George Hui

Efficacy of Two Alternate Vaccines Based on Plasmodium falciparum Merozoite Surface Protein 1 in an Aotus Challenge Trial

ABSTRACT In an attempt to produce a more defined, clinical-grade version of a vaccine based on Plasmodium falciparum merozoite surface protein 1 (MSP1), we evaluated the efficacy of two recombinant forms of MSP1 in an Aotus nancymai challenge model system. One recombinant vaccine, bvMSP142, based on the 42-kDa C-terminal portion of MSP1, was expressed as a secreted protein in baculovirus-infected insect cells. A highly pure baculovirus product could be reproducibly expressed and purified at yields in excess of 8 mg of pure protein per liter of culture. This protein, when tested for efficacy in the Aotus challenge model, gave significant protection, with only one of seven monkeys requiring treatment for uncontrolled parasitemia after challenge with P. falciparum. The second recombinant protein, P30P2MSP119, has been used in previous studies and is based on the smaller, C-terminal 19-kDa portion of MSP1 expressed inSaccharomyces cerevisiae. Substantial changes were made in its production process to optimize expression. The optimum form of this vaccine antigen (as judged by in vitro and in vivo indicators) was then evaluated, along with bvMSP142, for efficacy in theA. nancymai system. The new formulation of P30P3MSP119 performed significantly worse than bvMSP142 and appeared to be less efficacious than we have found in the past, with four of seven monkeys in the vaccinated group requiring treatment for uncontrolled parasitemia. With both antigens, protection was seen only when high antibody levels were obtained by formulation of the vaccines in Freunds adjuvant. Vaccine formulation in an alternate adjuvant, MF59, resulted in significantly lower antibody titers and no protection.

Expression and antigenicity of Plasmodium falciparum major merozoite surface protein (MSP119) variants secreted from Saccharomyces cerevisiae

Four antigenic variants of the 19-kDa carboxy terminal fragment of Plasmodium falciparum merozoite surface protein, MSP1 (MSP1(19)), were expressed in Saccharomyces cerevisiae as a histidine-tagged, secreted polypeptides (rMSP1(19)s). Structural analysis of the rMSP1(19)s indicated that a single amino acid change (E to Q) in the first EGF-like domain of the yeast-secreted rMSP1(19) proteins caused a significant change in their disulfide bond-dependent conformation. The antigenicity of the rMSP1(19)s were qualitatively and quantitatively analyzed by direct and competitive binding ELISAs. The data indicate that conserved and variant B cell determinants of MSP1(19), as well as epitopes that are known targets of protective antibodies, were recreated authentically in the rMSP1(19)s. Secretion of histidine-tagged rMSP1(19)s using the expression system described may be an efficient and effective means of producing a properly folded immunogen for a human vaccine against the blood stages of P. falciparum.

Characterization of a Plasmodium falciparum polypeptide associated with membrane vesicles in the infected erythrocytes

A Plasmodium falciparum polypeptide (46 kDa) associated with the infected erythrocytes of all asexual stages as well as immature gametocytes was identified by the monoclonal antibody (Mab) 30B8.3. The expression of this protein was not dependent upon the knobby phenotype and was detected in parasites grown either in human or Aotus erythrocytes. The antigen was heatstable, did not label with [14C]glucosamine, and was not sensitive to periodate oxidation. Immunofluorescent staining patterns of Mab 30B8.3 on in vitro cultured parasites varied from punctate (rings and trophozoites) to patchy (trophozoites and schizonts) fluorescence. The Mab 30B8.3 antigen was not detected on the infected erythrocyte surface by conventional wet-mount IFA procedure. However, when parasites were cultured in the presence of Mab 30B8.3, the epitope was detected by the monoclonal antibodies present in the culture medium. Differential extraction of the polypeptide from infected erythrocytes and immune electron microscopy of cryosectioned parasites localized the 30B8.3 epitope primarily on membranes of Maurers clefts within the infected erythrocytes cytosol. This 46 kDa polypeptide is unique because it seemed to be an integral membrane protein of the Maurers clefts/vesicles and it was not secreted into the culture medium nor deposited on the infected erythrocyte membrane. Previous studies indicate that several parasite proteins, excreted extracellularly or deposited on infected erythrocyte membrane, are found to be associated with Maurers cleft membranes and vesicles. The 46 kDa polypeptide described in this study may play an important role in the transport of the parasite antigens.

In vivo expression and immunological studies of the 42-kilodalton carboxyl-terminal processing fragment of Plasmodium falciparum merozoite surface protein 1 in the baculovirus-silkworm system.

