ABSTRACT
Dengue human infection models present an opportunity to explore the potential of a vaccine, anti-viral or immuno-compound for clinical benefit in a controlled setting. Here we report the outcome of a phase 1 open-label assessment of a low-dose dengue virus 3 (DENV-3) challenge model (NCT04298138), in which nine participants received a subcutaneous inoculation with 0.5 ml of a 1.4 × 103 plaque-forming unit per ml suspension of the attenuated DENV-3 strain CH53489. The primary and secondary endpoints of the study were to assess the safety of this DENV-3 strain in healthy flavivirus-seronegative individuals. All participants developed RNAaemia within 7 days after inoculation with peak titre ranging from 3.13 × 104 to 7.02 × 108 genome equivalents per ml. Solicited symptoms such as fever and rash, clinical laboratory abnormalities such as lymphopenia and thrombocytopenia, and self-reported symptoms such as myalgia were consistent with mild-to-moderate dengue in all volunteers. DENV-3-specific seroconversion and memory T cell responses were observed within 14 days after inoculation as assessed by enzyme-linked immunosorbent assay and interferon-gamma-based enzyme-linked immunospot. RNA sequencing and serum cytokine analysis revealed anti-viral responses that overlapped with the period of viraemia. The magnitude and frequency of clinical and immunologic endpoints correlated with an individual's peak viral titre.
Subject(s)
Antibodies, Viral , Dengue Vaccines , Dengue Virus , Dengue , Viremia , Humans , Dengue Virus/immunology , Dengue/immunology , Dengue/virology , Adult , Dengue Vaccines/immunology , Dengue Vaccines/administration & dosage , Dengue Vaccines/adverse effects , Male , Antibodies, Viral/blood , Antibodies, Viral/immunology , Female , Young Adult , Cytokines/blood , Cytokines/metabolism , RNA, Viral/blood , Seroconversion , Memory T Cells/immunology , Middle AgedABSTRACT
BACKGROUND: A vaccine to prevent infection and disease caused by Plasmodium vivax is needed both to reduce the morbidity caused by this parasite and as a key component in efforts to eradicate malaria worldwide. Vivax malaria protein 1 (VMP001), a novel chimeric protein that incorporates the amino- and carboxy- terminal regions of the circumsporozoite protein (CSP) and a truncated repeat region that contains repeat sequences from both the VK210 (type 1) and the VK247 (type 2) parasites, was developed as a vaccine candidate for global use. METHODS: We conducted a first-in-human Phase 1 dose escalation vaccine study with controlled human malaria infection (CHMI) of VMP001 formulated in the GSK Adjuvant System AS01B. A total of 30 volunteers divided into 3 groups (10 per group) were given 3 intramuscular injections of 15 µg, 30 µg, or 60 µg respectively of VMP001, all formulated in 500 µL of AS01B at each immunization. All vaccinated volunteers participated in a P. vivax CHMI 14 days following the third immunization. Six non-vaccinated subjects served as infectivity controls. RESULTS: The vaccine was shown to be well tolerated and immunogenic. All volunteers generated robust humoral and cellular immune responses to the vaccine antigen. Vaccination did not induce sterile protection; however, a small but significant delay in time to parasitemia was seen in 59% of vaccinated subjects compared to the control group. An association was identified between levels of anti-type 1 repeat antibodies and prepatent period. SIGNIFICANCE: This trial was the first to assess the efficacy of a P. vivax CSP vaccine candidate by CHMI. The association of type 1 repeat-specific antibody responses with delay in the prepatency period suggests that augmenting the immune responses to this domain may improve strain-specific vaccine efficacy. The availability of a P. vivax CHMI model will accelerate the process of P. vivax vaccine development, allowing better selection of candidate vaccines for advancement to field trials.
