Search

Plasmodium parasites are the causative agent of human malaria, and the development of a highly effective vaccine against infection, disease and transmission remains a key priority. It is widely established that multiple stages of the parasite's complex lifecycle within the human host and mosquito vector are susceptible to vaccine-induced antibodies. The mainstay approach to antibody induction by subunit vaccination has been the delivery of protein antigen formulated in adjuvant. Extensive efforts have been made in this endeavor with respect to malaria vaccine development, especially with regard to target antigen discovery, protein expression platforms, adjuvant testing, and development of soluble and virus-like particle (VLP) delivery platforms. The breadth of approaches to protein-based vaccines is continuing to expand as innovative new concepts in next-generation subunit design are explored, with the prospects for the development of a highly effective multi-component/multi-stage/multi-antigen formulation seeming ever more likely. This review will focus on recent progress in protein vaccine design, development and/or clinical testing for a number of leading malaria antigens from the sporozoite-, merozoite- and sexual-stages of the parasite's lifecycle-including PfCelTOS, PfMSP1, PfAMA1, PfRH5, PfSERA5, PfGLURP, PfMSP3, Pfs48/45 and Pfs25. Future prospects and challenges for the development, production, human delivery and assessment of protein-based malaria vaccines are discussed.

P. falciparum Merozoite Surface Protein-1 (MSP-1) is a leading erythrocytic-stage vaccine candidate. It is proteolytically processed to several fragments, and may play a role in binding and/or invasion of erythrocytes. During erythrocyte invasion secondary processing on MSP-1(42) gives a 33kDa and a 19kDa fragment (MSP-1(19)). Although, MSP-1(19) is a target for parasite inhibitory mAbs and protective immunity, it appears to lack T-helper epitopes. Since antibody is likely the effector mechanism induced by MSP-1-(19), it is important to insure that recombinant vaccines based upon this antigen be folded correctly and contain T-helper epitopes that will enhance induction of humoral responses. We developed recombinant MSP-1(42) that was structurally correct based on positive reactivities with conformation-dependent mAbs, including 12.10,12.8. 7.5, 7.2, and I El. Purification over three chromatographic steps that include nickel affinity chromatography, Q-substituted and CM-substituted resins, yielded protein that was 95% pure and exceeded FDA endotoxin standards. Mice seroconverted following immunization with recombinant MSP-1(42). Safety and immunogenicity testing in Rhesus monkeys revealed that MSP-1(42) /SBAS2, was safe and induced MSP-1(42)-specific antibody responses.

P. falciparum Merozoite Surface Protein-1 (MSP-1) is a leading erythrocytic stage vaccine candidate. Following secondary proteolytic processing, it may play a role in binding and/or invasion of erythrocytes. The secondary processing gives a 33kD and 19kD fragment. The MSP1-19 is a target for parasite inhibitory antibodies and protective immunity. However, it appears to lack T-helper epitopes. Since antibody is likely the effector mechanism induced by MSP1-19, it is important to insure that recombinant vaccines based on this antigen be folded correctly and contain T-helper epitopes that will enhance induction of humoral responses. We have developed recombinant MSP1-42 molecules that are structurally correct based on positive reactivities with conformation-dependent mAbs. These proteins have been produced to greater than 95% purity and exceed FDA endotoxin standards. Immunogenicity studies in mice and Rhesus monkeys reveal that good antibody responses are induced to MSP1-42 when these animals are immunized with the MSP1-42 plus SBAS2 adjuvant., Original contains color plates: All DTIC reproductions will be in black and white.

