High-level artemisinin-resistance with quinine co-resistance emerges in P. falciparum malaria under in vivo artesunate pressure.

BACKGROUND: Humanity has become largely dependent on artemisinin derivatives for both the treatment and control of malaria, with few alternatives available. A Plasmodium falciparum phenotype with delayed parasite clearance during artemisinin-based combination therapy has established in Southeast Asia, and is emerging elsewhere. Therefore, we must know how fast, and by how much, artemisinin-resistance can strengthen. METHODS: P. falciparum was subjected to discontinuous in vivo artemisinin drug pressure by capitalizing on a novel model that allows for long-lasting, high-parasite loads. Intravenous artesunate was administered, using either single flash-doses or a 2-day regimen, to P. falciparum-infected humanized NOD/SCID IL-2Rγ-/-immunocompromised mice, with progressive dose increments as parasites recovered. The parasite's response to artemisinins and other available anti-malarial compounds was characterized in vivo and in vitro. RESULTS: Artemisinin resistance evolved very rapidly up to extreme, near-lethal doses of artesunate (240 mg/kg), an increase of > 3000-fold in the effective in vivo dose, far above resistance levels reported from the field. Artemisinin resistance selection was reproducible, occurring in 80% and 41% of mice treated with flash-dose and 2-day regimens, respectively, and the resistance phenotype was stable. Measuring in vitro sensitivity proved inappropriate as an early marker of resistance, as IC50 remained stable despite in vivo resistance up to 30 mg/kg (ART-S: 10.7 nM (95% CI 10.2-11.2) vs. ART-R30: 11.5 nM (6.6-16.9), F = 0.525, p = 0.47). However, when in vivo resistance strengthened further, IC50 increased 10-fold (ART-R240 100.3 nM (92.9-118.4), F = 304.8, p < 0.0001), reaching a level much higher than ever seen in clinical samples. Artemisinin resistance in this African P. falciparum strain was not associated with mutations in kelch-13, casting doubt over the universality of this genetic marker for resistance screening. Remarkably, despite exclusive exposure to artesunate, full resistance to quinine, the only other drug sufficiently fast-acting to deal with severe malaria, evolved independently in two parasite lines exposed to different artesunate regimens in vivo, and was confirmed in vitro. CONCLUSION: P. falciparum has the potential to evolve extreme artemisinin resistance and more complex patterns of multidrug resistance than anticipated. If resistance in the field continues to advance along this trajectory, we will be left with a limited choice of suboptimal treatments for acute malaria, and no satisfactory option for severe malaria.

One hundred malaria attacks since birth. A longitudinal study of African children and young adults exposed to high malaria transmission.

Background: Despite significant progress in malaria control over the past twenty years, malaria remains a leading cause of child morbidity and mortality in Tropical Africa. As most patients do not consult any health facility much uncertainty persists about the true burden of the disease and the range of individual differences in susceptibility to malaria. Methods: Over a 25-years period, from 1990 to 2015, the inhabitants of Dielmo village, Senegal, an area of intense malaria transmission, have been monitored daily for their presence in the village and the occurrence of diseases. In case of fever thick blood films were systematically examined through microscopy for malaria parasites and patients received prompt diagnosis and treatment. Findings: We analysed data collected in 111 children and young adults monitored for at least 10 years (mean 17.3 years, maximum 25 years) enrolled either at birth (95 persons) or during the two first years of life. A total of 11,599 episodes of fever were documented, including 5268 malaria attacks. The maximum number of malaria attacks in a single person was 112. Three other persons suffered one hundred or more malaria attacks during follow-up. The minimum number of malaria attacks in a single person was 11. The mean numbers of malaria attacks in children reaching their 4th, 7th, and 10th birthdays were 23.0, 37.7, and 43.6 attacks since birth, respectively. Sixteen children (14.4%) suffered ten or more malaria attacks each year at ages 1-3 years, and six children (5.4%) each year at age 4-6 years. Interpretation: Long-term close monitoring shows that in highly endemic areas the malaria burden is higher than expected. Susceptibility to the disease may vary up to 10-fold, and for most children childhood is an endless history of malaria fever episodes. No other parasitic, bacterial or viral infection in human populations has such an impact on health. Funding: The Pasteur Institutes of Dakar and Paris, the Institut de Recherche pour le Développement, and the French Ministry of Cooperation provided funding.

Use of a colorimetric (DELI) test for the evaluation of chemoresistance of Plasmodium falciparum and Plasmodium vivax to commonly used anti-plasmodial drugs in the Brazilian Amazon.

