Limited polymorphism in Plasmodium falciparum ookinete surface antigen, von Willebrand factor A domain-related protein from clinical isolates

BACKGROUND: As malaria becomes increasingly drug resistant and more costly to treat, there is increasing urgency to develop effective vaccines. In comparison to other stages of the malaria lifecycle, sexual stage antigens are under less immune selection pressure and hence are likely to have limited antigenic diversity. METHODS: Clinical isolates from a wide range of geographical regions were collected. Direct sequencing of PCR products was then used to determine the extent of polymorphisms for the novel Plasmodium falciparum sexual stage antigen von Willebrand Factor A domain-related Protein (PfWARP). These isolates were also used to confirm the extent of diversity of sexual stage antigen Pfs28. RESULTS: PfWARP was shown to have non-synonymous substitutions at 3 positions and Pfs28 was confirmed to have a single non-synonymous substitution as previously described. CONCLUSION: This study demonstrates the limited antigenic diversity of two prospective P. falciparum sexual stage antigens, PfWARP and Pfs28. This provides further encouragement for the proceeding with vaccine trials based on these antigens

Natural Regulatory T Cells in Malaria: Host or Parasite Allies?

Plasmodium falciparum malaria causes 500 million clinical cases with approximately one million deaths each year. After many years of exposure, individuals living in endemic areas develop a form of clinical immunity to disease known as premunition, which is characterised by low parasite burdens rather than sterilising immunity. The reason why malaria parasites persist under a state of premunition is unknown but it has been suggested that suppression of protective immunity might be a mechanism leading to parasite persistence. Although acquired immunity limits the clinical impact of infection and provides protection against parasite replication, experimental evidence indicates that cell-mediated immune responses also result in detrimental inflammation and contribute to the aetiology of severe disease. Thus, an appropriate regulatory balance between protective immune responses and immune-mediated pathology is required for a favourable outcome of infection. As natural regulatory T (Treg) cells are identified as an immunosuppressive lineage able to modulate the magnitude of effector responses, several studies have investigated whether this cell population plays a role in balancing protective immunity and pathogenesis during malaria. The main findings to date are summarised in this review and the implication for the induction of pathogenesis and immunity to malaria is discussed

IL-10 from CD4+CD25−Foxp3−CD127− Adaptive Regulatory T Cells Modulates Parasite Clearance and Pathology during Malaria Infection

The outcome of malaria infection is determined, in part, by the balance of pro-inflammatory and regulatory immune responses. Failure to develop an effective pro-inflammatory response can lead to unrestricted parasite replication, whilst failure to regulate this response leads to the development of severe immunopathology. IL-10 and TGF-β are known to be important components of the regulatory response, but the cellular source of these cytokines is still unknown. Here we have examined the role of natural and adaptive regulatory T cells in the control of malaria infection and find that classical CD4+CD25hi (and Foxp3+) regulatory T cells do not significantly influence the outcome of infections with the lethal (17XL) strain of Plasmodium yoelii (PyL). In contrast, we find that adaptive IL-10-producing, CD4+ T cells (which are CD25−, Foxp3−, and CD127− and do not produce Th1, Th2, or Th17 associated cytokines) that are generated during both PyL and non-lethal P. yoelii 17X (PyNL) infections are able to down-regulate pro-inflammatory responses and impede parasite clearance. In summary, we have identified a population of induced Foxp3− regulatory (Tr1) T cells, characterised by production of IL-10 and down regulation of IL-7Rα, that modulates the inflammatory response to malaria

A Conserved Multi-Gene Family Induces Cross-Reactive Antibodies Effective in Defense against Plasmodium falciparum

BACKGROUND: Two related merozoite surface proteins, MSP3 and MSP6, have previously been identified as targets of antibody-dependent cellular inhibition (ADCI), a protective mechanism against Plasmodium falciparum malaria. Both MSP3 and MSP6 share a common characteristic small N-terminal signature amino-acid stretch (NLRNA/G), a feature similar to MSP3-like orthologs identified in other human and primate malaria parasites. METHODS/RESULTS: This signature amino-acid sequence led to the identification of eight ORFs contiguously located on P. falciparum chromosome 10. Our subsequent investigations on their expression, localization, sequence conservation, epitope sharing, immunogenicity and the functional role of antibodies in defense are reported here. Six members of P. falciparum MSP3-multigene family share similar sequence organization within their C-terminal regions, are simultaneously expressed as merozoite surface proteins and are highly conserved among parasite isolates. Each of these proteins is a target of naturally occurring antibodies effective at parasite killing in ADCI assays. Moreover, both naturally occurring antibodies and those generated by immunization display cross-reactivity with other members of the family and exhibit varied binding avidities. CONCLUSIONS/SIGNIFICANCE: The unusual characteristics of the MSP3 multi-gene family lead us to hypothesize that the simultaneous expression of targets eliciting cross-reactive antibody responses capable of controlling parasite densities could represent an immune process selected through evolution to maintain homeostasis between P. falciparum and human hosts; a process that allows the continuous transmission of the parasite without killing the host. Our observations also have practical consequences for vaccine development by suggesting MSP3 vaccine efficacy might be improved when combined with the various C-terminus regions of the MSP3 family members to generate a wider range of antibodies acting and to increase vaccine immunogenicity in varied human genetic backgrounds

