Antibodies to pre-erythrocytic Plasmodium falciparum antigens and risk of clinical malaria in Kenyan children

BACKGROUND: IgG antibodies to pre-erythrocytic antigens are involved in prevention of infection and disease in animal models of malaria but have not been associated with protection against disease in human malaria. METHODS: Levels of IgG antibodies to circumsporozoite protein (CSP), liver-stage antigen type 1 (LSA-1), and thrombospondin-related adhesive protein (TRAP) were measured in 86 children in a malaria-holoendemic area of Kenya. The children were then monitored for episodes of clinical malaria for 52 weeks. RESULTS: Children with high levels of IgG antibodies to CSP, LSA-1, and TRAP had a decreased risk of clinical malaria (adjusted hazard ratio, 0.29; 95% confidence interval 0.10-0.81; P = .02), a lower incidence of clinical malaria (P=.006), protection from clinical malaria with a parasite level of \u3e or =4000 parasites/microL (P= .03), and a higher hemoglobin level at enrollment (P= .009), compared with children with lower antibody levels. Protection against malaria morbidity was associated primarily with antibodies to CSP and LSA-1. CONCLUSIONS: Kenyan children with high levels of IgG antibodies to the pre-erythrocytic antigens CSP, LSA-1, and TRAP have a lower risk of developing clinical malaria than children without high levels of these antibodies. The decreased risk of clinical malaria may be mediated in part by prevention of high-density parasitemia

Correlation of high levels of antibodies to multiple pre-erythrocytic Plasmodium falciparum antigens and protection from infection

High levels of antibodies to multiple antigens may be more strongly associated with protection from infection than antibodies to a single antigen. Antibody-associated protection against Plasmodium falciparum infection was assessed in a cohort of 68 adults living in an area of holoendemic malaria in Kenya. Antibodies to the pre-erythrocytic antigens circumsporozoite protein (CSP), liver-stage antigen-1 (LSA-1), thrombospondin-related adhesive protein (TRAP), and blood-stage antigens apical membrane antigen-1 (AMA-1), erythrocyte binding antigen-175 (EBA-175), and merozoite surface protein 1 (MSP-1) were tested. Peptides were used for CSP (NANP repeat) and LSA-1 (central repeat), and recombinant antigens were used for TRAP (aa D(48)-K(394)), AMA-1 (ectodomain, non-glycosylated), EBA-175 (non-glycosylated), and MSP-1 (MSP-1(19)). Weekly microscopy testing for P. falciparum infection was performed over a 12-week period after drug-mediated clearance of P. falciparum parasitemia. Individuals with high levels of IgG antibodies (\u3e 2 arbitrary units) to CSP, LSA-1, and TRAP had a 57% decrease in the risk of infection (95% confidence interval = 20-77%, P = 0.016). This decreased risk remained significant after adjustment for age, prior parasitemia, bed net use, sickle cell trait, and village of residence. In contrast, protection against infection did not correlate with high levels of IgG antibodies to blood-stage antigens or IgM antibodies to pre-erythrocytic or blood-stage antigens. High levels of IgG antibodies to CSP, LSA-1, and TRAP may be useful immune correlates of protection against P. falciparum infection in malaria-endemic populations

Plasmodium falciparum liver stage antigen-1 is cross-linked by tissue transglutaminase

Background Plasmodium falciparum sporozoites injected by mosquitoes into the blood rapidly enter liver hepatocytes and undergo pre-erythrocytic developmental schizogony forming tens of thousands of merozoites per hepatocyte. Shortly after hepatocyte invasion, the parasite starts to produce Liver Stage Antigen-1 (LSA-1), which accumulates within the parasitophorous vacuole surrounding the mass of developing merozoites. The LSA-1 protein has been described as a flocculent mass, but its role in parasite development has not been determined. Methods Recombinant N-terminal, C-terminal or a construct containing both the N- and C- terminal regions flanking two 17 amino acid residue central repeat sequences (LSA-NRC) were subjected to in vitro modification by tissue transglutaminase-2 (TG2) to determine if cross-linking occurred. In addition, tissue sections of P. falciparum-infected human hepatocytes were probed with monoclonal antibodies to the isopeptide ε-(γ-glutamyl)lysine cross-bridge formed by TG2 enzymatic activity to determine if these antibodies co-localized with antibodies to LSA-1 in the growing liver schizonts. Results This study identified a substrate motif for (TG2) and a putative casein kinase 2 phosphorylation site within the central repeat region of LSA-1. The function of TG2 is the post-translational modification of proteins by the formation of a unique isopeptide ε-(γ-glutamyl)lysine cross-bridge between glutamine and lysine residues. When recombinant LSA-1 protein was crosslinked in vitro by purified TG2 in a calcium dependent reaction, a flocculent mass of protein was formed that was highly resistant to degradation. The cross-linking was not detectably affected by phosphorylation with plasmodial CK2 in vitro. Monoclonal antibodies specific to the very unique TG2 catalyzed ε- lysine cross-bridge co-localized with antibodies to LSA-1 in infected human hepatocytes providing visual evidence that LSA-1 was cross-linked in vivo. Conclusions While the role of LSA-1 is still unknown these results suggest that it becomes highly cross-linked which may aid in the protection of the parasite as it develo