BACKGROUND: The emergence and spread of Plasmodium falciparum and Plasmodium vivax resistance to available anti-malarial drugs represents a major drawback in the control of malaria and its associated morbidity and mortality. The aim of this study was to evaluate the chemoresistance profile of P. falciparum and P. vivax to commonly used anti-plasmodial drugs in a malaria-endemic area in the Brazilian Amazon. METHODS: The study was carried out in Manaus (Amazonas state), in the Brazilian Amazon. A total of 88 P. falciparum and 178 P. vivax isolates was collected from 2004 to 2007. The sensitivity of P. falciparum isolates was determined to chloroquine, quinine, mefloquine and artesunate and the sensitivity of P. vivax isolates was determined to chloroquine and mefloquine, by using the colorimetric DELI test. RESULTS: As expected, a high prevalence of P. falciparum isolates resistant to chloroquine (78.1%) was observed. The prevalence of isolates with profile of resistance or decreased sensitivity for quinine, mefloquine and artesunate was 12.7, 21.2 and 11.7%, respectively. In the case of P. vivax, the prevalence of isolates with profile of resistance for chloroquine and mefloquine was 9.8 and 28%, respectively. No differences in the frequencies of isolates with profile of resistance or geometric mean IC50s were seen when comparing the data obtained in 2004, 2005, 2006 and 2007, for all tested anti-malarials. CONCLUSIONS: The great majority of P. falciparum isolates in the Brazilian malaria-endemic area remain resistant to chloroquine, and the decreased sensitivity to quinine, mefloquine and artesunate observed in 10-20% of the isolates must be taken with concern, especially for artesunate. Plasmodium vivax isolates also showed a significant proportion of isolates with decreased sensitivity to chloroquine (first-line drug) and mainly to mefloquine. The data presented here also confirm the usefulness of the DELI test to generate results able to impact on public health policies.

Epidemiology of malaria in an area of seasonal transmission in Niger and implications for the design of a seasonal malaria chemoprevention strategy.

BACKGROUND: Few data are available about malaria epidemiological situation in Niger. However, implementation of new strategies such as vaccination or seasonal treatment of a target population requires the knowledge of baseline epidemiological features of malaria. A population-based study was conducted to provide better characterization of malaria seasonal variations and population groups the most at risk in this particular area. METHODS: From July 2007 to December 2009, presumptive cases of malaria among a study population living in a typical Sahelian village of Niger were recorded, and confirmed by microscopic examination. In parallel, asymptomatic carriers were actively detected at the end of each dry season in 2007, 2008 and 2009. RESULTS: Among the 965 presumptive malaria cases recorded, 29% were confirmed by microscopic examination. The incidence of malaria was found to decrease significantly with age (p < 0.01). The mean annual incidence was 0.254. The results show that the risk of malaria was higher in children under ten years (p < 0.0001). The number of malaria episodes generally followed the temporal pattern of changes in precipitation levels, with a peak of transmission in August and September. One-thousand and ninety subjects were submitted to an active detection of asymptomatic carriage of whom 16% tested positive; asymptomatic carriage decreased with increasing age. A higher prevalence of gametocyte carriage among asymptomatic population was recorded in children aged two to ten years, though it did not reach significance. CONCLUSIONS: In Southern Niger, malaria transmission mostly occurs from July to October. Children aged two to ten years are the most at risk of malaria, and may also represent the main reservoir for gametocytes. Strategies such as intermittent preventive treatment in children (IPTc) could be of interest in this area, where malaria transmission is highly seasonal. Based on these preliminary data, a pilot study could be implemented in Zindarou using IPTc targeting children aged two to ten years, during the three months of malaria transmission, together with an accurate monitoring of drug resistance.

A distinct peripheral blood monocyte phenotype is associated with parasite inhibitory activity in acute uncomplicated Plasmodium falciparum malaria.