TLR9 polymorphisms in African populations: no association with severe malaria, but evidence of cis-variants acting on gene expression

BACKGROUND: During malaria infection the Toll-like receptor 9 (TLR9) is activated through induction with plasmodium DNA or another malaria motif not yet identified. Although TLR9 activation by malaria parasites is well reported, the implication to the susceptibility to severe malaria is not clear. The aim of this study was to assess the contribution of genetic variation at TLR9 to severe malaria. METHODS: This study explores the contribution of TLR9 genetic variants to severe malaria using two approaches. First, an association study of four common single nucleotide polymorphisms was performed on both family- and population-based studies from Malawian and Gambian populations (n>6000 individual). Subsequently, it was assessed whether TLR9 expression is affected by cis-acting variants and if these variants could be mapped. For this work, an allele specific expression (ASE) assay on a panel of HapMap cell lines was carried out. RESULTS: No convincing association was found with polymorphisms in TLR9 for malaria severity, in either Gambian or Malawian populations, using both case-control and family based study designs. Using an allele specific expression assay it was observed that TLR9 expression is affected by cis-acting variants, these results were replicated in a second experiment using biological replicates. CONCLUSION: By using the largest cohorts analysed to date, as well as a standardized phenotype definition and study design, no association of TLR9 genetic variants with severe malaria was found. This analysis considered all common variants in the region, but it is remains possible that there are rare variants with association signals. This report also shows that TLR9 expression is potentially modulated through cis-regulatory variants, which may lead to differential inflammatory responses to infection between individuals

Evaluation of immunological escape mechanisms in a mouse model of colorectal liver metastases

<p>Abstract</p> <p>Background</p> <p>The local and systemic activation and regulation of the immune system by malignant cells during carcinogenesis is highly complex with involvement of the innate and acquired immune system. Despite the fact that malignant cells do have antigenic properties their immunogenic effects are minor suggesting tumor induced mechanisms to circumvent cancer immunosurveillance. The aim of this study is the analysis of tumor immune escape mechanisms in a colorectal liver metastases mouse model at different points in time during tumor growth.</p> <p>Methods</p> <p>CT26.WT murine colon carcinoma cells were injected intraportally in Balb/c mice after median laparotomy using a standardized injection technique. Metastatic tumor growth in the liver was examined by standard histological procedures at defined points in time during metastatic growth. Liver tissue with metastases was additionally analyzed for cytokines, T cell markers and Fas/Fas-L expression using immunohistochemistry, immunofluorescence and RT-PCR. Comparisons were performed by analysis of variance or paired and unpaired <it>t </it>test when appropriate.</p> <p>Results</p> <p>Intraportal injection of colon carcinoma cells resulted in a gradual and time dependent metastatic growth. T cells of regulatory phenotype (CD4+CD25+Foxp3+) which might play a role in protumoral immune response were found to infiltrate peritumoral tissue increasingly during carcinogenesis. Expression of cytokines IL-10, TGF-β and TNF-α were increased during tumor growth whereas IFN-γ showed a decrease of the expression from day 10 on following an initial increase. Moreover, liver metastases of murine colon carcinoma show an up-regulation of FAS-L on tumor cell surface with a decreased expression of FAS from day 10 on. CD8+ T cells express FAS and show an increased rate of apoptosis at perimetastatic location.</p> <p>Conclusions</p> <p>This study describes cellular and macromolecular changes contributing to immunological escape mechanisms during metastatic growth in a colorectal liver metastases mouse model simulating the situation in human cancer.</p

Regulatory T Cell Induction during Plasmodium chabaudi Infection Modifies the Clinical Course of Experimental Autoimmune Encephalomyelitis

BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) is used as an animal model for human multiple sclerosis (MS), which is an inflammatory demyelinating autoimmune disease of the central nervous system characterized by activation of Th1 and/or Th17 cells. Human autoimmune diseases can be either exacerbated or suppressed by infectious agents. Recent studies have shown that regulatory T cells play a crucial role in the escape mechanism of Plasmodium spp. both in humans and in experimental models. These cells suppress the Th1 response against the parasite and prevent its elimination. Regulatory T cells have been largely associated with protection or amelioration in several autoimmune diseases, mainly by their capacity to suppress proinflammatory response. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we verified that CD4(+)CD25(+) regulatory T cells (T regs) generated during malaria infection (6 days after EAE induction) interfere with the evolution of EAE. We observed a positive correlation between the reduction of EAE clinical symptoms and an increase of parasitemia levels. Suppression of the disease was also accompanied by a decrease in the expression of IL-17 and IFN-γ and increases in the expression of IL-10 and TGF-β1 relative to EAE control mice. The adoptive transfer of CD4(+)CD25(+) cells from P. chabaudi-infected mice reduced the clinical evolution of EAE, confirming the role of these T regs. CONCLUSIONS/SIGNIFICANCE: These data corroborate previous findings showing that infections interfere with the prevalence and evolution of autoimmune diseases by inducing regulatory T cells, which regulate EAE in an apparently non-specific manner

