Malaria vaccines based on the Plasmodium falciparum merozoite surface protein 3--should we avoid amino acid sequence polymorphisms or embrace them?
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Amino Acid Sequence↗
Biomedical subjects
Allan Saul
Publications and source records attributed to Allan Saul.
Sustained high-titer antibody responses induced by conjugating a malarial vaccine candidate to outer-membrane protein complex.
The development of protein subunit vaccines to combat some of the world's deadliest pathogens such as a malaria parasite, Plasmodium falciparum, is stalled, due in part to the inability to induce and sustain high-titer antibody responses. Here, we show the induction of persistent, high-titer antibody responses to recombinant Pfs25H, a human malarial transmission-blocking protein vaccine candidate, after chemical conjugation to the outer-membrane protein complex (OMPC) of Neisseria meningitidis serogroup B and adsorption to aluminum hydroxyphosphate. In mice, the Pfs25H-OMPC conjugate vaccine was >1,000 times more potent in generating anti-Pfs25H ELISA reactivity than a similar 0.5-microg dose of Pfs25H alone in Montanide ISA720, a water-in-oil adjuvant. The immune enhancement requires covalent conjugation between Pfs25H and the OMPC, given that physically mixed Pfs25H and OMPC on aluminum hydroxyphosphate failed to induce greater activity than the nonconjugated Pfs25H on aluminum hydroxyphosphate. The conjugate vaccine Pfs25H-OMPC also was highly immunogenic in rabbits and rhesus monkeys. In rhesus monkeys, the antibody responses were sustained over 18 months, at which time another vaccination with nonconjugated Pfs25H induced strong anamnestic responses. The vaccine-induced anti-Pfs25-specific antibodies in all animal species blocked the transmission of parasites to mosquitoes. Protein antigen conjugation to OMPC or other protein carrier may have general application to a spectrum of protein subunit vaccines to increase immunogenicity without the need for potentially reactogenic adjuvants.
Aluminum Hydroxide↗
The use of transgenic Plasmodium berghei expressing the Plasmodium vivax antigen P25 to determine the transmission-blocking activity of sera from malaria vaccine trials.
P25 is a major surface protein of Plasmodium ookinetes. Antibodies against P25 prevent the formation of oocysts in the mosquito and thereby block transmission of the parasite through an endemic population. Plasmodium vivax transmission-blocking vaccines based on Pv25 have undergone human trials and inhibit transmission significantly. The current assay to determine transmission-blocking activity (TBA) of these sera, the 'standard membrane feeding assay', is complex and can be performed by few groups worldwide that require both mosquito breeding facilities and access to volunteers naturally infected with P.vivax--a costly, and uncontrolled source of parasites. Here we report the development of novel assays to determine TBA using two clones (Pv25DR and Pv25DR3) of transgenic rodent parasites (Plasmodium berghei) expressing Pv25. We show that oocyst development of the transgenic parasites is inhibited by monoclonal antibody against Pv25 with the same kinetics exhibited by wild type parasites when exposed to mouse monoclonal antibodies targeted to a paralogous protein P28. Human transmission-blocking sera from a clinical vaccine trial of Pv25 inhibited oocyst development of Pv25DR and Pv25DR3, whereas non-blocking sera did not. We further show transmission-blocking activity can be determined in a simple assays of ookinete development in vitro, assays that obviate the need for mosquito colonies. These results demonstrate that transgenic rodent malarias expressing proteins from human Plasmodium species can be cheap, safe, and simple tools for testing TBA from sera. To this end the cloned lines have been deposited with, and are freely available from, MR4.
Animals↗
Production and characterization of clinical grade Escherichia coli derived Plasmodium falciparum 42 kDa merozoite surface protein 1 (MSP1(42)) in the absence of an affinity tag.
