidemics, increasing the risk of vector and possibly transfusion transmission. We investigated the risk of transfusion transmission in blood banks from endemic and non-endemic areas of Venezuela by examining blood donations for evidence of malaria infection. For this, commercial kits were used to detect both malaria-specific antibodies (all species) and malaria antigen (Plasmodium falciparum only) in samples from Venezuelan blood donors (n = 762). All samples were further studied by microscopy and polymerase chain reaction (PCR). The antibody results showed that P. falciparum-infected patients had a lower sample/cut-off ratio than Plasmodium vivax-infected patients. Conversely, a higher ratio for antigen was observed among all P. falciparum-infected individuals. Sensitivity and specificity were higher for malarial antigens (100 and 99.8%) than for antibodies (82.2 and 97.4%). Antibody-positive donors were observed in Caracas, Ciudad Bolívar, Puerto Ayacucho and Cumaná, with prevalences of 1.02, 1.60, 3.23 and 3.63%, respectively. No PCR-positive samples were observed among the donors. However, our results show significant levels of seropositivity in blood donors, suggesting that more effective measures are required to ensure that transfusion transmission does not occur.

An experimental protocol for the establishment of dogs with long-term cellular immune reactions to Leishmania antigens

Domestic dogs are considered to be the main reservoirs of zoonotic visceral leishmaniasis. In this work, we evaluated a protocol to induce Leishmania infantum/Leishmania chagasi-specific cellular and humoral immune responses in dogs, which consisted of two injections of Leishmania promastigote lysate followed by a subcutaneous inoculation of viable promastigotes. The primary objective was to establish a canine experimental model to provide positive controls for testing immune responses to Leishmania in laboratory conditions. After inoculation of viable promastigotes, specific proliferative responses of peripheral blood mononuclear cells (PBMCs) to either Leishmania lysate or recombinant proteins, the in vitro production of interferon-γ by antigen-stimulated PBMCs and a significant increase in circulating levels of anti-Leishmania antibodies were observed. The immunized dogs also displayed positive delayed-type hypersensitivity reactions to Leishmania crude antigens and to purified recombinant proteins. An important finding that supports the suitability of the dogs as positive controls is that they remained healthy for the entire observation period, i.e., more than seven years after infection. Following the Leishmania antigen lysate injections, the infection of dogs by the subcutaneous route appears to induce a sustained cellular immune response, leading to an asymptomatic infection. This provides a useful model for both the selection of immunogenic Leishmania antigens and for immunobiological studies on their possible immunoprotective activities.

Schistosoma mansoni triggers Dectin-2, which activates the Nlrp3 inflammasome and alters adaptive immune responses.

The propensity of helminths, such as schistosomes, to immunomodulate the host's immune system is an essential aspect of their survival. Previous research has demonstrated how soluble schistosomal egg antigens (SEA) dampen TLR-signaling during innate immune responses. We show here that the suppressive effect by SEA on TLR signaling is simultaneously coupled to the activation of the Nlrp3 (NLR family, pyrin domain containing 3) inflammasome and thus IL-1β production. Therefore, the responsible protein component of SEA contains the second signal that is required to trigger proteolytic pro-IL-1β processing. Moreover, the SEA component binds to the Dectin-2/FcRγ (Fc receptor γ chain) complex and activates the Syk kinase signaling pathway to induce reactive oxygen species and potassium efflux. As IL-1β has been shown to be an essential orchestrator against several pathogens we studied the in vivo consequences of Schistosoma mansoni infection in mice deficient in the central inflammasome adapter ASC and Nlrp3 molecule. These mice failed to induce local IL-1β levels in the liver and showed decreased immunopathology. Interestingly, antigen-specific Th1, Th2, and Th17 responses were down-regulated. Overall, these data imply that component(s) within SEA induce IL-1β production and unravel a crucial role of Nlrp3 during S. mansoni infection.

Identification of candidate antigens from adult stages of Toxocara canis for the serodiagnosis of human toxocariasis

In the present work, we identified adult Toxocara canis antigens through sodium dodecyl sulfate-polyacrylamide gel electrophoresis for potential use in human toxocariasis immunodiagnosis. The sensitivity and specificity of several semi-purified antigens, as well as their cross-reactivity with other parasitic infections, were assessed by IgM and IgG-enzime linked immunosorbent assay. Whilst we found that the crude extract of the parasite presented limited sensitivity, specificity and high cross-reactivity against other parasites, we identified 42, 58, 68 and 97-kDa semi-purified antigens as the most promising candidates for immunodiagnosis. Moreover, the 58 and 68-kDa antigens presented the lowest IgM cross-reactivity. When tested as a combination, a mixture of the 58 and 68-kDa antigens presented 100% sensitivity and specificity, as well as minor cross-reactivity. Although the combination of the 42, 58, 68 and 97-kDa antigens presented 100% sensitivity at a dilution of 1:40, the low specificity and high cross-reactivity observed suggested a limited use for diagnostic purposes. Our data suggested that the 58 and 68-kDa antigens might be most suitable for the immunodiagnosis of human toxocariasis.