High Antibody Titer against Apical Membrane Antigen-1 Is Required to Protect against Malaria in the Aotus Model

A Plasmodium falciparum 3D7 strain Apical Membrane Antigen-1 (AMA1) vaccine, formulated with AS02A adjuvant, slowed parasite growth in a recent Phase 1/2a trial, however sterile protection was not observed. We tested this AS02A, and a Montanide ISA720 (ISA) formulation of 3D7 AMA1 in Aotus monkeys. The 3D7 parasite does not invade Aotus erythrocytes, hence two heterologous strains, FCH/4 and FVO, were used for challenge, FCH/4 AMA1 being more homologous to 3D7 than FVO AMA1. Following three vaccinations, the monkeys were challenged with 50,000 FCH/4 or 10,000 FVO parasites. Three of the six animals in the AMA+ISA group were protected against FCH/4 challenge. One monkey did not become parasitemic, another showed only a short period of low level parasitemia that self-cured, and a third animal showed a delay before exhibiting its parasitemic phase. This is the first protection shown in primates with a recombinant P. falciparum AMA1 without formulation in Freund's complete adjuvant. No animals in the AMA+AS02A group were protected, but this group exhibited a trend towards reduced growth rate. A second group of monkeys vaccinated with AMA+ISA vaccine was not protected against FVO challenge, suggesting strain-specificity of AMA1-based protection. Protection against FCH/4 strain correlated with the quantity of induced antibodies, as the protected animals were the only ones to have in vitro parasite growth inhibitory activity of >70% at 1∶10 serum dilution; immuno-fluorescence titers >8,000; ELISA titers against full-length AMA1 >300,000 and ELISA titer against AMA1 domains1+2 >100,000. A negative correlation between log ELISA titer and day 11 cumulative parasitemia (Spearman rank r = −0.780, p value = 0.0001), further confirmed the relationship between antibody titer and protection. High titers of cross-strain inhibitory antibodies against AMA1 are therefore critical to confer solid protection, and the Aotus model can be used to down-select future AMA1 formulations, prior to advanced human trials

Elevated anti-Zta IgG levels and EBV viral load are associated with site of tumor presentation in endemic Burkitt's lymphoma patients: a case control study

<p>Abstract</p> <p>Background</p> <p>Endemic Burkitt's lymphoma (BL) is an extranodal tumor appearing predominantly in the jaw in younger children while abdominal tumors predominate with increasing age. Previous studies have identified elevated levels of antibodies to <it>Plasmodium falciparum </it>schizont extracts and Epstein-Barr virus (EBV) viral capsid antigens (VCA) in endemic BL relative to malaria exposed controls. However, these studies have neither determined if there were any differences based on the site of clinical presentation of the tumor nor examined a broader panel of EBV and <it>P. falciparum </it>antigens.</p> <p>Methods</p> <p>We used a suspension bead Luminex assay to measure the IgG levels against EBV antigens, VCA, EAd, EBNA-1 and Zta as well as <it>P. falciparum </it>MSP-1, LSA-1, and AMA-1 antigens in children with BL (n = 32) and in population-based age-and sex-matched controls (n = 25) from a malaria endemic region in Western Kenya with high incidence of BL. EBV viral load in plasma was determined by quantitative PCR.</p> <p>Results</p> <p>Relative to healthy controls, BL patients had significantly increased anti-Zta (<it>p </it>= 0.0017) and VCA IgG levels (<it>p </it>< 0.0001) and plasma EBV viral loads (<it>p </it>< 0.0001). In contrast, comparable IgG levels to all <it>P. falciparum </it>antigens tested were observed in BL patients compared to controls. Interestingly, when we grouped BL patients into those presenting with abdominal tumors or with jaw tumors, we observed significantly higher levels of anti-Zta IgG levels (<it>p </it>< 0.0065) and plasma EBV viral loads (<it>p </it>< 0.033) in patients with abdominal tumors compared to patients with jaw tumors.</p> <p>Conclusion</p> <p>Elevated antibodies to Zta and elevated plasma EBV viral load could be relevant biomarkers for BL and could also be used to confirm BL presenting in the abdominal region.</p