The 42 kDa cleavage product from the carboxyl end of the Plasmodium falciparum merozoite surface protein 1 (MSP1(42)) is an important blood-stage malaria vaccine target. Several recombinant protein expression systems have been used for production of MSP1(42) including yeast (Saccharomyces cerevisiae and Pichia pastoris), Escherichia coli, baculovirus and transgenic animals. To date, all of the reported recombinant proteins include a 6 x His affinity tag to facilitate purification, including three MSP1(42) clinical grade proteins currently in human trials. Under some circumstances, the presence of the 6 x His tag may not be desirable. Therefore, we were interested to produce clinical grade MSP1(42) without a 6 x His affinity tag from E. coli inclusion bodies. We produced a recombinant MSP1(42) with a P. falciparum FUP (Uganda-Palo Alto) phenotype which accounts for a substantial proportion of the MSP1(42) protein observed in African isolates. EcMSP1(42)-FUP was produced in E. coli inclusion bodies by high cell mass induction with IPTG using 5 L and 60 L bioreactors. Isolated inclusion bodies were solubilized in 8M guanidine-HCl and the EcMSP1(42)-FUP protein refolded by rapid dilution. Refolded EcMSP1(42)-FUP was purified using hydrophobic interaction chromatography, anion exchange chromatography, and size exclusion chromatography, and subject to biochemical characterization for integrity, identity, and purity. Endotoxin and host cell protein levels were within acceptable limits for human use. The process was successfully transferred to pilot-scale production in a cGMP environment. A final recovery of 87.8 mg of clinical-grade material per liter of fermentation broth was achieved. The EcMSP1(42)-FUP clinical antigen is available for preclinical evaluation and human studies.
Animals↗
Potency assay design for adjuvanted recombinant proteins as malaria vaccines.
Many licensed vaccines are composed of live, attenuated or inactivated whole-cell microorganisms, or they comprise purified components from whole-cell extracts or culture supernatants. For some diseases, pathology is fairly well understood, and there may be known correlates of protection that provide obvious parameters for assessment of vaccine potency. However, this is not always the case, and some effective vaccines are routinely used even though the mechanisms or correlates of protection are unknown. Some more modern vaccine approaches employ purified recombinant proteins, based on molecules that appear on the surface of the pathogen. This is one of the strategies that has been adopted in the quest to develop a malaria vaccine. Use of these parasite antigens as vaccine candidates is supported by substantial epidemiological data, and some have demonstrated the ability to elicit protective responses in animal models of malaria infection. However, there is as yet no immunological correlate of protection and no functional assays or animal models that have demonstrated the ability to predict efficacy in humans. There is little precedence for the most appropriate and practical method for assessing potency of vaccines based on these recombinant molecules for malaria vaccines. This is likely because the majority of malaria vaccine candidates have only recently entered clinical evaluation. The PATH Malaria Vaccine Initiative (MVI) convened a panel with expertise in potency assay design from industry, governmental institutions, and regulatory bodies to discuss and review the rationale, available methods, and best approaches for assessing the potency of recombinant proteins, specifically for their use as malarial vaccines. The aim of this meeting was to produce a discussion document on the practical potency assessment of recombinant protein malaria vaccines, focusing on early phase potency assay development.
Adjuvants, Immunologic↗
Toll-like receptor agonists influence the magnitude and quality of memory T cell responses after prime-boost immunization in nonhuman primates.
There is a remarkable heterogeneity in the functional profile (quality) of T cell responses. Importantly, the magnitude and/or quality of a response required for protection may be different depending on the infection. Here, we assessed the capacity of different Toll like receptor (TLR)-binding compounds to influence T helper cell (Th)1 and CD8+ T cell responses when used as adjuvants in nonhuman primates (NHP) with HIV Gag as a model antigen. NHP were immunized with HIV Gag protein emulsified in Montanide ISA 51, an oil-based adjuvant, with or without a TLR7/8 agonist, a TLR8 agonist, or the TLR9 ligand cytosine phosphate guanosine oligodeoxynucleotides (CpG ODN), and boosted 12 wk later with a replication-defective adenovirus-expressing HIV-Gag (rAD-Gag). Animals vaccinated with HIV Gag protein/Montanide and CpG ODN or the TLR7/8 agonist had higher frequencies of Th1 responses after primary immunization compared to all other vaccine groups. Although the rAD-Gag boost did not elevate the frequency of Th1 memory cytokine responses, there was a striking increase in HIV Gag-specific CD8+ T cell responses after the boost in all animals that had received a primary immunization with any of the TLR adjuvants. Importantly, the presence and type of TLR adjuvant used during primary immunization conferred stability and dramatically influenced the magnitude and quality of the Th1 and CD8+ T cell responses after the rAD-Gag boost. These data provide insights for designing prime-boost immunization regimens to optimize Th1 and CD8+ T cell responses.
Adjuvants, Immunologic↗
Enhancement of functional antibody responses to AMA1-C1/Alhydrogel, a Plasmodium falciparum malaria vaccine, with CpG oligodeoxynucleotide.