Malarial blood stage antigens can produce humoral immunity by induction of protective antibodies to epitopes on surface antigens on parasites. The merozoite surface protein-1 (MSP1) of P. falciparum is a major blood-stage vaccine candidate. MSP1 may play a role in binding and/or infection of erythrocytes by merozoites (parasites) during red blood cell invasion. The C-terminal fragment of MSP1, MSP1(sub 19), is highly conserved among all known strains of P. falciparum. immunization with recombinant proteins that have native-like conformations may elicit long-lasting protective immunity that mimics the natural immunity. We have used bacterial expression to tightly regulate transcription and translation of MSP1(sub 42). Plasmids that encode MSP1(sub 42) were prepared and tested for their ability to express recombinant protein. A three column purification scheme that included Ni(+2) metal chelate chromatography, anion and then cation exchange chromatography, yielded greater than 95% pure MSP1(sub 42). Immunoreactivity with mAbs showed that recombinant MSP1(sub 42) was conformationally similar to native MSP1. The objectives were to develop recombinant MSP1(sub 42) molecules that were structurally correct, to develop fermentation and purification processes that could be scaled-up for large-scale processes, and ultimately, to advance these products into human clinical trials.

Malarial erythrocytic-stage antigens produce humoral immunity by inducing protective antibodies to epitopes on surface antigens on the parasites. The merozoite surface protein-1 (MSP1) of P. falciparum is a major malaria erythrocytic-stage vaccine candidate. MSP1 may play a role in binding and/or infection of erythrocytes by merozoites (parasites) during red blood cell invasion. The C-terminal fragment of MSP1, MSP1(42), is highly conserved among all known strains of P. falciparum. However, it is folded into a complex structure containing six possible disulfide bridges. Immunization with recombinant proteins that have native-like conformations may elicit long-lasting protective immunity that mimics the natural development of humoral immunity. We have used a novel bacterial expression system that utilizes tightly regulatable transcription and translation signals to control expression of potentially toxic heterologous proteins. Several plasmids encoding MSP1(42) were constructed and tested for their ability to express recombinant protein. These constructs were designed for purification using Ni(+2)-chelating chromatography. Our results show that the recombinant proteins contained conformationally similar structures to the native parasites. The objectives were to develop recombinant MSP1(42) molecules that were structurally correct, (b) to develop fermentation and purification processes that could be scaled-up for large-scale processes, and ultimately (c) to advance these products into human clinical trials.

Vaccine development efforts will be guided by algorithms that predict immunogenic epitopes. Such prediction methods rely on classification-based algorithms that are trained against curated data sets of known B and T cell epitopes. It is unclear whether this empirical approach can be applied prospectively to predict epitopes associated with protective immunity for novel antigens. We present a comprehensive comparison of in silico B and T cell epitope predictions with in vivo validation using an previously uncharacterized malaria antigen, CelTOS. CelTOS has no known conserved structural elements with any known proteins, and thus is not represented in any epitope databases used to train prediction algorithms. This analysis represents a blind assessment of this approach in the context of a novel, immunologically relevant antigen. The limited accuracy of the tested algorithms to predict the in vivo immune responses emphasizes the need to improve their predictive capabilities for use as tools in vaccine design., Published in PLOS ONE, v8 n8 srticle ID e71610, 16 Aug 2013. Prepared in collaboration with the Malaria Vaccine Branch, U.S. Military Malaria Vaccine Program, Walter Reed Army Institute of Research, Silver Spring. MD and the U.S. Military HIV Research Program, Division of Retrovirology, Silver Spring, MD. Prepared in cooperation with Vital Probes, Inc., Mayfield, PA. Sponsored in part by USAID.

BackgroundTo guide malaria elimination efforts in Swaziland and other countries, accurate assessments of transmission are critical. Pooled-PCR has potential to efficiently improve sensitivity to detect infections; serology may clarify temporal and spatial trends in exposure.Methodology/principal findingsUsing a stratified two-stage cluster, cross-sectional design, subjects were recruited from the malaria endemic region of Swaziland. Blood was collected for rapid diagnostic testing (RDT), pooled PCR, and ELISA detecting antibodies to Plasmodium falciparum surface antigens. Of 4330 participants tested, three were RDT-positive yet false positives by PCR. Pooled PCR led to the identification of one P. falciparum and one P. malariae infection among RDT-negative participants. The P. falciparum-infected participant reported recent travel to Mozambique. Compared to performing individual testing on thousands of samples, PCR pooling reduced labor and consumable costs by 95.5%. Seropositivity was associated with age ≥20 years (11·7% vs 1·9%, P<0.001), recent travel to Mozambique (OR 4.4 [95% CI 1.0-19.0]) and residence in southeast Swaziland (RR 3.78, P<0.001).ConclusionsThe prevalence of malaria infection and recent exposure in Swaziland are extremely low, suggesting elimination is feasible. Future efforts should address imported malaria and target remaining foci of transmission. Pooled PCR and ELISA are valuable surveillance tools for guiding elimination efforts.