ABSTRACT The 42-kDa carboxyl-terminal processing fragment of Plasmodium falciparum merozoite surface protein 1 (MSP-142) is an anti-erythrocytic stage malaria vaccine candidate. In this study, MSP-142 was expressed by using the Bombyx mori nuclear polyhedrosis virus-silkworm expression system, and the antigenicity and immmunogenicity of the recombinant protein, Bmp42, were evaluated. The average yield of Bmp42, as determined by a sandwich enzyme-linked immunosorbent assay (ELISA), was 379 μg/ml of infected silkworm hemolymph, which was >100-fold higher than the level attainable in cell culture medium. N-terminal amino acid sequencing revealed that Bmp42 was correctly processed in silkworm cells. Data from immunoblotting, as well as from the inhibition ELISA, suggested that the conformational B-cell epitopes of MSP-142 were recreated in Bmp42. Immunization of rabbits with Bmp42 in complete Freunds adjuvant generated high-titer antibody responses against the immunogen. Specificity analyses of the anti-Bmp42 antibodies using several recombinant MSP-119 proteins expressing variant and conserved B-cell epitopes suggested that the anti-Bmp42 antibodies recognized primarily conserved epitopes on MSP-119. Furthermore, the anti-Bmp42 antibodies were highly effective in inhibiting the in vitro growth of parasites carrying homologous or heterologous MSP-142. Our results demonstrated that the baculovirus-silkworm expression system could be employed to express biologically and immunologically active recombinant MSP-142 at elevated levels; thus, it is an attractive alternative for producing a protective MSP-142 vaccine for human use.

Iron oxide nanoparticles as a clinically acceptable delivery platform for a recombinant blood-stage human malaria vaccine

This study explored the novel use of iron oxide (IO) nanoparticles (<20 nm) as a vaccine delivery platform without additional adjuvants. A recombinant malaria vaccine antigen, the merozoite surface protein 1 (rMSP1), was conjugated to IO nanoparticles (rMSP1‐IO). Immunizations in outbred mice with rMSP1‐IO achieved 100% responsiveness with antibody titers comparable to those obtained with rMSP1 formulated with a clinically acceptable adjuvant, Montanide ISA51 (2.7×10–3 vs. 1.6×10–3; respectively). Only rMSP1‐1O could induce significant levels (80%) of parasite inhibitory antibodies. The rMSP1‐IO was highly stable at 4°C and was amenable to lyophilization, maintaining its antigenicity, immunogenicity, and ability to induce inhibitory antibodies. Further testing in nonhuman primates, Aotus monkeys, also elicited 100% immune responsiveness and high levels of parasite inhibitory antibodies (55–100% inhibition). No apparent local or systemic toxicity was associated with IO immunizations. Murine macrophages and dendritic cells efficiently (>90%) internalized IO nanoparticles, but only the latter were significantly activated, with elevated expression/secretion of CD86, cytokines (IL‐6, TNF‐α, IL1‐b, IFN‐γ, and IL‐12), and chemokines (CXCL1, CXCL2, CCL2, CCL3, CCL4, and CXCL10). Thus, the IO nanoparticles is a novel, safe, and effective vaccine platform, with built‐in adjuvancy, that is highly stable and field deployable for cost‐effective vaccine delivery.—Pusic, K., Aguilar, Z., McLoughlin, J., Kobuch, S., Xu, H., Tsang, M., Wang, A., Hui, G. Iron oxide nanoparticles as a clinically acceptable delivery platform for a recombinant blood‐stage human malaria vaccine. FASEB J. 27, 1153–1166 (2013). www.fasebj.org

An experimental vaccine cocktail for Plasmodium falciparum malaria.

Surface proteins from several different life-cycle stages of the malaria parasite Plasmodium falciparum were expressed at high levels in the yeast Saccharomyces cerevisiae. Purified proteins, both individually and in cocktails, were used to immunize mice and goats in conjunction with either Freunds adjuvant or a muramyl tripeptide-based adjuvant. Immune responses were measured by enzyme-linked immunosorbent assays and by the ability of antisera to inhibit (1) the invasion of hepatocytes by live sporozoites, (2) in vitro invasion of human erythrocytes by live merozoites, and (3) the development of oocytes in the mosquito vector. These results suggest that cocktails of different stage-specific antigens can provide the components necessary to block the development of the malaria parasite at multiple stages of its life cycle.

Blood stage merozoite surface protein conjugated to nanoparticles induce potent parasite inhibitory antibodies.

In this proof-of-concept study we report the use of <15 nm, water soluble, inorganic nanoparticles as a vaccine delivery system for a blood stage malaria vaccine. The recombinant malarial antigen, Merozoite Surface Protein 1 (rMSP1) of Plasmodium falciparum served as the model vaccine. The rMSP1 was covalently conjugated to polymer-coated quantum dot CdSe/ZnS nanoparticles (QDs) via surface carboxyl groups, forming rMSP1-QDs. Anti-MSP1 antibody responses induced by rMSP1-QDs were found to have 2-3 log higher titers than those obtained with rMSP1 administered with the conventional adjuvants, Montanide ISA51 and CFA. Moreover, the immune responsiveness and the induction of parasite inhibitory antibodies were significantly superior in mice injected with rMSP1-QDs. The rMSP1-QDs delivered via intra-peritoneal (i.p.), intra-muscular (i.m.), and subcutaneous (s.c.) routes were equally efficacious. The high level of immunogenicity exhibited by the rMSP1-QDs was achieved without further addition of other adjuvant components. Bone marrow derived dendritic cells were shown to efficiently take up the nanoparticles leading to their activation and the expression/secretion of key cytokines, suggesting that this may be a mode of action for the enhanced immunogenicity. This study provides promising results for the use of water soluble, inorganic nanoparticles (<15 nm) as potent vehicles/platforms to enhance the immunogenicity of polypeptide antigens in adjuvant-free immunizations.