Apical membrane antigen 1 (AMA1) has been shown to be a promising malaria vaccine candidate. The multiallelic AMA1-C1 vaccine currently in Phase 1 trials in the US and Mali contains an equal mixture of the ectodomain portion of recombinant AMA1 from the FVO and 3D7 clones of Plasmodium falciparum, formulated on Alhydrogel. It is hoped that inclusion of a human-optimized CpG oligodeoxynucleotide (ODN) (CPG 7909) with our existing AMA1-C1/Alhydrogel vaccine will lead to a higher concentration of functional AMA1-C1 antibodies. Preclinical studies were performed in mice, rats and guinea pigs to assess the safety, immunogenicity and functionality of the immune response to AMA1-C1 with Alhydrogel + CPG 7909 compared to antigen with Alhydrogel alone. Day 42 mean anti-AMA1 ELISA titer values derived from individual animals were compared between Alhydrogel and Alhydrogel + CPG 7909 groups at each antigen dose for each species. Sera from Alhydrogel + CPG 7909 groups displayed significantly higher antibody titers (P < 0.025) than their comparable Alhydrogel alone group. Mouse IgG isotype analysis showed that AMA1-C1/Alhydrogel induced a predominately Th2 type response while AMA1-C1/Alhydrogel + CPG 7909 gave a mixed Th1/Th2 type response. When tested for functional activity by in vitro inhibition of parasite invasion, IgG isolated from serum pools of AMA1-C1/Alhydrogel + CPG 7909 animals was more effective against both FVO and 3D7 parasites than an equal concentration of IgG from animals receiving vaccines adjuvanted with Alhydrogel alone. These promising preclinical results have recently led to the start of a Phase 1 trial in the US.
Aluminum Hydroxide↗
Assessment of transmission-blocking activity of candidate Pvs25 vaccine using gametocytes from chimpanzees.
Macaca mulatta monkeys were immunized with the candidate transmission-blocking vaccine against Plasmodium vivax, Pvs25, combined with alum or Montanide ISA 720. Efficacy was measured by combining post-immunization sera with gametocytes obtained from infections induced in chimpanzees using membrane-feeding techniques. The results indicate that immunization of M. mulatta monkeys with Pvs25 and Montanide ISA 720 was more effective than with alum in efficacy and resulted in the maintenance of a lasting transmission-blocking immunity to P. vivax. This was evident two weeks after the second immunization, and more strongly demonstrable 62 and 152 days after the second immunization. This transmission-blocking activity was strongly reinforced by a third immunization given 181 days after the primary immunization, as measured three weeks later by indirect membrane feeding. The use of gametocytes of P. vivax derived from infections induced in chimpanzees can contribute to the selection of appropriate constructs, formulations, and immunization regimens for the development of effective transmission-blocking vaccines.
Alum Compounds↗
Improved yield of recombinant merozoite Surface protein 3 (MSP3) from Pichia pastoris using chemically defined media.
Plasmodium falciparum merozoite surface protein 3 (MSP3) is a leading blood-stage malaria vaccine candidate. Vaccination with Pichia pastoris derived recombinant MSP3 protected Aotus nacymai monkeys from the parasite's lethal challenge and the post-challenge antibody titer against MSP3 correlated with protection. In our preliminary attempts to produce this vaccine in fermentors, little or no expression of MSP3 was observed in chemically defined media, although the same P. pastoris strain produced MSP3 in complex media. Our goal is to develop a Phase I/II clinical manufacturing process in completely chemically defined media because of the concern of potential prion contamination in complex media containing animal derived products. Here, we report our investigations into various factors to improve the yield of MSP3 in defined media. We found that an induction pH (pH(i)) 6.8 yielded MSP3 at 434 mg/L whereas there was no product at pH(i)< or = 5, though cell growth was the same in all pH(i) levels examined. High levels of NH(4) (+) consumed at pH(i) 6.8 were directly correlated to the enhanced MSP3 production. Furthermore, an additional 3.5-fold increase in the yield of MSP3 was obtained by addition of casamino acids at pH(i) 6.8. No direct correlation was observed between protease activity in the culture supernatants and lack of MSP3 expression. Neither high P. pastoris biomass generated at a high specific growth rate (0.04/h) nor low induction temperatures during induction improved yield. Nitrogen source was the most important factor affecting expression of MSP3 in defined media.
Antigens, Protozoan↗
Phase 1 vaccine trial of Pvs25H: a transmission blocking vaccine for Plasmodium vivax malaria.