Background Assessment of malaria endemicity at different altitudes and transmission intensities, in the era of dwindling vector densities in the highlands, will provide valuable information for malaria control and surveillance. Measurement of serum anti-malarial antibodies is a useful marker of malaria exposure that indicates long-term transmission potential. We studied the serologic evidence of malaria endemicity at two highland sites along a transmission intensity cline. An improved understanding of the micro-geographic variation in malaria exposure in the highland ecosystems will be relevant in planning effective malaria control. Methods Total IgG levels to Plasmodium falciparum MSP-119 were measured in an age-stratified cohort (< 5, 5-14 and ≥ 15 years) in 795 participants from an uphill and valley bottom residents during low and high malaria transmission seasons. Antibody prevalence and level was compared between different localities. Regression analysis was performed to examine the association between antibody prevalence and parasite prevalence. Age-specific MSP-119 seroprevalence data was fitted to a simple reversible catalytic model to investigate the relationship between parasite exposure and age. Results Higher MSP-119 seroprevalence and density were observed in the valley residents than in the uphill dwellers. Adults (> 15 years) recorded high and stable immune response in spite of changing seasons. Lower responses were observed in children (≤ 15 years), which, fluctuated with changing seasons particularly in the valley residents. In the uphill population, annual seroconversion rate (SCR) was 8.3% and reversion rate was 3.0%, with seroprevalence reaching a plateau of 73.3% by age of 20. Contrary, in the valley bottom population, the annual SCR was 35.8% and the annual seroreversion rate was 3.5%, and seroprevalence in the population had reached 91.2% by age 10. Conclusion The study reveals the micro-geographic variation in malaria endemicit

Background Assessment of malaria endemicity at different altitudes and transmission intensities, in the era of dwindling vector densities in the highlands, will provide valuable information for malaria control and surveillance. Measurement of serum anti-malarial antibodies is a useful marker of malaria exposure that indicates long-term transmission potential. We studied the serologic evidence of malaria endemicity at two highland sites along a transmission intensity cline. An improved understanding of the micro-geographic variation in malaria exposure in the highland ecosystems will be relevant in planning effective malaria control. Methods Total IgG levels to Plasmodium falciparum MSP-119 were measured in an age-stratified cohort (< 5, 5-14 and ≥ 15 years) in 795 participants from an uphill and valley bottom residents during low and high malaria transmission seasons. Antibody prevalence and level was compared between different localities. Regression analysis was performed to examine the association between antibody prevalence and parasite prevalence. Age-specific MSP-119 seroprevalence data was fitted to a simple reversible catalytic model to investigate the relationship between parasite exposure and age. Results Higher MSP-119 seroprevalence and density were observed in the valley residents than in the uphill dwellers. Adults (> 15 years) recorded high and stable immune response in spite of changing seasons. Lower responses were observed in children (≤ 15 years), which, fluctuated with changing seasons particularly in the valley residents. In the uphill population, annual seroconversion rate (SCR) was 8.3% and reversion rate was 3.0%, with seroprevalence reaching a plateau of 73.3% by age of 20. Contrary, in the valley bottom population, the annual SCR was 35.8% and the annual seroreversion rate was 3.5%, and seroprevalence in the population had reached 91.2% by age 10. Conclusion The study reveals the micro-geographic variation in malaria endemicit

