Jean-Louis Pérignon

Long-term clinical protection from falciparum malaria is strongly associated with IgG3 antibodies to merozoite surface protein 3.

Background Surrogate markers of protective immunity to malaria in humans are needed to rationalize malaria vaccine discovery and development. In an effort to identify such markers, and thereby provide a clue to the complex equation malaria vaccine development is facing, we investigated the relationship between protection acquired through exposure in the field with naturally occurring immune responses (i.e., induced by the parasite) to molecules that are considered as valuable vaccine candidates. Methods and Findings We analyzed, under comparative conditions, the antibody responses of each of six isotypes to five leading malaria vaccine candidates in relation to protection acquired by exposure to natural challenges in 217 of the 247 inhabitants of the African village of Dielmo, Senegal (96 children and 121 older adolescents and adults). The status of susceptibility or resistance to malaria was determined by active case detection performed daily by medical doctors over 6 y from a unique follow-up study of this village. Of the 30 immune responses measured, only one, antibodies of the IgG3 isotype directed to merozoite surface protein 3 (MSP3), was strongly associated with clinical protection against malaria in all age groups, i.e., independently of age. This immunological parameter had a higher statistical significance than the sickle cell trait, the strongest factor of protection known against Plasmodium falciparum. A single determination of antibody was significantly associated with the clinical outcome over six consecutive years in children submitted to massive natural parasite challenges by mosquitoes (over three parasite inoculations per week). Finally, the target epitopes of these antibodies were found to be fully conserved. Conclusions Since anti-MSP3 IgG3 antibodies can naturally develop along with protection against P. falciparum infection in young children, our results provide the encouraging indication that these antibodies should be possible to elicit by vaccination early in life. Since these antibodies have been found to achieve parasite killing under in vitro and in vivo conditions, and since they can be readily elicited by immunisation in naïve volunteers, our immunoepidemiological findings support the further development of MSP3-based vaccine formulations.

A hypothesis about the chronicity of malaria infection

It is generally accepted that malaria evolves as a chronic blood infection by escaping the immune responses directed against a series of antigens that express variable epitopes and/or by selecting parasite populations with distinct polymorphic antigens. However, exacting in vitro studies, performed with clinically well-defined biological material, have correlated the state of protection of African adults (in whom low-grade infection persists) with an indirect defence mechanism where the antibodies are effective owing to their ability to cooperate with blood monocytes. Further studies showed that the antibody bridges the parasite (at the merozoite stage) with a monocyte and triggers the release of mediators which have a parasitistatic, reversible and non-antigen-specific effect. The fact that the parasite directly triggers the antiparasite effect leads Pierre Druilhe and Jean-Louis Pérignon to formulate here an alternative hypothesis for the chronicity of malaria infection, which would rely on conserved antigenic targets and, in contrast with direct mechanisms, would not select emerging mutated parasites. The above two mechanisms are discussed in the context of their fitness with clinical and parasitological observations. It is proposed that they are not mutually exclusive but, rather, may come into play successively as patients gradually evolve from high-grade symptomatic to low-grade asymptomatic parasitic infection.

Mechanisms of defense against P. falciparum asexual blood stages in humans.

Based on the observation that host-parasite immune relationships depend not only on the parasite species but also on the host, we consider that P. falciparum can be best studied in humans. In vivo observations suggest that there are in humans a series of immunities towards P. falciparum: besides innate non-antigen-specific defenses, which play a major role, additional defense mechanisms are acquired after consecutive malaria attacks. They contribute to realize such various states of immunity as acquired resistance to cerebral malaria, anti-disease immunity, strain-specific immunity, and, after a long time of exposure in hyper- or holoendemic areas, the host-parasite equilibrium characteristic of premunition. Passive transfer experiments have established that IgG play a major role in premunition. We review, on the one hand, in vitro evidences that protective antibodies have the characteristic capacity of promoting a monocyte-dependent inhibition of parasite growth (ADCI) and, on the other hand, the in vivo observations that suggest that this mechanism of defense could be one of the immune foundations of the state of premunition.

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.

Cellular Immune Deficiency in Two Siblings with Hereditary Orotic Aciduria

HEREDITARY orotic aciduria is a rare inborn error of pyrimidine metabolism that is associated with autosomal recessive inheritance.1 The disease is usually characterized by retarded growth and deve...