Protective Antibody and CD8+ T-Cell Responses to the Plasmodium falciparum Circumsporozoite Protein Induced by a Nanoparticle Vaccine

Background The worldwide burden of malaria remains a major public health problem due, in part, to the lack of an effective vaccine against the Plasmodium falciparum parasite. An effective vaccine will most likely require the induction of antigen specific CD8+ and CD4+ T-cells as well as long-lasting antibody responses all working in concert to eliminate the infection. We report here the effective modification of a self-assembling protein nanoparticle (SAPN) vaccine previously proven effective in control of a P. berghei infection in a rodent model to now present B- and T-cell epitopes of the human malaria parasite P. falciparum in a platform capable of being used in human subjects. Methodology/Principal Findings To establish the basis for a SAPN-based vaccine, B- and CD8+ T-cell epitopes from the P. falciparum circumsporozoite protein (PfCSP) and the universal CD4 T-helper epitope PADRE were engineered into a versatile small protein (∼125 amino acids) that self-assembles into a spherical nanoparticle repetitively displaying the selected epitopes. P. falciparum epitope specific immune responses were evaluated in mice using a transgenic P. berghei malaria parasite of mice expressing the human malaria full-length P. falciparum circumsporozoite protein (Tg-Pb/PfCSP). We show that SAPN constructs, delivered in saline, can induce high-titer, long-lasting (1 year) protective antibody and poly-functional (IFNγ+, IL-2+) long-lived central memory CD8+ T-cells. Furthermore, we demonstrated that these Ab or CD8+ T–cells can independently provide sterile protection against a lethal challenge of the transgenic parasites. Conclusion The SAPN construct induces long-lasting antibody and cellular immune responses to epitope specific sequences of the P. falciparum circumsporozoite protein (PfCSP) and prevents infection in mice by a transgenic P. berghei parasite displaying the full length PfCSP

The use of a P. falciparum specific coiled-coil domain to construct a self-assembling protein nanoparticle vaccine to prevent malaria.

The parasitic disease malaria remains a major global public health concern and no truly effective vaccine exists. One approach to the development of a malaria vaccine is to target the asexual blood stage that results in clinical symptoms. Most attempts have failed. New antigens such as P27A and P27 have emerged as potential new vaccine candidates. Multiple studies have demonstrated that antigens are more immunogenic and are better correlated with protection when presented on particulate delivery systems. One such particulate delivery system is the self-assembling protein nanoparticle (SAPN) that relies on coiled-coil domains of proteins to form stable nanoparticles. In the past we have used de novo designed amino acid domains to drive the formation of the coiled-coil scaffolds which present the antigenic epitopes on the particle surface. Here we use naturally occurring domains found in the tex1 protein to form the coiled-coil scaffolding of the nanoparticle. Thus, by engineering P27A and a new extended form of the coiled-coil domain P27 onto the N and C terminus of the SAPN protein monomer we have developed a particulate delivery system that effectively displays both antigens on a single particle that uses malaria tex1 sequences to form the nanoparticle scaffold. These particles are immunogenic in a murine model and induce immune responses similar to the ones observed in seropositive individuals in malaria endemic regions. We demonstrate that our P27/P27A-SAPNs induce an immune response akin to the one in seropositive individuals in Burkina Faso. Since P27 is highly conserved among different Plasmodium species, these novel SAPNs may even provide cross-protection between Plasmodium falciparum and Plasmodium vivax the two major human malaria pathogens. As the SAPNs are also easy to manufacture and store they can be delivered to the population in need without complication thus providing a low cost malaria vaccine