Expression of Plasmodium falciparum erythrocyte membrane protein 1 in experimentally infected humans

BACKGROUND: Parasites causing severe malaria in non-immune patients express a restricted subset of variant surface antigens (VSA), which are better recognized by immune sera than VSA expressed during non-severe disease in semi-immune individuals. The most prominent VSA are the var gene-encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family, which is expressed on the surface of infected erythrocytes where it mediates binding to endothelial receptors. Thus, severe malaria may be caused by parasites expressing PfEMP1 variants that afford parasites optimal sequestration in immunologically naïve individuals and high effective multiplication rates. METHODS: var gene transcription was analysed using real time PCR and PfEMP1 expression by western blots as well as immune plasma recognition of parasite cultures established from non-immune volunteers shortly after infection with NF54 sporozoites. RESULTS: In cultures representing the first generation of parasites after hepatic release, all var genes were transcribed, but GroupA var genes were transcribed at the lowest levels. In cultures established from second or third generation blood stage parasites of volunteers with high in vivo parasite multiplication rates, the var gene transcription pattern differed markedly from the transcription pattern of the cultures representing first generation parasites. This indicated that parasites expressing specific var genes, mainly belonging to group A and B, had expanded more effectively in vivo compared to parasites expressing other var genes. The differential expression of PfEMP1 was confirmed at the protein level by immunoblot analysis. In addition, serological typing showed that immune sera more often recognized second and third generation parasites than first generation parasites. CONCLUSION: In conclusion, the results presented here support the hypothesis that parasites causing severe malaria express a subset of PfEMP1, which bestows high parasite growth rates in individuals with limited pre-existing immunity

Bacterium-like particles as multi-epitope delivery platform for Plasmodium berghei circumsporozoite protein induce complete protection against malaria in mice

Contains fulltext : 110364.pdf (publisher's version ) (Open Access)BACKGROUND: Virus-like particles have been regularly used as an antigen delivery system for a number of Plasmodium peptides or proteins. The present study reports the immunogenicity and protective efficacy of bacterium-like particles (BLPs) generated from Lactococcus lactis and loaded with Plasmodium berghei circumsporozoite protein (PbCSP) peptides. METHODS: A panel of BLP-PbCSP formulations differing in composition and quantity of B-cell, CD4+ and CD8+ T-cell epitopes of PbCSP were tested in BALB/c mice. RESULTS: BLP-PbCSP1 induced specific humoral responses but no IFN-gamma ELISPOT response, protecting 30-40% of the immunized mice. BLP-PbCSP2, with reduced length of the non-immunogenic part of the T-cell-epitopes construct, increased induction of IFN-gamma responses as well as protection up to 60-70%. Compared to controls, lower parasitaemia was observed in unprotected mice immunized with BLP-PbCSP1 or 2, suggestive for partial immunity. Finally, further increase of the number of B-cell epitopes and codon optimization (BLP-PbCSP4) induced the highest anti-CSP antibody levels and number of IFN-gamma spots, resulting in sterile immunity in 100% of the immunized mice. CONCLUSION: Presentation of Plasmodium-derived antigens using BLPs as a delivery system induced complete protection in a murine malaria model. Eventually, BLPs have the potential to be used as a novel versatile delivery platform in malaria vaccine development

Erratum to: Diagnosis and treatment based on quantitative PCR after controlled human malaria infection

Immunogenetics and cellular immunology of bacterial infectious disease

Extended Malaria Parasite Clearance Time in African Children Following Artemisinincombination Therapy Enhances Transmission\ud to Anopheles Mosquitoes

Artemisinin resistance was recently shown to have spread or emerged on the Thailand/Myanmar border. Evidence is accumulating that the parasite clearance time after artemisinin-based combination therapy (ACT) is increasing in settings in Asia and Africa. It is currently unknown if an extended parasite clearance time after ACTs has consequences for the individual patient or confers a higher malaria transmission potential. 298 children in Mbita, Western Kenya, with uncomplicated falciparum malaria were randomized to artemether-lumefantrine (AL, n = 153) ordihydroartemisinin-piperaquine (DP, n = 145). Parasite carriage post-treatment was determined by microscopy and qPCR, gametocyte carriage by quantitative nucleic acid sequence based amplication. Infectiousness to mosquitoes was determined by mosquito membrane feeding assays. Both drugs were efficacious as judged by standard trial outcomes. Sub-patent residual parasitaemia on day 3 was detected by qPCR in 36.11% (95% CI 25.11 - 48.29) of children treated with AL, and in 30.16% (95% CI 19.23 - 43.02) of children treated with DP. After adjustment for age, treatment arm and enrolment parasite density, children with an extended parasite clearance time were significantly more likely to have microscopically detected recurrent parasitaemia during follow-up (Odds Ratio: 19.51, 95% CI 5.24 - 72.71, p < 0.001). Children with an extended parasite clearance time were also more likely to be infectious to mosquitoes (Odds Ratio 2.76; 95% CI 1.14 - 6.67, p = 0.02) and gave rise to a higher oocyst load in mosquitoes (Incidence Rate Ratio 2.80, 95% CI 1.49 - 5.24, p = 0.001). Our findings indicate that an extended parasite clearance time after ACTs has consequences for the individual patient and for the population at large due to higher transmission potential. The high prevalence of residual subpatent parasitaemia after treatment may be due to novel parasite genotypes with reduced drug sensitivity, inadequate population-level immunity, or the higher sensitivity of qPCR for detection of persisting parasites.\u

