The Plasmodium falciparum circumsporozoite protein (CSP) is critical for sporozoite function and invasion of hepatocytes. Given its critical nature, a phase III human CSP malaria vaccine trial is ongoing. The CSP is composed of three regions as follows: an N terminus that binds heparin sulfate proteoglycans, a four amino acid repeat region (NANP), and a C terminus that contains a thrombospondin-like type I repeat (TSR) domain. Despite the importance of CSP, little is known about its structure. Therefore, recombinant forms of CSP were produced by expression in both Escherichia coli (Ec) and then refolded (EcCSP) or in the methylotrophic yeast Pichia pastoris (PpCSP) for structural analyses. To analyze the TSR domain of recombinant CSP, conformation-dependent monoclonal antibodies that recognized unfixed P. falciparum sporozoites and inhibited sporozoite invasion of HepG2 cells in vitro were identified. These monoclonal antibodies recognized all recombinant CSPs, indicating the recombinant CSPs contain a properly folded TSR domain structure. Characterization of both EcCSP and PpCSP by dynamic light scattering and velocity sedimentation demonstrated that both forms of CSP appeared as highly extended proteins (R h 4.2 and 4.58 nm, respectively). Furthermore, high resolution atomic force microscopy revealed flexible, rod-like structures with a ribbon-like appearance. Using this information, we modeled the NANP repeat and TSR domain of CSP. Consistent with the biochemical and biophysical results, the repeat region formed a rod-like structure about 21-25 nm in length and 1.5 nm in width. Thus native CSP appears as a glycosylphosphatidylinositol-anchored, flexible rod-like protein on the sporozoite surface.

Development of a Plasmodium falciparum (Pf) transmission blocking vaccine (TBV) has the potential to significantly impact malaria control. Antibodies elicited against sexual stage proteins in the human bloodstream are taken up with the blood meal of the mosquitoes and inactivate parasite development in the mosquito. In a phase 1 trial, a leading TBV identified as Pfs25-EPA/Alhydrogel appeared safe and immunogenic, however, the level of Pfs25-specific antibodies were likely too low for an effective vaccine. Pfs230, a 230-kDa sexual stage protein expressed in gametocytes is an alternative vaccine candidate. A unique 6-cysteine-rich domain structure within Pfs230 have thwarted its recombinant expression and characterization for clinical evaluation for nearly a quarter of a century. Here, we report on the identification, biochemical, biophysical, and immunological characterization of recombinant Pfs230 domains. Rabbit antibodies generated against recombinant Pfs230 domains blocked mosquito transmission of a laboratory strain and two field isolates using an ex vivo assay. A planned clinical trial of the Pfs230 vaccine is a significant step toward the potential development of a transmission blocking vaccine to eliminate malaria.Development of a malaria vaccine that effectively protects against parasite infection of both the natural host, Anopheles mosquitoes, and its secondary host, man, would effectively disrupt transmission and clinical disease. The most well known investigational vaccine against Plasmodium falciparum malaria that recently received a positive scientific opinion from the Committee for Medicinal Products for Human Use of the European Medicines Agency in July 2015, is identified as RTS,S (Mosquirix TM ). This vaccine targets the circumsporozoite protein that is present on the surface of the sporozoite, the parasite stage that infects man (1). RTS,S, a virus like-particle-based vaccine, is protective against clinical disease in about 30% of the young children who participated in a phase 3 trial (1, 2).Efforts toward development of a vaccine to disrupt parasite infection of the mosquito host, also identified as a transmission blocking vaccine have to date only been able to evaluate a sexual stage-specific protein, Pfs25, 2 which is a 25-kDa protein expressed on the zygote and ookinete surfaces. A phase 1 study demonstrated that human antibodies raised against a recombinant Pfs25 (Pfs25H) protein formulated in Montanide ISA 51, a water-in-oil adjuvant formulation, were biologically active in an ex vivo feeding assay (3), however, this formulation was not deemed suitable for a public health vaccine. More recently, in preclinical studies, Pfs25H has been shown to have enhanced immunogenic properties when chemically conjugated to a carrier molecule such as Neisseria meningitis outer membrane protein complex (4), or Pseudomonas aeruginosa ExoProtein A (EPA) (5, 6). In particular, the chemically conjugated Pfs25-EPA has the biophysical features of a nanoparticle with a diameter of about 25 m in solution, simil...