Human CD8(+) T cells engineered with supraphysiological TCRs are functionally inhibited via PD-1 and SHP-1 phosphatase

Purpose/Objective: Protective CD8+ T cell responses rely on TCRdependent recognition of immunogenic peptides presented by MHC I. Cytolytic T lymphocytes directed against self/tumor antigens express TCRs of lower affinity/avidity than pathogen-derived T lymphocytes and elicit less protective immune responses due to mechanisms of central and peripheral tolerance. Anti-tumor T cell reactivity can be improved by increasing the TCR-pMHC affinity within physiological limits, while intriguingly further increase in the supraphysiological range (KD < 1 lM) leads to drastic functional declines. We aim at identifying the molecular mechanisms underlying the loss of T cell responsiveness associated with supraphysiological TCRpMHC affinities in order to improve effectiveness of TCR-engineered T cells used in adoptive cell transfer (ACT) cancer immunotherapy. Materials and methods: Using a panel of human CD8+ T cells engineered with TCRs of incremental affinity for the HLA-A2-resticted tumor cancer testis antigen NY-ESO-1, we performed comparative gene expression microarray and TCR-mediated signaling analysis together with membrane receptors level analysis. Results: As compared to cells expressing TCR affinities generating optimal function (KD from 5to 1 lM), those with supraphysiological affinity (KD from 1 lM to 15 nM) had an overall reduced expression of genes implied in signaling, cell activation and proliferation, and showed impaired proximal and distal TCR signaling capacity. This correlated with a decline in surface expression of CD8b, CD28 and activatory TNFR superfamily members. Importantly, expression of inhibitory receptor PD-1 and SHP-1 phosphatase was upregulated in a TCR affinity-dependent manner. Consequently, PD-L1 and SHP-1 blockade restored the function of T cells with high TCRs affinity. Moreover, SHP-1 inhibition also augmented functional efficacy of T cells with TCRs of optimal affinity. Conclusions: Our findings indicate that TCR affinity-associated regulatory mechanisms control T cells responsiveness at various levels to limit potential auto-reactive cytotoxic effects. They also support the development of ACT therapies combined with blockade of inhibitory molecules such as SHP-1 to enhance effectiveness of T cell immunotherapy.

The NK cell receptor repertoire: formation, adaptation and exploitation.

The identification of NK cell receptors specific for MHC class I molecules has greatly improved our knowledge of NK cell reactivity and specificity. Inhibitory receptors prevent NK cell activation directed against cells expressing self-MHC class I molecules. Consequently, diseased cells that do not express self-MHC class I molecules become susceptible to NK cell-mediated attack. Because of the specificity and distribution of inhibitory NK cell receptors, cells that express non-self (allogeneic) MHC class I molecules are also susceptible to NK cell reactions. This feature has been exploited in a clinical setting to treat leukemia patients.

Methods for the ex vivo characterization of human CD8+ T subsets based on gene expression and replicative history analysis.

The generation of an antigen-specific T-lymphocyte response is a complex multi-step process. Upon T-cell receptor-mediated recognition of antigen presented by activated dendritic cells, naive T-lymphocytes enter a program of proliferation and differentiation, during the course of which they acquire effector functions and may ultimately become memory T-cells. A major goal of modern immunology is to precisely identify and characterize effector and memory T-cell subpopulations that may be most efficient in disease protection. Sensitive methods are required to address these questions in exceedingly low numbers of antigen-specific lymphocytes recovered from clinical samples, and not manipulated in vitro. We have developed new techniques to dissect immune responses against viral or tumor antigens. These allow the isolation of various subsets of antigen-specific T-cells (with major histocompatibility complex [MHC]-peptide multimers and five-color FACS sorting) and the monitoring of gene expression in individual cells (by five-cell reverse transcription-polymerase chain reaction [RT-PCR]). We can also follow their proliferative life history by flow-fluorescence in situ hybridization (FISH) analysis of average telomere length. Recently, using these tools, we have identified subpopulations of CD8+ T-lymphocytes with distinct proliferative history and partial effector-like properties. Our data suggest that these subsets descend from recently activated T-cells and are committed to become differentiated effector T-lymphocytes.