Premunition against Plasmodium falciparum in a malaria hyperendemic village in Myanmar

Premunition, naturally acquired protective immunity against Plasmodium falciparum, has been described in hyperendemic areas of Africa and Papua New Guinea. However, its occurrence in Asia is debatable. In order to elucidate this question, a longitudinal study was undertaken in Oo-Do, a malaria endemic village in Myanmar [Burma] in 1995-97. Only 2 species, Plasmodium falciparum and P. vivax, were detected, with the former predominating. Data from 116 subjects showed that all were infected at one time or another, over a period of 3 years, with a 38% reinfection rate after eradication of patent parasitaemia. The high rate of prevalence (90-100%) of parasite-specific antibodies in the indirect immunofluorescence antibody test and the presence of the primary vector (Anopheles minimus) and 15 other species of Anopheles throughout the year indicated a high level of transmission. The spleen rate was 70% in 5-9 years old children and was inversely related with age. The incidence of parasitaemia was maximal (49%) in children aged 2-4 years, and then declined marginally with age. There was a significant difference (P = 0.001) between the asymptomatic and febrile parasitaemia levels. Also, malarial episodes occurred more frequently in children than in adults (P = 0.001). Taken together, all these facts indicated that the inhabitants of Oo-Do had progressively developed non-sterile partial protective immunity against P. falciparum malaria, or premunition. To our knowledge, this is the first detailed clinico-epidemiological study to document the occurrence of premunition in Myanmar.

In vivo veritas: lessons from immunoglobulin-transfer experiments in malaria patients

In most fields of medicine, experimentation starts with studies in vitro, moves to animal models and eventually proceeds to research on humans. Malaria provides a good example of the limits of this progression. The most important malarial parasite of man, Plasmodium falciparum, only infects man. The specificity of this relationship accounts for the many differences which exist between artificial models of falciparum malaria and natural infections. Ultimately, human infections appear to be the sole, relevant ‘model’ for the study of human-Plasmodium interactions. Immunoglobulin-transfer experiments, for example, clearly indicated that antibodies mediated the state of acquired immunity called premunition. However, further studies in vitro or in animal models led to conflicting results about how the antibodies acted. Transfer experiments in human volunteers, appropriately coupled to in-vitro studies, seemed the only way to help solve this issue. The design of these investigations, with its technical and ethi...

Understanding human-Plasmodium falciparum immune interactions uncovers the immunological role of worms.

Background Former studies have pointed to a monocyte-dependant effect of antibodies in protection against malaria and thereby to cytophilic antibodies IgG1 and IgG3, which trigger monocyte receptors. Field investigations have further documented that a switch from non-cytophilic to cytophilic classes of antimalarial antibodies was associated with protection. The hypothesis that the non-cytophilic isotype imbalance could be related to concomittant helminthic infections was supported by several interventions and case-control studies. Methods and Findings We investigated here the hypothesis that the delayed acquisition of immunity to malaria could be related to a worm-induced Th2 drive on antimalarial immune responses. IgG1 to IgG4 responses against 6 different parasite-derived antigens were analyzed in sera from 203 Senegalese children, half carrying intestinal worms, presenting 421 clinical malaria attacks over 51 months. Results show a significant correlation between the occurrence of malaria attacks, worm carriage (particularly that of hookworms) and a decrease in cytophilic IgG1 and IgG3 responses and an increase in non-cytophilic IgG4 response to the merozoite stage protein 3 (MSP3) vaccine candidate. Conclusion The results confirm the association with protection of anti-MSP3 cytophilic responses, confirm in one additional setting that worms increase malaria morbidity and show a Th2 worm-driven pattern of anti-malarial immune responses. They document why large anthelminthic mass treatments may be worth being assessed as malaria control policies.

Toward the Rational Design of a Malaria Vaccine Construct Using the MSP3 Family as an Example: Contribution of Immunogenicity Studies in Models

ABSTRACT Plasmodium falciparum merozoite surface protein 3 (MSP3), the target of antibodies that mediate parasite killing in cooperation with blood monocytes and are associated with protection in exposed populations, is a vaccine candidate under development. It belongs to a family of six structurally related genes. To optimize immunogenicity, we attempted to improve its design based on knowledge of antigenicity of various regions from the conserved C terminus of the six proteins and an analysis of the immunogenicity of “tailored” constructs. The immunogenicity studies were conducted in BALB/c and C57BL/6J mice, using MSP3 (referred to here as MSP3-1) as a model. Four constructs were designed in order to assess the effect of sequences flanking the 69-amino-acid region of MSP3-1 previously shown to be the target of biologically active antibodies. The results indicate major beneficial effects of removing (i) the subregion downstream from the 69-amino-acid sequence, since antibody titers increased by 2 orders of magnitude, and (ii) the upstream subregion which, although it defines a T-helper cell epitope, is not the target of antibodies. The construct, excluding both flanking sequences, was able to induce Th1-like responses, with a dominance of cytophilic antibodies. This led to design a multigenic construct based on these results, combining the six members of the MSP3 family. This new construction was immunogenic in mice, induced antibodies that recognized the parasite native proteins, and inhibited parasite growth in the functional antibody-dependent cellular inhibition assay, thus satisfying the preclinical criteria for a valuable vaccine candidate.

Analysis of innate defences against Plasmodium falciparum in immunodeficient mice

BackgroundMice 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.MethodsNOD/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.ResultsResults 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.ConclusionsDespite 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.