Improving statistical inference on pathogen densities estimated by quantitative molecular methods: malaria gametocytaemia as a case study

BACKGROUND: Quantitative molecular methods (QMMs) such as quantitative real-time polymerase chain reaction (q-PCR), reverse-transcriptase PCR (qRT-PCR) and quantitative nucleic acid sequence-based amplification (QT-NASBA) are increasingly used to estimate pathogen density in a variety of clinical and epidemiological contexts. These methods are often classified as semi-quantitative, yet estimates of reliability or sensitivity are seldom reported. Here, a statistical framework is developed for assessing the reliability (uncertainty) of pathogen densities estimated using QMMs and the associated diagnostic sensitivity. The method is illustrated with quantification of Plasmodium falciparum gametocytaemia by QT-NASBA. RESULTS: The reliability of pathogen (e.g. gametocyte) densities, and the accompanying diagnostic sensitivity, estimated by two contrasting statistical calibration techniques, are compared; a traditional method and a mixed model Bayesian approach. The latter accounts for statistical dependence of QMM assays run under identical laboratory protocols and permits structural modelling of experimental measurements, allowing precision to vary with pathogen density. Traditional calibration cannot account for inter-assay variability arising from imperfect QMMs and generates estimates of pathogen density that have poor reliability, are variable among assays and inaccurately reflect diagnostic sensitivity. The Bayesian mixed model approach assimilates information from replica QMM assays, improving reliability and inter-assay homogeneity, providing an accurate appraisal of quantitative and diagnostic performance. CONCLUSIONS: Bayesian mixed model statistical calibration supersedes traditional techniques in the context of QMM-derived estimates of pathogen density, offering the potential to improve substantially the depth and quality of clinical and epidemiological inference for a wide variety of pathogens

Modest heterologous protection after Plasmodium falciparum sporozoite immunization: a double-blind randomized controlled clinical trial.

BACKGROUND: A highly efficacious vaccine is needed for malaria control and eradication. Immunization with Plasmodium falciparum NF54 parasites under chemoprophylaxis (chemoprophylaxis and sporozoite (CPS)-immunization) induces the most efficient long-lasting protection against a homologous parasite. However, parasite genetic diversity is a major hurdle for protection against heterologous strains. METHODS: We conducted a double-blind, randomized controlled trial in 39 healthy participants of NF54-CPS immunization by bites of 45 NF54-infected (n = 24 volunteers) or uninfected mosquitoes (placebo; n = 15 volunteers) against a controlled human malaria infection with the homologous NF54 or the genetically distinct NF135.C10 and NF166.C8 clones. Cellular and humoral immune assays were performed as well as genetic characterization of the parasite clones. RESULTS: NF54-CPS immunization induced complete protection in 5/5 volunteers against NF54 challenge infection at 14 weeks post-immunization, but sterilely protected only 2/10 and 1/9 volunteers against NF135.C10 and NF166.C8 challenge infection, respectively. Post-immunization plasma showed a significantly lower capacity to block heterologous parasite development in primary human hepatocytes compared to NF54. Whole genome sequencing showed that NF135.C10 and NF166.C8 have amino acid changes in multiple antigens targeted by CPS-induced antibodies. Volunteers protected against heterologous challenge were among the stronger immune responders to in vitro parasite stimulation. CONCLUSIONS: Although highly protective against homologous parasites, NF54-CPS-induced immunity is less effective against heterologous parasite clones both in vivo and in vitro. Our data indicate that whole sporozoite-based vaccine approaches require more potent immune responses for heterologous protection. TRIAL REGISTRATION: This trial is registered in clinicaltrials.gov, under identifier NCT02098590

A randomized feasibility trial comparing four antimalarial drug regimens to induce Plasmodium falciparum gametocytemia in the controlled human malaria infection model.