Acquired antibody levels to Plasmodium falciparum merozoite surface antigen 1 in residents of a highly endemic area of Papua New Guinea

The prevalence and concentration of antibodies to a yeast-expressed N-terminal region (195A) and a baculo-virus-expressed C-terminal region (BVp42) of merozoite surface antigen 1 (MSA-1) were measured during a cross-sectional survey in the Wosera area of East Sepik Province, Papua New Guinea, in order to obtain baseline data on naturally acquired antibody response to this antigen in preparation for a vaccine trial. Overall, the seropositivity rate was 78% for 195A and 91% for BVp42. Although antibody prevalence to both molecules increased with age, higher antibody prevalence rates were observed for BVp42 in all age groups studied. In children, significant positive associations were found between parasite prevalence and antibody prevalence for both regions of MSA-1 and between spleen rates and anti-BVp42 antibody prevalence. Concentration of antibody against both regions increased significantly with age, but was always higher for BVp42. In children, antibody levels to both regions of MSA-1 were significantly higher in those infected (symptomatic and asymptomatic), while in adults no significant difference in antibody concentration was observed between those infected and those uninfected. However, enlarged spleens were associated with higher antibody concentration to both regions of MSA-1 in both children and adults. The C-terminal of MSA-1 appeared to be more recognized than the N-terminal, in terms of both antibody prevalence and concentration.

A large-scale in vitro production system for Plasmodium falciparum.

near their center so that the anterior and posterior ends were juxtaposed (Fig. 1). Folded sporozoites were most numerous 32 HAI but were present up to 43 HAI. At 32 HAI, many small round meronts were seen near the basement membrane of the crypt enterocytes, below or beside the host cell nucleus. Several nuclei were scattered ramdomly throughout the developing meront and an eosinophilic refractile body was present (Fig. 2). The meronts increased in size during 32 to 40 HAI, the nuclei became peripherally arranged and the refractile body persisted. As the merozoites developed, they appeared to bud in a whorled pattern from a central residuum. Nuclei of the merozoites were arranged side by side (Fig. 3). Mature meronts were first seen 40 HAI. Twenty-five meronts were 16-27 X 12.8-19 gtm (mean 20.8 X 15.6 tm) and in cross section contained about 22 merozoites that were arranged randomly in the parasitophorous vacuole (Fig. 4). The first generation merozoites were approximately 7.6 X 1.3 Atm and had centrally located nuclei. Mature meronts were most numerous 43 HAI but were still present in sections from animals killed 51 HAI. The first generation meront differs from the second generation meront in the time of maturation and in the size of the meront and merozoites. The mature second generation meronts were 19.8 X 12.6 ,m (mean) and contained about 39 merozoites that were 17.4 X 2 ,im (mean). The third generation meront was smaller (mean 13.1 X 11.2 ,um) then the first generation meront and did not mature until 120 HAI. Merozoites of the first generation meronts appeared to bud from a central residual body. This is similar to that reported for the third generation meront (Todd and Lepp, 1971, loc. cit.).

Biological Activities of Anti-Merozoite Surface Protein-1 Antibodies Induced by Adjuvant-Assisted Immunizations in Mice with Different Immune Gene Knockouts

ABSTRACT Immunizations with Plasmodium falciparum MSP1-42 or MSP1-19 induce antibodies that inhibit parasites in vitro, which correlates with in vivo protective immunity by vaccination. We previously showed that several adjuvant formulations can induce anti-MSP1-19 antibodies in interleukin-6, intercellular adhesion molecule 1, CD80, and CD86 knockout (KO) mice and at levels similar to those obtained in the healthy uninfected hosts. Here, we determine whether these immune gene KOs or the immunopotentiating activities of the adjuvants have a more important influence on the induction of parasite-inhibitory anti-MSP1-19 antibodies. Results showed that the biological activities of the anti-MSP1-19 antibodies induced by these adjuvants were not affected by the immune gene KOs. All adjuvant formulations that induced significant inhibitory antibody responses (i.e., >50% inhibition of parasite growth) contained monophosphoryl lipid A (MPL) in emulsion carriers, whereas MPL or emulsion carriers alone were ineffective. The ability to retain vaccine efficacy by the MSP1-19 and adjuvant formulations in the altered immunological background is a valuable and significant attribute in light of many instances of skewed immune status in the targeted vaccine populations.