Subject(s)
Malaria Vaccines/immunology , Malaria, Vivax/prevention & control , Plasmodium vivax/immunology , Protozoan Proteins/immunology , Adolescent , Adult , Antibodies, Protozoan/immunology , Female , Humans , Malaria Vaccines/administration & dosage , Malaria Vaccines/adverse effects , Malaria, Vivax/immunology , Malaria, Vivax/parasitology , Male , Middle Aged , Protozoan Proteins/administration & dosage , Protozoan Proteins/adverse effects , Vaccination , Young AdultABSTRACT
Plasmodium falciparum Liver Stage Antigen 1 (LSA-1) is a pre-erythrocytic stage antigen. Our LSA-1 vaccine candidate is a recombinant protein with full-length C- and N-terminal flanking domains and two of the 17 amino acid repeats from the central repeat region termed "LSA-NRC." We describe the first Phase I/II study of this recombinant LSA-NRC protein formulated with either the AS01 or AS02 adjuvant system. We conducted an open-label Phase I/II study. Thirty-six healthy malaria-naïve adults received one of four formulations by intra-deltoid injection on a 0 and 1 month schedule; low dose (LD) LSA-NRC/AS01:10microg LSA-NRC/0.5ml AS01 (n=5), high dose (HD) LSA-NRC/AS01: 50microg LSA-NRC/0.5ml AS01 (n=13); LD LSA-NRC/AS02: 10microg LSA-NRC/0.5ml AS02 (n=5) and HD LSA-NRC/AS02: 50microg LSA-NRC/0.5ml AS02 (n=13). Two weeks post-second immunization, the high dose vaccinees and 6 non-immunized infectivity controls underwent experimental malaria sporozoite challenge. The vaccines showed a reassuring safety profile but were moderately reactogenic. There were no serious adverse events. All subjects seroconverted after the first immunization. Following the second immunization, LSA-1-specific CD4+ T cells producing two cytokines (IL-2 and IFN-gamma) were found by intra-cellular staining in all subjects in the LD LSA-NRC/AS01B group and in 3 of 5 subjects in the LD LSA-NRC/AS02 group. In contrast, the HD LSA-NRC/AS01 and HD LSA-NRC/AS02 group subjects had fewer LSA-1-specific CD4+ T cells, and minimal to no IFN-gamma responses. There was no increase in LSA-1-specific CD8+ T cells found in any group. Per protocol, 22 high dose vaccinees, but no low dose vaccinees, underwent P. falciparum homologous malaria challenge (3D7 clone). All vaccinees became parasitemic and there was no delay in their pre-patent period versus controls (p=0.95). LSA-NRC/AS01 and LSA-NRC/AS02 elicited antigen-specific antibody and CD4+ T cell responses, but elicited no protective immunity. Although the optimal antigen dose of LSA-NRC may not have been selected for the challenge portion of the protocol, further vaccine development based upon LSA-1 should not be excluded and should include alternative vaccine platforms able to elicit additional effector mechanisms such as CD8+ T cells.
Subject(s)
Antigens, Protozoan/immunology , CD4-Positive T-Lymphocytes/immunology , Malaria Vaccines/immunology , Malaria, Falciparum/prevention & control , Adjuvants, Immunologic/pharmacology , Adult , Antibodies, Protozoan/blood , Antibody Formation , Female , Humans , Immunity, Cellular , Immunity, Humoral , Immunization Schedule , Immunization, Secondary , Interferon-gamma/immunology , Interleukin-2/immunology , Malaria Vaccines/administration & dosage , Malaria Vaccines/adverse effects , Malaria, Falciparum/immunology , Male , Parasitemia/immunology , Plasmodium falciparum/immunology , Recombinant Proteins/immunology , Sporozoites/immunology , Young AdultABSTRACT
BACKGROUND: 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 microg/0.5 mL) in 5 adults, 30 malaria-naïve adults were randomly allocated to receive full dose (50 microg/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 microg/mL (103-371 microg/mL), full dose AMA-1/AS01B 279 microg/mL (210-369 microg/mL) and full dose AMA-1/AS02A 216 microg/mL (169-276 microg/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. SIGNIFICANCE: All three vaccine formulations were found to be safe and highly immunogenic. These immune responses did not translate into significant vaccine efficacy in malaria-naïve adults employing a primary sporozoite challenge model, but encouragingly, estimation of parasite growth rates from qPCR data may suggest a partial biological effect of the vaccine. Further evaluation of the immunogenicity and efficacy of the AMA-1/AS02A formulation is ongoing in a malaria-experienced pediatric population in Mali. TRIAL REGISTRATION: www.clinicaltrials.gov NCT00385047.
Subject(s)
Adjuvants, Immunologic/administration & dosage , Antigens, Protozoan/immunology , Lipid A/analogs & derivatives , Malaria Vaccines/administration & dosage , Malaria, Falciparum/prevention & control , Membrane Proteins/immunology , Protozoan Proteins/immunology , Saponins/administration & dosage , Adjuvants, Immunologic/pharmacology , Adolescent , Adult , Alleles , Animals , Antigens, Protozoan/administration & dosage , Antigens, Protozoan/genetics , Double-Blind Method , Drug Combinations , Enzyme-Linked Immunosorbent Assay , Humans , Lipid A/administration & dosage , Lipid A/pharmacology , Malaria Vaccines/adverse effects , Malaria Vaccines/immunology , Membrane Proteins/administration & dosage , Membrane Proteins/genetics , Middle Aged , Plasmodium falciparum/immunology , Plasmodium falciparum/metabolism , Protozoan Proteins/administration & dosage , Protozoan Proteins/genetics , Saponins/pharmacologyABSTRACT
The global eradication of malaria will require the development of vaccines to prevent infection cause by Plasmodium vivax in addition to Plasmodium falciparum. In an attempt to contribute to this effort we have previously reported the cloning and expression of a vaccine based on the circumsporozoite protein of P. vivax. The synthetic vaccine encodes for a full-length molecule encompassing the N-terminal and C-terminal regions flanking a chimeric repeat region representing VK210 and VK247, the two major alleles of P. vivax CSP. The vaccine, designated vivax malaria protein 001 (VMP001), was purified to >95% homogeneity using a three-column purification scheme and had low endotoxin levels and passed the rabbit pyrogenicity assay. The protein is recognized by monoclonal antibodies directed against the two repeat motifs, as well as polyclonal antibodies. Immunization with VMP001 induced high titer antibodies in mice using Montanide ISA 720. We currently have more than 10,000 doses of purified bulk and 1800 vials of formulated bulk vaccine available for clinical testing and VMP001 is currently undergoing further development as a candidate vaccine to prevent malaria in humans.
