Interferon-γ, a valuable surrogate marker of Plasmodium falciparum pre-erythrocytic stages protective immunity

Immunity against the pre-erythrocytic stages of malaria is the most promising, as it is strong and fully sterilizing. Yet, the underlying immune effectors against the human Plasmodium falciparum pre-erythrocytic stages remain surprisingly poorly known and have been little explored, which in turn prevents any rational vaccine progress. Evidence that has been gathered in vitro and in vivo, in higher primates and in humans, is reviewed here, emphasizing the significant role of IFN-γ, either as a critical immune mediator or at least as a valuable surrogate marker of protection. One may hope that these results will trigger investigations in volunteers immunized either by optimally irradiated or over-irradiated sporozoites, to quickly delineate better surrogates of protection, which are essential for the development of a successful malaria vaccine

Immune mechanisms underlying the premunition against Plasmodium falciparum malaria

The most unique characteristic of a parasite when it is in its normal host is the ability to make itself tolerated, which clearly indicates that it has sophisticated means to ensure the neutrality of its host. This is true also in the case of Plasmodium falciparum, since after numerous malaria attacks an equilibrium is reached with a chronic stage of infection, characterized by a relatively low parasitemia, and low or no disease (Sergent & Parrot 1935). We shall briefly review the main characteristics of this state of "premunition", and present data suggesting that the underlying mechanisms of defense rely on the cooperation between cell and antibodies, leading to an antibody dependent cellular inhibition of the intra-erythrocytic growth of the parasite

Identification of a Conserved Region of Plasmodium falciparum MSP3 Targeted by Biologically Active Antibodies to Improve Vaccine Design

Merozoite surface protein 3 (MSP3) is a target of antibody-dependent cellular inhibition (ADCI), a protective mechanism against Plasmodium falciparum malaria. From the C-terminal half of the molecule, 6 overlapping peptides were chosen to characterize human immune responses. Each peptide defined at least 1 non-crossreactive B cell epitope. Distinct patterns of antibody responses, by level and IgG subclass distribution, were observed in inhabitants of a malaria-endemic area. Antibodies affinity purified toward each peptide differed in their functional capacity to mediate parasite killing in ADCI assays: 3 of 6 overlapping peptides had a major inhibitory effect on parasite growth. This result was confirmed by the passive transfer of anti-MSP3 antibodies in vivo in a P. falciparum mouse model. T helper cell epitopes were identified in each peptide. Antigenicity and functional assays identified a 70-amino acid conserved domain of MSP3 as a target of biologically active antibodies to be included in future vaccine constructs based on MSP

Further Improvements of the P. falciparum Humanized Mouse Model

BACKGROUND: It has been shown previously that it is possible to obtain growth of Plasmodium falciparum in human erythrocytes grafted in mice lacking adaptive immune responses by controlling, to a certain extent, innate defences with liposomes containing clodronate (clo-lip). However, the reproducibility of those models is limited, with only a proportion of animals supporting longstanding parasitemia, due to strong inflammation induced by P. falciparum. Optimisation of the model is much needed for the study of new anti-malarial drugs, drug combinations, and candidate vaccines. MATERIALS/METHODS: We investigated the possibility of improving previous models by employing the intravenous route (IV) for delivery of both human erythrocytes (huRBC) and P. falciparum, instead of the intraperitoneal route (IP), by testing various immunosuppressive drugs that might help to control innate mouse defences, and by exploring the potential benefits of using immunodeficient mice with additional genetic defects, such as those with IL-2Rγ deficiency (NSG mice). RESULTS: We demonstrate here the role of aging, of inosine and of the IL-2 receptor γ mutation in controlling P. falciparum induced inflammation. IV delivery of huRBC and P. falciparum in clo-lip treated NSG mice led to successful infection in 100% of inoculated mice, rapid rise of parasitemia to high levels (up to 40%), long-lasting parasitemia, and consistent results from mouse-to-mouse. Characteristics were closer to human infection than in previous models, with evidence of synchronisation, partial sequestration, and receptivity to various P. falciparum strains without preliminary adaptation. However, results show that a major IL-12p70 inflammatory response remains prevalent. CONCLUSION: The combination of the NSG mouse, clodronate loaded liposomes, and IV delivery of huRBC has produced a reliable and more relevant model that better meets the needs of Malaria research

Analysis of innate defences against Plasmodium falciparum in immunodeficient mice