BackgroundTo guide malaria elimination efforts in Swaziland and other countries, accurate assessments of transmission are critical. Pooled-PCR has potential to efficiently improve sensitivity to detect infections; serology may clarify temporal and spatial trends in exposure.Methodology/principal findingsUsing a stratified two-stage cluster, cross-sectional design, subjects were recruited from the malaria endemic region of Swaziland. Blood was collected for rapid diagnostic testing (RDT), pooled PCR, and ELISA detecting antibodies to Plasmodium falciparum surface antigens. Of 4330 participants tested, three were RDT-positive yet false positives by PCR. Pooled PCR led to the identification of one P. falciparum and one P. malariae infection among RDT-negative participants. The P. falciparum-infected participant reported recent travel to Mozambique. Compared to performing individual testing on thousands of samples, PCR pooling reduced labor and consumable costs by 95.5%. Seropositivity was associated with age ≥20 years (11·7% vs 1·9%, P<0.001), recent travel to Mozambique (OR 4.4 [95% CI 1.0-19.0]) and residence in southeast Swaziland (RR 3.78, P<0.001).ConclusionsThe prevalence of malaria infection and recent exposure in Swaziland are extremely low, suggesting elimination is feasible. Future efforts should address imported malaria and target remaining foci of transmission. Pooled PCR and ELISA are valuable surveillance tools for guiding elimination efforts.

(See the article by Greenhouse et al, on pages 19-26.) Background. RTS,S/AS01(E) is the lead candidate malaria vaccine and confers pre-erythrocytic immunity. Vaccination may therefore impact acquired immunity to blood-stage malaria parasites after natural infection. Methods. We measured, by enzyme-linked immunosorbent assay, antibodies to 4 Plasmodium falciparum merozoite antigens (AMA-1, MSP-1(42), EBA-175, and MSP-3) and by growth inhibitory activity (GIA) using 2 parasite clones (FV0 and 3D7) at 4 times on 860 children who were randomized to receive with RTS,S/AS01(E) or a control vaccine. Results. Antibody concentrations to AMA-1, EBA-175, and MSP-1(42) decreased with age during the first year of life, then increased to 32 months of age. Anti-MSP-3 antibody concentrations gradually increased, and GIA gradually decreased up to 32 months. Vaccination with RTS,S/AS01(E) resulted in modest reductions in AMA-1, EBA-175, MSP-1(42), and MSP-3 antibody concentrations and no significant change in GIA. Increasing anti-merozoite antibody concentrations and GIA were prospectively associated with increased risk of clinical malaria. Conclusions. Vaccination with RTS,S/AS01E reduces exposure to blood-stage parasites and, thus, reduces anti-merozoite antigen antibody concentrations. However, in this study, these antibodies were not correlates of clinical immunity to malaria. Instead, heterogeneous exposure led to confounded, positive associations between increasing antibody concentration and increasing risk of clinical malaria.

(See the article by Greenhouse et al, on pages 19-26.) Background. RTS,S/AS01(E) is the lead candidate malaria vaccine and confers pre-erythrocytic immunity. Vaccination may therefore impact acquired immunity to blood-stage malaria parasites after natural infection. Methods. We measured, by enzyme-linked immunosorbent assay, antibodies to 4 Plasmodium falciparum merozoite antigens (AMA-1, MSP-1(42), EBA-175, and MSP-3) and by growth inhibitory activity (GIA) using 2 parasite clones (FV0 and 3D7) at 4 times on 860 children who were randomized to receive with RTS,S/AS01(E) or a control vaccine. Results. Antibody concentrations to AMA-1, EBA-175, and MSP-1(42) decreased with age during the first year of life, then increased to 32 months of age. Anti-MSP-3 antibody concentrations gradually increased, and GIA gradually decreased up to 32 months. Vaccination with RTS,S/AS01(E) resulted in modest reductions in AMA-1, EBA-175, MSP-1(42), and MSP-3 antibody concentrations and no significant change in GIA. Increasing anti-merozoite antibody concentrations and GIA were prospectively associated with increased risk of clinical malaria. Conclusions. Vaccination with RTS,S/AS01E reduces exposure to blood-stage parasites and, thus, reduces anti-merozoite antigen antibody concentrations. However, in this study, these antibodies were not correlates of clinical immunity to malaria. Instead, heterogeneous exposure led to confounded, positive associations between increasing antibody concentration and increasing risk of clinical malaria.