Identification of Schistosoma mansoni candidate antigens for diagnosis of schistosomiasis

The development of a more sensitive diagnostic test for schistosomiasis is needed to overcome the limitations of the use of stool examination in low endemic areas. Using parasite antigens in enzyme linked immunosorbent assay is a promising strategy, however a more rational selection of parasite antigens is necessary. In this study we performed in silico analysis of the Schistosoma mansoni genome, using SchistoDB database and bioinformatic tools for screening immunogenic antigens. Based on evidence of expression in all parasite life stage within the definitive host, extracellular or plasmatic membrane localization, low similarity to human and other helminthic proteins and presence of predicted B cell epitopes, six candidates were selected: a glycosylphosphatidylinositol-anchored 200 kDa protein, two putative cytochrome oxidase subunits, two expressed proteins and one hypothetical protein. The recognition in unidimensional and bidimensional Western blot of protein with similar molecular weight and isoelectric point to the selected antigens by sera from S. mansoni infected mice indicate a good correlation between these two approaches in selecting immunogenic proteins.

Schistosoma mansoni antigens alter the cytokine response in vitro during cutaneous leishmaniasis

Schistosoma mansoni infection or associated products are able to down-modulate the type 1 CD4+ T cell inflammatory response characteristic of autoimmune diseases. In this study, we evaluated how S. mansoni antigens altered the immune response that was induced by the soluble Leishmania antigen (SLA) from cutaneous leishmaniasis (CL) patients. Cytokines were measured from the supernatants of peripheral blood mononuclear cell cultures stimulated with SLA. This was performed using the sandwich enzyme linked immunosorbent assay technique in the presence or absence of S. mansoni recombinant antigens Sm29, SmTSP-2 and PIII. The addition of S. mansoni antigens to the cultures resulted in the reduction of interferon gamma (IFN-γ) levels in 37-50% of patients. Although to a lesser extent, the antigens were also able to decrease the production of tumour necrosis factor-alpha (TNF-α). We compared patients that either had or did not have reduction in IFN-γ and TNF-α production in cultures stimulated with SLA in the presence of S. mansoni antigens. We found that there was no significant difference in the levels of interleukin (IL)-10 and IL-5 in response to S. mansoni antigens between the groups. The antigens used in this study down-modulated the in vitro proinflammatory response induced by SLA in a group of CL patients through a currently undefined mechanism.

DRB1*03:01 haplotypes: differential contribution to multiple sclerosis risk and specific association with the presence of intrathecal IgM bands.

BACKGROUND Multiple sclerosis (MS) is a multifactorial disease with a genetic basis. The strongest associations with the disease lie in the Human Leukocyte Antigen (HLA) region. However, except for the DRB1*15:01 allele, the main risk factor associated to MS so far, no consistent effect has been described for any other variant. One example is HLA-DRB1*03:01, with a heterogeneous effect across populations and studies. We postulate that those discrepancies could be due to differences in the diverse haplotypes bearing that allele. Thus, we aimed at studying the association of DRB1*03:01 with MS susceptibility considering this allele globally and stratified by haplotypes. We also evaluated the association with the presence of oligoclonal IgM bands against myelin lipids (OCMB) in cerebrospinal fluid. METHODS Genotyping of HLA-B, -DRB1 and -DQA1 was performed in 1068 MS patients and 624 ethnically matched healthy controls. One hundred and thirty-nine MS patients were classified according to the presence (M+, 58 patients)/absence (M-, 81 patients) of OCMB. Comparisons between groups (MS patients vs. controls and M+ vs. M-) were performed with the chi-square test or the Fisher exact test. RESULTS Association of DRB1*03:01 with MS susceptibility was observed but with different haplotypic contribution, being the ancestral haplotype (AH) 18.2 the one causing the highest risk. Comparisons between M+, M- and controls showed that the AH 18.2 was affecting only M+ individuals, conferring a risk similar to that caused by DRB1*15:01. CONCLUSIONS The diverse DRB1*03:01-containing haplotypes contribute with different risk to MS susceptibility. The AH 18.2 causes the highest risk and affects only to individuals showing OCMB.

Molecular markers and genetic diversity of Plasmodium vivax

Enhanced understanding of the transmission dynamics and population genetics for Plasmodium vivax is crucial in predicting the emergence and spread of novel parasite phenotypes with major public health implications, such as new relapsing patterns, drug resistance and increased virulence. Suitable molecular markers are required for these population genetic studies. Here, we focus on two groups of molecular markers that are commonly used to analyse natural populations of P. vivax. We use markers under selective pressure, for instance, antigen-coding polymorphic genes, and markers that are not under strong natural selection, such as most minisatellite and microsatellite loci. First, we review data obtained using genes encoding for P. vivax antigens: circumsporozoite protein, merozoite surface proteins 1 and 3α, apical membrane antigen 1 and Duffy binding antigen. We next address neutral or nearly neutral molecular markers, especially microsatellite loci, providing a complete list of markers that have already been used in P. vivax populations studies. We also analyse the microsatellite loci identified in the P. vivax genome project. Finally, we discuss some practical uses for P. vivax genotyping, for example, detecting multiple-clone infections and tracking the geographic origin of isolates.