Background: Mice with genetic deficiencies in adaptive immunity are used for the grafting of human cells or pathogens, to study human diseases, however, the innate immune responses to xenografts in these mice has received little attention. Using the NOD/SCID Plasmodium falciparum mouse model an analysis of innate defences responsible for the substantial control of P. falciparum which remains in such mice, was performed. Methods: NOD/SCID mice undergoing an immunomodulatory protocol that includes, clodronate-loaded liposomes to deplete macrophages and an anti-polymorphonuclear leukocytes antibody, were grafted with human red blood cells and P. falciparum. The systematic and kinetic analysis of the remaining innate immune responses included the number and phenotype of peripheral blood leukocytes as well as inflammatory cytokines/chemokines released in periphery. The innate responses towards the murine parasite Plasmodium yoelii were used as a control. Results: Results show that 1) P. falciparum induces a strong inflammation characterized by an increase in circulating leukocytes and the release of inflammatory cytokines; 2) in contrast, the rodent parasite P. yoelii, induces a far more moderate inflammation; 3) human red blood cells and the anti-inflammatory agents employed induce low-grade inflammation; and 4) macrophages seem to bear the most critical function in controlling P. falciparum survival in those mice, whereas polymorphonuclear and NK cells have only a minor role. Conclusions: Despite the use of an immunomodulatory treatment, immunodeficient NOD/SCID mice are still able to mount substantial innate responses that seem to be correlated with parasite clearance. Those results bring new insights on the ability of innate immunity from immunodeficient mice to control xenografts of cells of human origin and human pathogens

Satisfactory safety and immunogenicity of MSP3 malaria vaccine candidate in Tanzanian children aged 12-24 months.

BACKGROUND: Development and deployment of an effective malaria vaccine would complement existing malaria control measures. A blood stage malaria vaccine candidate, Merozoite Surface Protein-3 (MSP3), produced as a long synthetic peptide, has been shown to be safe in non-immune and semi-immune adults. A phase Ib dose-escalating study was conducted to assess the vaccine's safety and immunogenicity in children aged 12 to 24 months in Korogwe, Tanzania (ClinicalTrials.gov number: NCT00469651). METHODS: This was a double-blind, randomized, controlled, dose escalation phase Ib trial, in which children were given one of two different doses of the MSP3 antigen (15 microg or 30 microg) or a control vaccine (Engerix B). Children were randomly allocated either to the MSP3 candidate malaria vaccine or the control vaccine administered at a schedule of 0, 1, and 2 months. Immunization with lower and higher doses was staggered for safety reasons starting with the lower dose. The primary endpoint was safety and reactogenicity within 28 days post-vaccination. Blood samples were obtained at different time points to measure immunological responses. Results are presented up to 84 days post-vaccination. RESULTS: A total of 45 children were enrolled, 15 in each of the two MSP3 dose groups and 15 in the Engerix B group. There were no important differences in reactogenicity between the two MSP3 groups and Engerix B. Grade 3 adverse events were infrequent; only five were detected throughout the study, all of which were transient and resolved without sequelae. No serious adverse event reported was considered to be related to MSP3 vaccine. Both MSP3 dose regimens elicited strong cytophilic IgG responses (subclasses IgG1 and IgG3), the isotypes involved in the monocyte-dependant mechanism of Plasmodium falciparum parasite-killing. The titers reached are similar to those from African adults having reached a state of premunition. Furthermore, vaccination induced seroconversion in all vaccinees. CONCLUSION: The MSP3 malaria vaccine candidate was safe, well tolerated and immunogenic in children aged 12-24 months living in a malaria endemic community. Given the vaccine's safety and its induction of cytophilic IgG responses, its efficacy against P. falciparum infection and disease needs to be evaluated in Phase 2 studies

Increase of malaria attacks among children presenting concomitant infection by Schistosoma mansoni in Senegal

Helminthic infections concomitant with malaria are common in inter-tropical areas. A recent study showed that mice co-infected with Schistosoma mansoni and Plasmodium chabaudi develop higher P. chabaudi parasitaemia and had a higher mortality rate. This important observation deserved to be further investigated among human populations. Malaria attacks were recorded in 512 children aged 6–15 years living in Richard Toll (Northern Senegal) among whom 336 were infected by S. mansoni, and 175 were not. The incidence rate of malaria attacks was significantly higher among S. mansoni-infected individuals, particularly those carrying the highest worm loads, as compared to uninfected subjects (26.6% versus 16,4 %). In contrast, the rate of malaria attacks was lower, without reaching significance, in medium grade S. mansoni infections. Thus, infection by S. mansoni affects susceptibility to malaria, but this can vary according to the intensity of parasite load. The immunological mechanisms underlying this dual effect need to be further explored

A Malaria Vaccine That Elicits in Humans Antibodies Able to Kill Plasmodium falciparum