Background: Malaria elimination strategies require a thorough understanding of parasite transmission from human to mosquito. A clinical model to induce gametocytes to understand their dynamics and evaluate transmission-blocking interventions (TBI) is currently unavailable. Here, we explore the use of the well-established Controlled Human Malaria Infection model (CHMI) to induce gametocyte carriage with different antimalarial drug regimens. Methods: In a single centre, open-label randomised trial, healthy malaria-naive participants (aged 18–35 years) were infected with Plasmodium falciparum by bites of infected Anopheles mosquitoes. Participants were randomly allocated to four different treatment arms (n = 4 per arm) comprising low-dose (LD) piperaquine (PIP) or sulfadoxine-pyrimethamine (SP), followed by a curative regimen upon recrudescence. Male and female gametocyte densities were determined by molecular assays. Results: Mature gametocytes were observed in all participants (16/16, 100%). Gametocytes appeared 8.5–12 days after the first detection of asexual parasites. Peak gametocyte densities and gametocyte burden was highest in the LD-PIP/SP arm, and associated with the preceding asexual parasite biomass (p=0.026). Male gametocytes had a mean estimated circulation time of 2.7 days (95% CI 1.5–3.9) compared to 5.1 days (95% CI 4.1–6.1) for female gametocytes. Exploratory mosquito feeding assays showed successful sporadic mosquito infections. There were no serious adverse events or significant differences in the occurrence and severity of adverse events between study arms (p=0.49 and p=0.28). Conclusions: The early appearance of gametocytes indicates gametocyte commitment during the first wave of asexual parasites emerging from the liver. Treatment by LD-PIP followed by a curative SP regimen, results in the highest gametocyte densities and the largest number of gametocyte-positive days. This model can be used to evaluate the effect of drugs and vaccines on gametocyte dynamics, and lays the foundation for fulfilling the critical unmet need to evaluate transmission-blocking interventions against falciparum malaria for downstream selection and clinical development. Funding: Funded by PATH Malaria Vaccine Initiative (MVI). Clinical trial number: NCT02836002

Long Term Protection after Immunization with P. berghei Sporozoites Correlates with Sustained IFNγ Responses of Hepatic CD8+ Memory T Cells

Protection against P. berghei malaria can successfully be induced in mice by immunization with both radiation attenuated sporozoites (RAS) arresting early during liver stage development, or sporozoites combined with chloroquine chemoprophylaxis (CPS), resulting in complete intra-hepatic parasite development before killing of blood-stages by chloroquine takes place. We assessed the longevity of protective cellular immune responses by RAS and CPS P. berghei immunization of C57BL/6j mice. Strong effector and memory (TEM) CD8+ T cell responses were induced predominantly in the liver of both RAS and CPS immunized mice while CD4+ T cells with memory phenotype remained at base line levels. Compared to unprotected naïve mice, we found high sporozoite-specific IFNγ ex vivo responses that associated with induced levels of in vivo CD8+ TEM cells in the liver but not spleen. Long term evaluation over a period of 9 months showed a decline of malaria-specific IFNγ responses in RAS and CPS mice that significantly correlated with loss of protection (r2 = 0.60, p<0.0001). The reducing IFNγ response by hepatic memory CD8+ T cells could be boosted by re-exposure to wild-type sporozoites. Our data show that sustainable protection against malaria associates with distinct intra-hepatic immune responses characterized by strong IFNγ producing CD8+ memory T cells

Phase 1/2a Study of the Malaria Vaccine Candidate Apical Membrane Antigen-1 (AMA-1) Administered in Adjuvant System AS01B or AS02A

Contains fulltext : 79496.pdf (publisher's version ) (Open Access)BACKGROUND: This Phase 1/2a study evaluated the safety, immunogenicity, and efficacy of an experimental malaria vaccine comprised of the recombinant Plasmodium falciparum protein apical membrane antigen-1 (AMA-1) representing the 3D7 allele formulated with either the AS01B or AS02A Adjuvant Systems. METHODOLOGY/PRINCIPAL FINDINGS: After a preliminary safety evaluation of low dose AMA-1/AS01B (10 microg/0.5 mL) in 5 adults, 30 malaria-naive adults were randomly allocated to receive full dose (50 microg/0.5 mL) of AMA-1/AS01B (n = 15) or AMA-1/AS02A (n = 15), followed by a malaria challenge. All vaccinations were administered intramuscularly on a 0-, 1-, 2-month schedule. All volunteers experienced transient injection site erythema, swelling and pain. Two weeks post-third vaccination, anti-AMA-1 Geometric Mean Antibody Concentrations (GMCs) with 95% Confidence Intervals (CIs) were high: low dose AMA-1/AS01B 196 microg/mL (103-371 microg/mL), full dose AMA-1/AS01B 279 microg/mL (210-369 microg/mL) and full dose AMA-1/AS02A 216 microg/mL (169-276 microg/mL) with no significant difference among the 3 groups. The three vaccine formulations elicited equivalent functional antibody responses, as measured by growth inhibition assay (GIA), against homologous but not against heterologous (FVO) parasites as well as demonstrable interferon-gamma (IFN-gamma) responses. To assess efficacy, volunteers were challenged with P. falciparum-infected mosquitoes, and all became parasitemic, with no significant difference in the prepatent period by either light microscopy or quantitative polymerase chain reaction (qPCR). However, a small but significant reduction of parasitemia in the AMA-1/AS02A group was seen with a statistical model employing qPCR measurements. SIGNIFICANCE: All three vaccine formulations were found to be safe and highly immunogenic. These immune responses did not translate into significant vaccine efficacy in malaria-naive adults employing a primary sporozoite challenge model, but encouragingly, estimation of parasite growth rates from qPCR data may suggest a partial biological effect of the vaccine. Further evaluation of the immunogenicity and efficacy of the AMA-1/AS02A formulation is ongoing in a malaria-experienced pediatric population in Mali. TRIAL REGISTRATION: www.clinicaltrials.gov NCT00385047