Parasite-Dependent Expansion of TNF Receptor II–Positive Regulatory T Cells with Enhanced Suppressive Activity in Adults with Severe Malaria

Severe Plasmodium falciparum malaria is a major cause of global mortality, yet the immunological factors underlying progression to severe disease remain unclear. CD4+CD25+ regulatory T cells (Treg cells) are associated with impaired T cell control of Plasmodium spp infection. We investigated the relationship between Treg cells, parasite biomass, and P. falciparum malaria disease severity in adults living in a malaria-endemic region of Indonesia. CD4+CD25+Foxp3+CD127lo Treg cells were significantly elevated in patients with uncomplicated (UM; n = 17) and severe malaria (SM; n = 16) relative to exposed asymptomatic controls (AC; n = 10). In patients with SM, Treg cell frequency correlated positively with parasitemia (r = 0.79, p = 0.0003) and total parasite biomass (r = 0.87, p<0.001), both major determinants for the development of severe and fatal malaria, and Treg cells were significantly increased in hyperparasitemia. There was a further significant correlation between Treg cell frequency and plasma concentrations of soluble tumor necrosis factor receptor II (TNFRII) in SM. A subset of TNFRII+ Treg cells with high expression of Foxp3 was increased in severe relative to uncomplicated malaria. In vitro, P. falciparum–infected red blood cells dose dependently induced TNFRII+Foxp3hi Treg cells in PBMC from malaria-unexposed donors which showed greater suppressive activity than TNFRII− Treg cells. The selective enrichment of the Treg cell compartment for a maximally suppressive TNFRII+Foxp3hi Treg subset in severe malaria provides a potential link between immune suppression, increased parasite biomass, and malaria disease severity. The findings caution against the induction of TNFRII+Foxp3hi Treg cells when developing effective malaria vaccines

Suppression of HIV-Specific and Allogeneic T Cell Activation by Human Regulatory T Cells Is Dependent on the Strength of Signals

Regulatory T cells (Tregs) suppress immune responses against both self and non-self antigens. Tregs require activation through the T cell receptor (TCR) and IL-2 to exert their suppressive functions. However, how strength of TCR signals modulate the potency of Treg-mediated suppression of antigen-specific T cell activation remain unclear. We found that both strength of TCR signals and ratios of Tregs to target cells, either through superantigen, allogeneic antigens or HIV-specific peptides, modified the suppressive ability of Tregs. While human Tregs were able to mediate suppression in the presence of only autologous antigen-presenting cells, this was much less efficient as compared to when Tregs were activated by allogeneic dendritic cells. In another physiologically relevant system, we show that the strength of peptide stimulation, high frequency of responder CD8+ T cells or presence of high IL-2 can override the suppression of HIV-specific CD8+ T cells by Tregs. These findings suggest that ratios and TCR activation of human Tregs, are important parameters to overcome robust immune responses to pathogens or allogeneic antigens. Modulating the strength of T cell signals and selective enhancement or depletion of antigen-specific Tregs thus may have implications for designing potent vaccines and regulating immune responses during allogeneic transplantation and chronic infections

AFCo1, a meningococcal B-derived cochleate adjuvant, strongly enhances antibody and T-cell immunity against Plasmodium falciparum merozoite surface protein 4 and 5

<p>Abstract</p> <p>Background</p> <p>Whilst a large number of malaria antigens are being tested as candidate malaria vaccines, a major barrier to the development of an effective vaccine is the lack of a suitable human adjuvant capable of inducing a strong and long lasting immune response. In this study, the ability of AFCo1, a potent T and B cell adjuvant based on cochleate structures derived from meningococcal B outer membrane proteoliposomes (MBOMP), to boost the immune response against two <it>Plasmodium falciparum </it>antigens, merozoite surface protein 4 (MSP4) and 5 (MSP5), was evaluated.</p> <p>Methods</p> <p>Complete Freund's adjuvant (CFA), which is able to confer protection against malaria in animal MSP4/5 vaccine challenge models, was used as positive control adjuvant. MSP4 and 5-specific IgG, delayed-type hypersensitivity (DTH), T-cell proliferation, and cytokine production were evaluated in parallel in mice immunized three times intramuscularly with MSP4 or MSP5 incorporated into AFCo1, synthetic cochleate structures, CFA or phosphate buffered saline.</p> <p>Results</p> <p>AFCo1 significantly enhanced the IgG and T-cell response against MSP4 and MSP5, with a potency equivalent to CFA, with the response being characterized by both IgG1 and IgG2a isotypes, increased interferon gamma production and a strong DTH response, consistent with the ability of AFCo1 to induce Th1-like immune responses.</p> <p>Conclusion</p> <p>Given the proven safety of MBOMP, which is already in use in a licensed human vaccine, AFCo1 could assist the development of human malaria vaccines that require a potent and safe adjuvant.</p