Plasmodium vivax is responsible for the majority of malaria cases outside of Africa, and results in substantial morbidity. Transmission blocking vaccines are a potentially powerful component of a multi-faceted public health approach to controlling or eliminating malaria. We report the first phase 1 clinical trial of a P. vivax transmission blocking vaccine in humans. The Pvs25H vaccine is a recombinant protein derived from the Pvs25 surface antigen of P. vivax ookinetes. The protein was expressed in Saccharomyces cerevisiae, purified, and adsorbed onto Alhydrogel. Ten volunteers in each of three dose groups (5, 20, or 80 microg) were vaccinated by intramuscular injection in an open-label study at 0, 28 and 180 days. No vaccine-related serious adverse events were observed. The majority of adverse events causally related to vaccination were mild or moderate in severity. Injection site tenderness was the most commonly observed adverse event. Anti-Pvs25H antibody levels measured by ELISA peaked after the third vaccination. Vaccine-induced antibody is functionally active as evidenced by significant transmission blocking activity in the membrane feeding assay. Correlation between antibody concentration and degree of inhibition was observed. Pvs25H generates transmission blocking immunity in humans against P. vivax demonstrating the potential of this antigen as a component of a transmission blocking vaccine.
Adolescent↗
A human phase 1 vaccine clinical trial of the Plasmodium falciparum malaria vaccine candidate apical membrane antigen 1 in Montanide ISA720 adjuvant.
A dose escalating, placebo-controlled phase 1 trial was conducted to test the safety and immunogenicity of a vaccine containing recombinant Plasmodium falciparum apical membrane antigen 1 (AMA1) formulated in Montanide ISA720. Three groups of volunteers were vaccinated intramuscularly with 5 microg, 20 microg or 80 microg of AMA1, respectively, in 0.5 mL of formulation at 0, 3 and 6 months. Anti-AMA1 antibody levels and T cell stimulation indices were measured before and after each vaccination. No vaccine-related serious adverse events were recorded. Most subjects generated a mild to moderate, transient local reaction after the first vaccination. Three subjects developed a local reaction approximately 10 days following vaccination. Six of the 29 subjects seroconverted. Only one of these developed a high antibody titre. However, the interpretation of this trial was compromised by a loss of potency of the formulated vaccine during the course of the study.
Adjuvants, Immunologic↗
Models of Phase 1 vaccine trials: optimization of trial design to minimize risks of multiple serious adverse events.
A mathematical model of Phase 1 vaccine trial design was used to investigate strategies for minimizing the number of serious adverse events (SAEs) that could be encountered in the first Phase 1 trials of new vaccine formulations. For a relatively standard dose escalation trial with three dose groups each with 10 subjects, an optimal balanced between risk of more than one serious adverse event and trial design is achieved by splitting each dose group into two subgroups of three and seven. Based on the modeling, for a two vaccination, dose-escalating Phase 1 trial, a design where all subjects receive the first vaccination before any subject receives a second vaccination generally carries a lower risk of multiple serious adverse events than other designs.
Clinical Trials, Phase I as Topic↗
Montanide ISA 720 vaccines: quality control of emulsions, stability of formulated antigens, and comparative immunogenicity of vaccine formulations.
Montanide ISA 720 is an experimental adjuvant, formulated as water-in-oil emulsions, that induces high antibody titers in several animal species. It has been used in human vaccine trials with malaria and HIV vaccines. The heightened response is likely due, in part, to the formation of a depot at the injection site. However, post-formulation modifications were seen with seven proteins tested during storage of ISA 720 formulations at 37 degrees C for 1 week and two proteins stored longer at 4 degrees C. Potency studies in mice, in which the stored vaccines were diluted into placebo emulsions for appropriate dosing, indicated that this instability could lead to loss of immunogenicity in the post-injection depot, limiting the allowable storage time of preformed vaccines. We describe point-of-injection formulation for ISA 720 vaccines that meets the requirement for in vitro stability. For preformed vaccines, addition of glycine or glycylglycine prevented antigen modification on storage at 37 degrees C, providing a potential way of stabilizing antigen/ISA 720 formulations for in vitro storage and the post-injection depot.
Adjuvants, Immunologic↗
Revisiting Freund's incomplete adjuvant for vaccines in the developing world.