Monocyte (MO) subpopulations display distinct phenotypes and functions which can drastically change during inflammatory states. We hypothesized that discrete MO subpopulations are induced during malaria infection and associated with anti-parasitic activity. We characterized the phenotype of blood MO from healthy malaria-exposed individuals and that of patients with acute uncomplicated malaria by flow cytometry. In addition, MO defense function was evaluated by an in vitro antibody dependent cellular inhibition (ADCI) assay. At the time of admission, the percentages and absolute numbers of CD16+ MO, and CCR2+CX3CR1+ MO, were high in a majority of patients. Remarkably, expression of CCR2 and CX3CR1 on the CD14(high (hi)) MO subset defined two subgroups of patients that also differed significantly in their functional ability to limit the parasite growth, through the ADCI mechanism. In the group of patients with the highest percentages and absolute numbers of CD14(hi)CCR2+CX3CR1+ MO and the highest mean levels of ADCI activity, blood parasitemias were lower (0.14+/-0.34%) than in the second group (1.30+/-3.34%; p = 0.0053). Data showed that, during a malaria attack, some patients' MO can exert a strong ADCI activity. These results bring new insight into the complex relationships between the phenotype and the functional activity of blood MO from patients and healthy malaria-exposed individuals and suggest discrete MO subpopulations are induced during malaria infection and are associated with anti-parasitic activity.

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.

Toward the rational design of a malaria vaccine construct using the MSP3 family as an example: contribution of antigenicity studies in humans.

Plasmodium falciparum merozoite surface protein (MSP3) is a main target of protective immunity against malaria that is currently undergoing vaccine development. It was shown recently to belong, together with MSP6, to a new multigene family whose C-terminal regions have a similar organization, contain both homologous and divergent regions, and are highly conserved across isolates. In an attempt to rationally design novel vaccine constructs, we extended the analysis of antigenicity and function of region-specific antibodies, previously performed with MSP3 and MSP6, to the remaining four proteins of the MSP3 family using four recombinant proteins and 24 synthetic peptides. Antibodies to each MSP3 family antigen were found to be highly prevalent among malaria-exposed individuals from the village of Dielmo (Senegal). Each of the 24 peptides was antigenic, defining at least one epitope mimicking that of the native proteins, with a distinct IgG isotype pattern for each, although with an overall predominance of the IgG3 subclass. Human antibodies affinity purified upon each of the 24 peptides exerted an antiparasite antibody-dependent cellular inhibition effect, which in most cases was as strong as that of IgG from protected African adults. The two regions with high homology were found to generate a broad network of cross-reactive antibodies with various avidities. A first multigenic construct was designed using these findings and those from related immunogenicity studies in mice and demonstrated valuable immunological properties. These results indicate that numerous regions from the MSP3 family play a role in protection and provide a rationale for the tailoring of new MSP3-derived malaria vaccines.

Toward the rational design of a malaria vaccine construct using the MSP3 family as an example: contribution of immunogenicity studies in models.

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.

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.

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

BACKGROUND: 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 Plasmodium falciparum 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 Anopheles gambiae, despite anti-malarial prophylactic treatment. METHODS: Ab responses to 13 pre-erythrocytic P. falciparum-specific peptides derived from the proteins Lsa1, Lsa3, Glurp, Salsa, Trap, Starp, CSP and Pf11.1, and to 2 peptides specific for the Anopheles gambiae 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. RESULTS: 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. CONCLUSION: 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.

Evidence for multiple B- and T-cell epitopes in Plasmodium falciparum liver-stage antigen 3.

Liver-stage antigen 3 (LSA-3) is a new vaccine candidate that can induce protection against Plasmodium falciparum sporozoite challenge. Using a series of long synthetic peptides (LSP) encompassing most of the 210-kDa LSA-3 protein, a study of the antigenicity of this protein was carried out in 203 inhabitants from the villages of Dielmo (n = 143) and Ndiop (n = 60) in Senegal (the level of malaria transmission differs in these two villages). Lymphocyte responses to each individual LSA-3 peptide were recorded, some at high prevalences (up to 43%). Antibodies were also detected to each of the 20 peptides, many at high prevalence (up to 84% of responders), and were directed to both nonrepeat and repeat regions. Immune responses to LSA-3 were detectable even in individuals of less than 5 years of age and increased with age and hence exposure to malaria, although they were not directly related to the level of malaria transmission. Thus, several valuable T- and B-cell epitopes were characterized all along the LSA-3 protein, supporting the antigenicity of this P. falciparum vaccine candidate. Finally, antibodies specific for peptide LSP10 located in a nonrepeat region of LSA-3 were found significantly associated with a lower risk of malaria attack over 1 year of daily clinical follow-up in children between the ages of 7 and 15 years, but not in older individuals.

Mild increases in serum hepcidin and interleukin-6 concentrations impair iron incorporation in haemoglobin during an experimental human malaria infection.