Conjugation of polysaccharides to carrier proteins has been a successful approach for producing safe and effective vaccines. In an attempt to increase the immunogenicity of two malarial vaccine candidate proteins of Plasmodium falciparum, apical membrane antigen 1 (AMA1) to a blood stage vaccine candidate and surface protein 25 (Pfs25) a mosquito stage vaccine candidate, were each independently chemically conjugated to the mutant, nontoxic Pseudomonas aeruginosa ExoProtein A (rEPA). AMA1 is a large (66kD) relatively good immunogen in mice; Pfs25 is a poorly immunogenic protein when presented on alum to mice. Mice were immunized on days 0 and 28 with AMA1- or Pfs25-rEPA conjugates or unconjugated AMA1 or Pfs25, all formulated on Alhydrogel. Remarkably, sera from mice 14 days after the second immunization with Pfs25-rEPA conjugates displayed over a 1000-fold higher antibody titers as compared to unconjugated Pfs25. In contrast, AMA1 conjugated under the same conditions induced only a three-fold increase in antibody titers. When tested for functional activity, antibodies elicited by the AMA1-rEPA inhibited invasion of erythrocytes by blood-stage parasites and antibodies elicited by the Pfs25-rEPA conjugates blocked the development of the sexual stage parasites in the mosquito midgut. These results demonstrate that conjugation to rEPA induces a marked improvement in the antibody titer in mice for the poor immunogen (Pfs25) and for the larger protein (AMA1). These conjugates now need to be tested in humans to determine if mice are predictive of the response in humans.

Successful efforts to control infectious diseases have often required the use of effective vaccines. The current global strategy for control of malaria, including elimination and eradication will also benefit from the development of an effective vaccine that interrupts malaria transmission. To this end, a vaccine that disrupts malaria transmission within the mosquito host has been investigated for several decades targeting a 25 kDa ookinete specific surface protein, identified as Pfs25. Phase 1 human trial results using a recombinant Pfs25H/Montanide ISA51 formulation demonstrated that human Pfs25 specific antibodies block parasite infectivity to mosquitoes; however, the extent of blocking was likely insufficient for an effective transmission blocking vaccine. To overcome the poor immunogenicity, processes to produce and characterize recombinant Pfs25H conjugated to a detoxified form of Pseudomonas aeruginosa exoprotein A (EPA) have been developed and used to manufacture a cGMP pilot lot for use in human clinical trials. The Pfs25-EPA conjugate appears as a nanoparticle with an average molar mass in solution of approximately 600 kDa by static light scattering with an average diameter 20 nm (range 10 to 40 nm) by dynamic light scattering. The molar ratio of Pfs25H to EPA is about 3 to 1 by amino acid analysis, respectively. Outbred mice immunized with the Pfs25-EPA conjugated nanoparticle formulated on Alhydrogel® had a 75 to 110 fold increase in Pfs25H specific antibodies when compared to an unconjugated Pfs25H/Alhydrogel® formulation. A phase 1 human trial using the Pfs25-EPA/Alhydrogel® formulation is ongoing in the United States.

Proteins Pfs230 and Pfs48/45 are Plasmodium falciparum transmission-blocking (TB) vaccine candidates that form a membrane-bound protein complex on gametes. The biological role of Pfs230 or the Pfs230-Pfs48/45 complex remains poorly understood. Here, we present the crystal structure of recombinant Pfs230 domain 1 (Pfs230D1M), a 6-cysteine domain, in complex with the Fab fragment of a TB monoclonal antibody (mAb) 4F12. We observed the arrangement of Pfs230 on the surface of macrogametes differed from that on microgametes, and that Pfs230, with no known membrane anchor, may exist on the membrane surface in the absence of Pfs48/45. 4F12 appears to sterically interfere with Pfs230 function. Combining mAbs against different epitopes of Pfs230D1 or of Pfs230D1 and Pfs48/45, significantly increased TB activity. These studies elucidate a mechanism of action of the Pfs230D1 vaccine, model the functional activity induced by a polyclonal antibody response and support the development of TB vaccines targeting Pfs230D1 and Pfs230D1-Pfs48/45.