Monoclonal auto-antibodies and sera of autoimmune patients react with Plasmodium falciparum and inhibit its in vitro growth

The relationship between autoimmunity and malaria is not well understood. To determine whether autoimmune responses have a protective role during malaria, we studied the pattern of reactivity to plasmodial antigens of sera from 93 patients with 14 different autoimmune diseases (AID) who were not previously exposed to malaria. Sera from patients with 13 different AID reacted against Plasmodium falciparum by indirect fluorescent antibody test with frequencies varying from 33-100%. In addition, sera from 37 AID patients were tested for reactivity against Plasmodium yoelii 17XNL and the asexual blood stage forms of three different P. falciparum strains. In general, the frequency of reactive sera was higher against young trophozoites than schizonts (p < 0.05 for 2 strains), indicating that the antigenic determinants targeted by the tested AID sera might be more highly expressed by the former stage. The ability of monoclonal auto-antibodies (auto-Ab) to inhibit P. falciparum growth in vitro was also tested. Thirteen of the 18 monoclonal auto-Ab tested (72%), but none of the control monoclonal antibodies, inhibited parasite growth, in some cases by greater than 40%. We conclude that autoimmune responses mediated by auto-Ab may present anti-plasmodial activity.

Platform for Plasmodium vivax vaccine discovery and development

Plasmodium vivax is the most prevalent malaria parasite on the American continent. It generates a global burden of 80-100 million cases annually and represents a tremendous public health problem, particularly in the American and Asian continents. A malaria vaccine would be considered the most cost-effective measure against this vector-borne disease and it would contribute to a reduction in malaria cases and to eventual eradication. Although significant progress has been achieved in the search for Plasmodium falciparum antigens that could be used in a vaccine, limited progress has been made in the search for P. vivax components that might be eligible for vaccine development. This is primarily due to the lack of in vitro cultures to serve as an antigen source and to inadequate funding. While the most advanced P. falciparum vaccine candidate is currently being tested in Phase III trials in Africa, the most advanced P. vivax candidates have only advanced to Phase I trials. Herein, we describe the overall strategy and progress in P. vivax vaccine research, from antigen discovery to preclinical and clinical development and we discuss the regional potential of Latin America to develop a comprehensive platform for vaccine development.

Synergism/complementarity of recombinant adenoviral vectors and other vaccination platforms during induction of protective immunity against malaria

The lack of immunogenicity of most malaria antigens and the complex immune responses required for achieving protective immunity against this infectious disease have traditionally hampered the development of an efficient human malaria vaccine. The current boom in development of recombinant viral vectors and their use in prime-boost protocols that result in enhanced immune outcomes have increased the number of malaria vaccine candidates that access pre-clinical and clinical trials. In the frontline, adenoviruses and poxviruses seem to be giving the best immunization results in experimental animals and their mutual combination, or their combination with recombinant proteins (formulated in adjuvants and given in sequence or being given as protein/virus admixtures), has been shown to reach unprecedented levels of anti-malaria immunity that predictably will be somehow reproduced in the human setting. However, all this optimism was previously seen in the malaria vaccine development field without many real applicable results to date. We describe here the current state-of-the-art in the field of recombinant adenovirus research for malaria vaccine development, in particular referring to their use in combination with other immunogens in heterologous prime-boost protocols, while trying to simultaneously show our contributions and point of view on this subject.

Characterization of the immunological properties of " Plasmodium falciparum " and " Plasmodium berghei " circumsporozoite proteins : antibody responses and recognition by specific CD8+ T cells