The correct selection of individuals who will benefit from iron supplements in malaria-endemic regions requires improved insight in the effects of malaria on host iron homeostasis and innovative biomarkers. We assessed sequential changes in serum hepcidin and in traditional biochemical iron status indicators during an experimental Plasmodium falciparum malaria infection with five adult volunteers. The haemoglobin content of reticulocytes (Ret-H(e)) and of mature red blood cells (RBC-H(e)) represented iron incorporation into haemoglobin. Low-density parasitaemia and its treatment induced a mild increase in interleukin (IL)-6 and serum hepcidin concentrations. Despite this only mild increase, a marked hypoferraemia with a strong increase in serum ferritin concentrations developed, which was associated with a sharp fall in Ret-H(e), while RBC-H(e) remained unchanged. The ratio of soluble transferrin receptor (sTfR) to log ferritin concentrations decreased to an average nadir of 63% of the baseline value. We concluded that even mild increases in serum hepcidin and IL-6 concentrations result in a disturbed host iron homeostasis. Serum hepcidin, Ret-H(e) and Delta-H(e) (Ret-H(e) minus RBC-H(e)) are promising biomarkers to select those individuals who will benefit from iron supplements in malaria endemic regions, while the sTfR/log ferritin ratio should be used with caution to assess iron status during malaria.

The malaria candidate vaccine liver stage antigen-3 is highly conserved in Plasmodium falciparum isolates from diverse geographical areas.

BACKGROUND: A high level of genetic stability has been formerly identified in segments of the gene coding for the liver stage antigen-3 (LSA-3), a subunit vaccine candidate against Plasmodium falciparum. The exploration of lsa-3 polymorphisms was extended to the whole sequence of this large antigen in 20 clinical isolates from four geographical areas; Senegal, Comoro islands, Brazil and Thailand. METHODS: The whole 4680 bp genomic sequence of lsa-3 was amplified by polymerase chain reaction and sequenced. The clinical isolate sequences were aligned on the sequence of the laboratory reference P. falciparum strain 3D7. RESULTS: The non-repeated sequence of lsa-3 was very well conserved with only a few allelic variations scattered along the sequence. Interestingly, a formerly identified immunodominant region, employed for the majority of pre-clinical vaccine development, was totally conserved at the genetic level. The most significant variations observed were in the number and organization of tetrapeptide repeated units, but not in their composition, resulting in different lengths of these repeated regions. The shorter repeated regions were from Brazilian origin. A correlation between the geographical distribution of the parasites with single nucleotide polymorphisms was not detected. CONCLUSION: The lack of correlation between allelic polymorphisms with a specific transmission pressure suggests that LSA-3 is a structurally constrained molecule. The unusual characteristics of the lsa-3 gene make the molecule an interesting candidate for a subunit vaccine against malaria.

Cardiac complication after experimental human malaria infection: a case report.

A 20 year-old healthy female volunteer participated in a clinical Phase I and IIa safety and efficacy trial with candidate malaria vaccine PfLSA-3-rec adjuvanted with aluminium hydroxide. Eleven weeks after the third and last immunization she was experimentally infected by bites of Plasmodium falciparum-infected mosquitoes. When the thick blood smear became positive, at day 11, she was treated with artemether/lumefantrine according to protocol. On day 16 post-infection i.e. two days after completion of treatment, she woke up with retrosternal chest pain. She was diagnosed as acute coronary syndrome and treated accordingly. She recovered quickly and her follow-up was uneventful. Whether the event was related to the study procedures such as the preceding vaccinations, malaria infection or antimalarial drugs remains elusive. However, the relation in time with the experimental malaria infection and apparent absence of an underlying condition makes the infection the most probable trigger. This is in striking contrast, however, with the millions of malaria cases each year and the fact that such complication has never been reported in the literature. The rare occurrence of cardiac events with any of the preceding study procedures may even support a coincidental finding. Apart from acute coronary syndrome, myocarditis can be considered as a final diagnosis, but the true nature and patho-physiological explanation of the event remain unclear.

Assessment of the relative success of sporozoite inoculations in individuals exposed to moderate seasonal transmission.