fThe extended rod-like Plasmodium falciparum circumsporozoite protein (CSP) is comprised of three primary domains: a charged N terminus that binds heparan sulfate proteoglycans, a central NANP repeat domain, and a C terminus containing a thrombospondin-like type I repeat (TSR) domain. Only the last two domains are incorporated in RTS,S, the leading malaria vaccine in phase 3 trials that, to date, protects about 50% of vaccinated children against clinical disease. A seroepidemiological study indicated that the N-terminal domain might improve the efficacy of a new CSP vaccine. Using a panel of CSP-specific monoclonal antibodies, well-characterized recombinant CSPs, label-free quantitative proteomics, and in vitro inhibition of sporozoite invasion, we show that native CSP is N-terminally processed in the mosquito host and undergoes a reversible conformational change to mask some epitopes in the N-and C-terminal domains until the sporozoite interacts with the liver hepatocyte. Our findings show the importance of understanding processing and the biophysical change in conformation, possibly due to a mechanical or molecular signal, and may aid in the development of a new CSP vaccine.T he development of a vaccine to aid in the control of malaria is critical, as Plasmodium falciparum has evolved resistance to all antimalarial drugs deployed so far, including artemisinin (1). The leading malaria vaccine (RTS,S), currently in phase 3 trials, contains a formulated virus-like particle that encompasses the central and carboxyl-terminal domains of the circumsporozoite protein (CSP) fused to the hepatitis B virus surface antigen (2) and protects approximately 30% to 50% of infants or children from clinical disease for a limited duration (3, 4). Naturally derived human antibodies against a portion of the N-terminal region, including region 1, are associated with a reduced risk of disease (5), providing a basis to design new CSP vaccines. This N-terminal region of the CSP is absent from RTS,S.The importance of understanding protein structure because of its impact on the induction of broadly neutralizing antibodies and subsequent vaccine design continues to be revealed in the HIV arena (6, 7). In malaria, the importance of protein conformation for the induction of neutralizing antibodies was recently shown in vivo for an orthologue of the leading asexual-stage malaria vaccine antigen apical membrane antigen-1 (AMA-1). Only a recombinant AMA-1 forming a stable complex with a constrained synthetic rhoptry neck protein-2 peptide induced protective antibodies in vivo against a lethal blood-stage challenge malaria parasite infection (8). When developing a novel CSP vaccine, these more recent developments need to be considered with regard to the potential for changes within the CSP, such as through in vivo processing or conformational changes (9, 10) in a protein with a known extended rod-like structure (11), that could mask the adhesion domains located at the N-and C-terminal domains (9).To address these questions, a panel of CSP-speci...

dApical membrane antigen 1 (AMA1) is a leading vaccine candidate, but the allelic polymorphism is a stumbling block for vaccine development. We previously showed that a global set of AMA1 haplotypes could be grouped into six genetic populations. Using this information, six recombinant AMA1 proteins representing each population were produced. Rabbits were immunized with either a single recombinant AMA1 protein or mixtures of recombinant AMA1 proteins (mixtures of 4, 5, or 6 AMA1 proteins). Antibody levels were measured by enzyme-linked immunosorbent assay (ELISA), and purified IgG from each rabbit was used for growth inhibition assay (GIA) with 12 different clones of parasites (a total of 108 immunogen-parasite combinations). Levels of antibodies to all six AMA1 proteins were similar when the antibodies were tested against homologous antigens. When the percent inhibitions in GIA were plotted against the number of ELISA units measured with homologous AMA1, all data points followed a sigmoid curve, regardless of the immunogen. In homologous combinations, there were no differences in the percent inhibition between the single-allele and allele mixture groups. However, all allele mixture groups showed significantly higher percent inhibition than the single-allele groups in heterologous combinations. The 5-allele-mixture group showed significantly higher inhibition to heterologous parasites than the 4-allele-mixture group. On the other hand, there was no difference between the 5-and 6-allele-mixture groups. These data indicate that mixtures with a limited number of alleles may cover a majority of the parasite population. In addition, using the data from 72 immunogen-parasite combinations, we mathematically identified 13 amino acid polymorphic sites which significantly impact GIA activities. These results could be a foundation for the rational design of a future AMA1 vaccine.

The function of the antigen-5/CAP family of proteins found in the salivary gland of bloodsucking animals has remained elusive for decades. Antigen-5 members from the hematophagous insects Dipetalogaster maxima (DMAV) and Triatoma infestans (TIAV) were expressed and discovered to attenuate platelet aggregation, ATP secretion, and thromboxane A2 generation by low doses of collagen (<1 μg/ml) but no other agonists. DMAV did not interact with collagen, glycoprotein VI, or integrin α2β1. This inhibitory profile resembles the effects of antioxidants Cu,Zn-superoxide dismutase (Cu,Zn-SOD) in platelet function. Accordingly, DMAV was found to inhibit cytochrome c reduction by O2⨪ generated by the xanthine/xanthine oxidase, implying that it exhibits antioxidant activity. Moreover, our results demonstrate that DMAV blunts the luminescence signal of O2⨪ generated by phorbol 12-myristate 13-acetate-stimulated neutrophils. Mechanistically, inductively coupled plasma mass spectrometry and fluorescence spectroscopy revealed that DMAV, like Cu,Zn-SOD, interacts with Cu2+, which provides redox potential for catalytic removal of O2⨪. Notably, surface plasmon resonance experiments (BIAcore) determined that DMAV binds sulfated glycosaminoglycans (e.g. heparin, KD ∼100 nmol/liter), as reported for extracellular SOD. Finally, fractions of the salivary gland of D. maxima with native DMAV contain Cu2+ and display metal-dependent antioxidant properties. Antigen-5/CAP emerges as novel family of Cu2+-dependent antioxidant enzymes that inhibit neutrophil oxidative burst and negatively modulate platelet aggregation by a unique salivary mechanism.