SUMMARY Interest in developing intervention strategies against malaria by targeting the liver stage of the Plasmodium life cycle has been fueled by studies which show that sterile protective immunity can be achieved by immunization with radiation-attenuated sporozoites. Anti-malarial drugs and insecticides have been widely used to control the disease, but in the hope of developing a more cost-effective intervention strategy, vaccine development has taken centre stage in malaria research. There is currently no vaccine against malaria. Attenuated sporozoite-induced immunity is achieved by antibodies and T cells against malaria liver stage antigens, the most abundant being the circumsporozoite protein (CSP), and many vaccine formulations aim at mimicking this immunity. However, the mechanisms by which the antibody and T cell immune responses are generated after infection by sporozoites, or after immunization with different vaccine formulations are still not well understood. The first part of this work aimed at determining the ability of primary hepatocytes from BALB/c mice to process and present CSP-derived peptides after infection with P. berghei sporozoites. Both infected hepatocytes and those traversed by sporozoites during migration were found to be capable of processing and presenting the CSP to specific CD8+ T cells in vitro. The pathway of processing and presentation involved the proteasome, aspartic proteases and transport through a post-Endoplasmic Reticulum (ER) compartment. These results suggest that in vivo, infected hepatocytes contribute to the elicitation and expansion of a T cell response. In the second part, the antibody responses of CB6F1 mice to synthetic peptides corresponding to the N- and C-terminal domains of P. berghei and P. falciparum CS proteins were characterized. Mice were immunized with single peptides or a combination of N- and C-terminal peptides. The peptides were immunogenic in mice and the antisera generated could recognize the native CSP on the sporozoite surface. Antisera generated against the N-terminal peptides or against the combinations inhibited sporozoite invasion of hepatocytes in vitro. In vivo, more mice immunized with single P. berghei peptides were protected from infection upon a challenge with P. berghei sporozoites, than mice immunized with a combination of N- and C-terminal peptides. Furthermore, P. falciparum N-terminal peptides were recognized by serum samples from people living in malaria-endemic areas. Importantly, recognition of a peptide from the N-terminal fragment of the P. falciparum CSP by sera from children living in a malaria-endemic region was associated with protection from disease. These results underline the potential of using such peptides as malaria vaccine candidates. RESUME L'intérêt de développer des stratégies d'intervention contre la malaria ciblant le stade pré-erythrocytaire a été alimenté par des études qui montrent qu'il est possible d'obtenir une immunité par l'injection de sporozoites irradiés. Les médicaments et les insecticides anti-paludiques ont été largement utilisés pour contrôler la maladie, mais dans l'espoir de développer une stratégie d'intervention plus rentable, le développement de vaccins a été placé au centre des recherches actuelles contre la malaria. A l'heure actuelle, il n'existe aucun vaccin contre la malaria. L'immunité induite par les sporozoites irradiés est due à l'effet combiné d'anticorps et de cellules T qui agissent contre les antigènes du stade hépatique dont le plus abondant est la protéine circumsporozoite (CSP). Beaucoup de formulations de vaccin visent à imiter l'immunité induite par les sporozoites irradiés. Cependant, les mécanismes par lesquels les anticorps et les cellules T sont génerés après infection par les sporozoites ou après immunisation avec des formulations de vaccin ne sont pas bien compris. La première partie de ce travail a visé à déterminer la capacité de hépatocytes primaires provenant de souris BALB/c à "processer" et à présenter des peptides dérivés de la CSP, après infection par des sporozoites de Plasmodium berghei. Nous avons montré que in vitro, les hépatocytes infectés et ceux traversés par les sporozoites pendant leur migration étaient capables de "processer" et de présenter la CSP aux cellules T CD8+ spécifiques. La voie de présentation implique le protéasome, les protéases de type aspartique et le transport à travers un compartiment post-reticulum endoplasmique. Ces résultats suggèrent que in vivo, les hépatocytes infectés contribuent à l'induction et à l'expansion d'une réponse immunitaire spécifique aux cellules T. Dans la deuxième partie, nous avons caractérisé les réponses anticorps chez les souris de la souche CB6F1 face aux peptides N- et C-terminaux des protéines circumsporozoites de Plasmodium berghei et Plasmodium falciparum. Les souris ont été immunisées avec les peptides individuellement ou en combinaison. Les peptides utilisés étaient immunogéniques chez les souris, et les anticorps produits pouvaient reconnaître la protéine CSP native à la surface des sporozoites. In vitro, les sera contre les peptides N-teminaux et les combinaisons étaient capables d'inhiber l'invasion de hépatocytes par les sporozoites. In vivo, plus de souris immunisées avec les peptides individuels de la CSP de P. berghei étaient protégées contre la malaria que les souris immunisées avec une combinaison de peptides N- et C-terminaux. De plus, les peptides N-terminaux de la CSP de P. falciparum ont été reconnus par les sera de personnes vivant dans des régions endémiques pour la malaria. Il est intéressant de voir que la reconnaissance d'un peptide N-terminal de P. falciparum par des sera d'enfants habitant dans des régions endémiques était associé à la protection contre la maladie. Ces résultats soulignent le potentiel de ces peptides comme candidats-vaccin contre la malaria.