The rightful emphasis on collaboration between the public and private sectors in solving the world's health problems, especially those of the poor in the developing world, relies on industry developing new technologies with the support of government and foundations. However, certain products, despite their potential to have a great impact on disease in the developing world, will not be developed by industry because they carry the risk of lawsuits owing to possible severe adverse reactions--risks that are not counterbalanced by potential profit of products that are of limited use in the developed world. Freund's incomplete adjuvant (FIA) is one such example that is better developed in the public sector. In this article, we present the evidence that has led to our interest in FIA, evidence of its potential benefit in vaccines against blood-stage malaria, and the way forward to make safe, effective and affordable vaccines for malaria and other serious diseases in the developing world.
Animals↗
Phase 1 clinical trial of apical membrane antigen 1: an asexual blood-stage vaccine for Plasmodium falciparum malaria.
Apical membrane antigen 1 (AMA1), a polymorphic merozoite surface protein, is a leading blood-stage malaria vaccine candidate. A phase 1 trial was conducted with 30 malaria-naive volunteers to assess the safety and immunogenicity of the AMA1-C1 malaria vaccine. AMA1-C1 contains an equal mixture of recombinant proteins based on sequences from the FVO and 3D7 clones of Plasmodium falciparum. The proteins were expressed in Pichia pastoris and adsorbed on Alhydrogel. Ten volunteers in each of three dose groups (5 mug, 20 mug, and 80 mug) were vaccinated in an open-label study at 0, 28, and 180 days. The vaccine was well tolerated, with pain at the injection site being the most commonly observed reaction. Anti-AMA1 immunoglobulin G (IgG) was detected by enzyme-linked immunosorbent assay (ELISA) in 15/28 (54%) volunteers after the second immunization and in 23/25 (92%) after the third immunization, with equal reactivity to both AMA1-FVO and AMA1-3D7 vaccine components. A significant dose-response relationship between antigen dose and antibody response by ELISA was observed, and the antibodies were predominantly of the IgG1 isotype. Confocal microscopic evaluation of sera from vaccinated volunteers demonstrated reactivity with P. falciparum schizonts in a pattern similar to native parasite AMA1. Antigen-specific in vitro inhibition of both FVO and 3D7 parasites was achieved with IgG purified from sera of vaccinees, demonstrating biological activity of the antibodies. To our knowledge, this is the first AMA1 vaccine candidate to elicit functional immune responses in malaria-naive humans, and our results support the further development of this vaccine.
Adult↗
posttranslational modification of recombinant Plasmodium falciparum apical membrane antigen 1: impact on functional immune responses to a malaria vaccine candidate.
Recombinant apical membrane antigen 1 (AMA1) is a leading vaccine candidate for Plasmodium falciparum malaria, as antibodies against recombinant P. falciparum AMA1 (PfAMA1) interrupt merozoite invasion into erythrocytes. In order to investigate the role of posttranslational modification in modulating the functional immune response to recombinant AMA1, two separate alleles of PfAMA1 (FVO and 3D7), in which native N-glycosylation sites have been mutated, were produced using Escherichia coli and a Pichia pastoris expression system. Recombinant Pichia pastoris AMA1-FVO (PpAMA1-FVO) and PpAMA1-3D7 are O-linked glycosylated, and 45% of PpAMA1-3D7 is nicked, though all four recombinant molecules react with conformation-specific monoclonal antibodies. To address the immunological effect of O-linked glycosylation, we compared the immunogenicity of E. coli AMA1-FVO (EcAMA1-FVO) and PpAMA1-FVO antigens, since both molecules are intact. The effect of antigen nicking was then investigated by comparing the immunogenicity of EcAMA1-3D7 and PpAMA1-3D7. Our data demonstrate that there is no significant difference in the rabbit antibody titer elicited towards EcAMA1-FVO and PpAMA1-FVO or to EcAMA1-3D7 and PpAMA1-3D7. Furthermore, we have demonstrated that recombinant AMA1 (FVO or 3D7), whether expressed and refolded from E. coli or produced from the Pichia expression system, is equivalent and mimics the functionality of the native protein in in vitro growth inhibition assay experiments. We conclude that in the case of recombinant AMA1, the E. coli- and P. pastoris-derived antigens are immunologically and functionally equivalent and are unaffected by the posttranslational modification resulting from expression in these two systems.
Amino Acid Sequence↗
Determining residual host cell antigen levels in purified recombinant proteins by slot blot and scanning laser densitometry.
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Animals↗
Extraction and characterization of vaccine antigens from water-in-oil adjuvant formulations.
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Adjuvants, Immunologic↗