(See the article by Greenhouse et al, on pages 19-26.) Background. RTS,S/AS01(E) is the lead candidate malaria vaccine and confers pre-erythrocytic immunity. Vaccination may therefore impact acquired immunity to blood-stage malaria parasites after natural infection. Methods. We measured, by enzyme-linked immunosorbent assay, antibodies to 4 Plasmodium falciparum merozoite antigens (AMA-1, MSP-1(42), EBA-175, and MSP-3) and by growth inhibitory activity (GIA) using 2 parasite clones (FV0 and 3D7) at 4 times on 860 children who were randomized to receive with RTS,S/AS01(E) or a control vaccine. Results. Antibody concentrations to AMA-1, EBA-175, and MSP-1(42) decreased with age during the first year of life, then increased to 32 months of age. Anti-MSP-3 antibody concentrations gradually increased, and GIA gradually decreased up to 32 months. Vaccination with RTS,S/AS01(E) resulted in modest reductions in AMA-1, EBA-175, MSP-1(42), and MSP-3 antibody concentrations and no significant change in GIA. Increasing anti-merozoite antibody concentrations and GIA were prospectively associated with increased risk of clinical malaria. Conclusions. Vaccination with RTS,S/AS01E reduces exposure to blood-stage parasites and, thus, reduces anti-merozoite antigen antibody concentrations. However, in this study, these antibodies were not correlates of clinical immunity to malaria. Instead, heterogeneous exposure led to confounded, positive associations between increasing antibody concentration and increasing risk of clinical malaria.

The basic premise of vaccination is the triggering of host immune responses leading to the induction of adaptive immunity having sufficient magnitude and duration to provide long term protection. This has been achieved by many licensed vaccines, the majority based on attenuated or Inactivated organisms, although often the protective antigens and underlying molecular -- mechanisms have not been Identified. However, this traditional approach has not led to the development of a licensed vaccine for malaria or for several other devastating infectious diseases. Recendy, substantial efforts have been focused on applying rational molecular design principles toward the development of novel vaccines for these refractory pathogens. In this review, we discuss the molecular aspects of antigen design, adjuvant advancement and the development of vaccine delivery systems as they are being applied to malaria vaccines., Pub in Human Vaccines, v6 n1 p54-77, January 2010.

The Plasmodium protein Cell-traversal protein for ookinetes and sporozoites (CelTOS) plays an important role in cell traversal of host cells in both, mosquito and vertebrates, and is required for successful malaria infections. CelTOS is highly conserved among the Plasmodium species, suggesting an important functional role across all species. Therefore targeting the immune response to this highly conserved protein and thus potentially interfering with its biological function may result in protection against infection even by heterologous species of Plasmodium. Methodology/Principal Findings: To test this hypothesis, we developed a recombinant codon-harmonized P. falciparum CelTOS protein that can be produced to high yields in the E. coli expression system. Inbred Balb/c and outbred CD-1 mice were immunized with various doses of the recombinant protein adjuvanted with Montanide ISA 720 and characterized using in vitro and in vivo analyses. Conclusions/Significance: Immunization with PfCelTOS resulted in potent humoral and cellular immune responses and most importantly induced sterile protection against a heterologous challenge with P. berghei sporozoites in a proportion of both inbred and outbred mice. The biological activity of CelTOS-specific antibodies against the malaria parasite is likely linked to the impairment of sporozoite motility and hepatocyte infectivity. The results underscore the potential of this antigen as a pre-erythrocytic vaccine candidate and demonstrate for the first time a malaria vaccine that is cross-protective between species., Pub. in PLoS One, v5 n8, p1-9, Aug 2010.

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 mg/0.5 mL) in 5 adults, 30 malaria-naive adults were randomly allocated to receive full dose (50 mg/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 mg/mL (103-371 mg/mL), full-dose AMA-1/AS01B 279 mg/mL (210-369 mg/mL) and full-dose AMA-1/AS02A 216 mg/mL (169-276 mg/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., The original document contains color images. Pub. in PLoS One, v4 n4, p1-13,-Apr 2009. Sponsored by National Institution of Health and Walter Reed Army Institution of Research.