BACKGROUND: The time necessary for malaria parasite to re-appear in the blood following treatment (re-infection time) is an indirect method for evaluating the immune defences operating against pre-erythrocytic and early erythrocytic malaria stages. Few longitudinal data are available in populations in whom malaria transmission level had also been measured. METHODS: One hundred and ten individuals from the village of Ndiop (Senegal), aged between one and 72 years, were cured of malaria by quinine (25 mg/day oral Quinimax in three equal daily doses, for seven days). Thereafter, thick blood films were examined to detect the reappearance of Plasmodium falciparum every week, for 11 weeks after treatment. Malaria transmission was simultaneously measured weekly by night collection of biting mosquitoes. RESULTS: Malaria transmission was on average 15.3 infective bites per person during the 77 days follow up. The median reappearance time for the whole study population was 46.8 days, whereas individuals would have received an average one infective bite every 5 days. At the end of the follow-up, after 77 days, 103 of the 110 individuals (93.6%; CI 95% [89.0-98.2]) had been re-infected with P. falciparum. The median reappearance time ('re-positivation') was longer in subjects with patent parasitaemia at enrolment than in parasitologically-negative individuals (58 days vs. 45.9; p = 0.03) and in adults > 30 years than in younger subjects (58.6 days vs. 42.7; p = 0.0002). In a multivariate Cox PH model controlling for the sickle cell trait, G6PD deficiency and the type of habitat, the presence of parasitaemia at enrolment and age >/= 30 years were independently predictive of a reduced risk of re-infection (PH = 0.5 [95% CI: 0.3-0.9] and 0.4; [95% CI: 0.2-0.6] respectively). CONCLUSION: Results indicate the existence of a substantial resistance to sporozoites inoculations, but which was ultimately overcome in almost every individual after 2 1/2 months of natural challenges. Such a study design and the results obtained suggest that, despite a small sample size, this approach can contribute to assess the impact of intervention methods, such as the efficacy vector-control measures or of malaria pre-erythrocytic stages vaccines.

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.

Pre-erythrocytic stage malaria vaccines: time for a change in path.

Vaccines against the pre-erythrocytic stages of malaria hold the greatest promise as an effective intervention tool against malaria, as shown by immunization with radiation-attenuated sporozoites over four decades ago. To date, however, the development of subunit vaccines, while generating high expectations and investment, has not lived up at all to the promise. This path has been characterized by insufficient research into both identification of key defense mechanisms in humans and the discovery of better antigens, focusing rather on a technological race of how to present mainly a single antigen. The lack of success has also led, perhaps from desperation, to a revival of the live attenuated sporozoite approach, handicapped, however, by major bottlenecks in production, safety, and regulatory issues. It should now be clear that the field can no longer continue to succeed in mice and fail in the clinic. We advocate here in favor of a third option, relying on an understanding of the basis of attenuated sporozoite immunity in humans, to provide leads to the discovery of critical immunogens and the use of models with validated relevance to the human situation in order to rationalize and renew the promise of pre-erythrocytic subunit vaccines.

Evaluating Human Immune Responses for Vaccine Development in a Novel Human Spleen Cell-Engrafted NOD-SCID-IL2rγNull Mouse Model.

The lack of preclinical models able to faithfully predict the immune responses which are later obtained in the clinic is a major hurdle for vaccines development as it increases markedly the delays and the costs required to perform clinical studies. We developed and evaluated the relevance to human immune responses of a novel humanized mouse model, humanized-spleen cells-NOD-SCID-gamma null (Hu-SPL-NSG), in which we grafted human spleen cells in immunodeficient NOD-SCID-IL-2rγnull (NSG) mice. We selected the malaria vaccine candidate, Liver Stage Antigen 3-Full Length, because we had previously observed a major discrepancy between preclinical and clinical results, and compared its immunogenicity with that of a shorter form of the molecule, LSA3-729. NSG mice engrafted with human spleen lymphocytes were immunized with either LSA3-FL or LSA3-729, both adjuvanted with montanide ISA720. We found that the shorter LSA3-729 triggered the production of human antibodies and a T-helper-type 1 cellular immune response associated with protection whereas LSA3-FL did not. Results were consistent in five groups receiving lymphocytes from five distinct human donors. We identified antigenic regions in the full-length molecule, but not in the shorter version, which induced T-regulatory type of cellular responses. These regions had failed to be predicted by previous preclinical experiments in a wide range of animal models, including primates. Results were reproducible using spleen cells from all five human donors. The findings in the Hu-SPL-NSG model were similar to the results obtained using LSA3-FL in the clinic and hence could have been used to predict them. The model does not present graft versus host reaction, low survival of engrafted B lymphocytes and difficulty to raise primary immune responses, all limitations previously reported in humanized immune-compromised mice. Results also point to the shorter construct, LSA3-729 as a more efficient vaccine candidate. In summary, our findings indicate that the Hu-SPL-NSG model could be a relevant and cost-saving choice for early selection of vaccine candidates before clinical development, and deserves being further evaluated.

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.