BACKGROUND: Plasmodium falciparum merozoite surface protein 3 is a malaria vaccine candidate that was identified, characterised, and developed based on a unique immuno-clinical approach. The vaccine construct was derived from regions fully conserved among various strains and containing B cell epitopes targeted by human antibodies (from malaria-immune adults) that are able to mediate a monocyte-dependent parasite killing effect. The corresponding long synthetic peptide was administered to 36 volunteers, with either alum or Montanide ISA720 as adjuvant. METHODS AND FINDINGS: Both formulations induced cellular and humoral immune responses. With alum, the responses lasted up to 12 mo. The vaccine-induced antibodies were predominantly of cytophilic classes, i.e., able to cooperate with effector cells. In vitro, the antibodies induced an inhibition of the P. falciparum erythrocytic growth in a monocyte-dependent manner, which was in most instances as high as or greater than that induced by natural antibodies from immune African adults. In vivo transfer of the volunteers' sera into P. falciparum–infected humanized SCID mice profoundly reduced or abrogated parasitaemia. These inhibitory effects were related to the antibody reactivity with the parasite native protein, which was seen in 60% of the volunteers, and remained in samples taken 12 mo postimmunisation. CONCLUSION: This is the first malaria vaccine clinical trial to clearly demonstrate antiparasitic activity by vaccine-induced antibodies by both in vitro and in vivo methods. The results, showing the induction of long-lasting antibodies directed to a fully conserved polypeptide, also challenge current concepts about malaria vaccines, such as unavoidable polymorphism, low antigenicity, and poor induction of immune memory

Pre-Clinical Assessment of Novel Multivalent MSP3 Malaria Vaccine Constructs

BACKGROUND: MSP3 has been shown to induce protection against malaria in African children. The characterization of a family of Plasmodium falciparum merozoite surface protein 3 (MSP3) antigens sharing a similar structural organization, simultaneously expressed on the merozoite surface and targeted by a cross-reactive network of protective antibodies, is intriguing and offers new perspectives for the development of subunit vaccines against malaria. METHODS: Eight recombinant polyproteins containing carefully selected regions of this family covalently linked in different combinations were all efficiently produced in Escherichia coli. The polyproteins consisted of one monovalent, one bivalent, one trivalent, two tetravalents, one hexavalent construct, and two tetravalents incorporating coiled-coil repeats regions from LSA3 and p27 vaccine candidates. RESULTS: All eight polyproteins induced a strong and homogeneous antibody response in mice of three distinct genotypes, with a dominance of cytophilic IgG subclasses, lasting up to six months after the last immunization. Vaccine-induced antibodies exerted a strong monocyte-mediated in vitro inhibition of P. falciparum growth. Naturally acquired antibodies from individuals living in an endemic area of Senegal recognized the polyproteins with a reactivity mainly constituted of cytophilic IgG subclasses. CONCLUSIONS: Combination of genetically conserved and antigenically related MSP3 proteins provides promising subunit vaccine constructs, with improved features as compared to the first generation construct employed in clinical trials (MSP3-LSP). These multivalent MSP3 vaccine constructs expand the epitope display of MSP3 family proteins, and lead to the efficient induction of a wider range of antibody subclasses, even in genetically different mice. These findings are promising for future immunization of genetically diverse human populations

A multiplex assay for the simultaneous detection of antibodies against 15 Plasmodium falciparum and Anopheles gambiae saliva antigens

<p>Abstract</p> <p>Background</p> <p>Assessment exposure and immunity to malaria is an important step in the fight against the disease. Increased malaria infection in non-immune travellers under anti-malarial chemoprophylaxis, as well as the implementation of malaria elimination programmes in endemic countries, raises new issues that pertain to these processes. Notably, monitoring malaria immunity has become more difficult in individuals showing low antibody (Ab) responses or taking medications against the <it>Plasmodium </it><it>falciparum </it>blood stages. Commonly available techniques in malaria seroepidemiology have limited sensitivity, both against pre-erythrocytic, as against blood stages of the parasite. Thus, the aim of this study was to develop a sensitive tool to assess the exposure to malaria or to bites from the vector <it>Anopheles gambiae</it>, despite anti-malarial prophylactic treatment.</p> <p>Methods</p> <p>Ab responses to 13 pre-erythrocytic <it>P. falciparum</it>-specific peptides derived from the proteins Lsa1, Lsa3, Glurp, Salsa, Trap, Starp, CSP and Pf11.1, and to 2 peptides specific for the <it>Anopheles gambiae </it>saliva protein gSG6 were tested. In this study, 253 individuals from three Senegalese areas with different transmission intensities and 124 European travellers exposed to malaria during a short period of time were included.</p> <p>Results</p> <p>The multiplex assay was optimized for most but not all of the antigens. It was rapid, reproducible and required a small volume of serum. Proportions of Ab-positive individuals, Ab levels and the mean number of antigens (Ags) recognized by each individual increased significantly with increases in the level of malaria exposure.</p> <p>Conclusion</p> <p>The multiplex assay developed here provides a useful tool to evaluate immune responses to multiple Ags in large populations, even when only small amounts of serum are available, or Ab titres are low, as in case of travellers. Finally, the relationship of Ab responses with malaria endemicity levels provides a way to monitor exposure in differentially exposed autochthonous individuals from various endemicity areas, as well as in travellers who are not immune, thus indirectly assessing the parasite transmission and malaria risk in the new eradication era.</p