Subject(s)
Escherichia coli/immunology , Malaria Vaccines/therapeutic use , Malaria, Vivax/immunology , Plasmodium vivax/immunology , Vaccines, Synthetic/immunology , Animals , Cloning, Molecular , Humans , Immunization/methods , Malaria, Falciparum/immunology , Plasmodium falciparum/immunology , Rabbits/immunology , Recombinant Fusion Proteins/immunologyABSTRACT
We report the first safety and immunogenicity trial of the Plasmodium falciparum vaccine candidate FMP2.1/AS02A, a recombinant E. coli-expressed protein based upon the apical membrane antigen-1 (AMA-1) of the 3D7 clone formulated with the AS02A adjuvant. We conducted an open-label, staggered-start, dose-escalating Phase I trial in 23 malaria-naïve volunteers who received 8, 20 or 40microg of FMP2.1 in a fixed volume of 0.5mL of AS02A on a 0, 1, and 2 month schedule. Nineteen of 23 volunteers received all three scheduled immunizations. The most frequent solicited local and systemic adverse events associated with immunization were injection site pain (68%) and headache (29%). There were no significant laboratory abnormalities or vaccine-related serious adverse events. All volunteers seroconverted after second immunization as determined by ELISA. Immune sera recognized sporozoites and merozoites by immunofluorescence assay (IFA), and exhibited both growth inhibition and processing inhibition activity against homologous (3D7) asexual stage parasites. Post-immunization, peripheral blood mononuculear cells exhibited FMP2.1-specific lymphoproliferation and IFN-gamma and IL-5 ELISPOT assay responses. This is the first PfAMA-1-based vaccine shown to elicit both potent humoral and cellular immunity in humans. Encouraged by the potential of FMP1/AS02A to target host immunity against PfAMA-1 that is known to be expressed by sporozoite, hepatic and erythrocytic stages, we have initiated field trials of FMP2.1/AS02A in an endemic population in the Republic of Mali.
Subject(s)
Antigens, Protozoan/immunology , Lipid A/analogs & derivatives , Malaria Vaccines/adverse effects , Malaria Vaccines/immunology , Membrane Proteins/immunology , Plasmodium falciparum/immunology , Protozoan Proteins/immunology , Saponins/immunology , Adjuvants, Immunologic , Adolescent , Adult , Animals , Antibodies, Protozoan/blood , Cell Line , Cell Proliferation , Cells, Cultured , Cricetinae , Drug Combinations , Enzyme-Linked Immunosorbent Assay , Escherichia coli/genetics , Female , Fluorescent Antibody Technique, Indirect , Headache , Humans , Immunization, Secondary , Interferon-gamma/biosynthesis , Interleukin-5/biosynthesis , Leukocytes, Mononuclear/immunology , Lipid A/immunology , Malaria Vaccines/administration & dosage , Male , Merozoites/immunology , Mesocricetus , Middle Aged , Pain , Plasmodium falciparum/growth & development , Sporozoites/immunology , Vaccines, Synthetic/immunologyABSTRACT
The RTS,S/AS02A protein-based vaccine consistently demonstrates significant protection against infection with Plasmodium falciparum malaria and also against clinical malaria and severe disease in children in areas of endemicity. Here we demonstrate with rhesus macaques that priming with a replication-defective human adenovirus serotype 35 (Ad35) vector encoding circumsporozoite protein (CS) (Ad35.CS), followed by boosting with RTS,S in an improved MPL- and QS21-based adjuvant formulation, AS01B, maintains antibody responses and dramatically increases levels of T cells producing gamma interferon and other Th1 cytokines in response to CS peptides. The increased T-cell responses induced by the combination of Ad35.CS and RTS,S/AS01B are sustained for at least 6 months postvaccination and may translate to improved and more durable protection against P. falciparum infection in humans.
Subject(s)
Adenoviridae/genetics , Immunization Schedule , Malaria Vaccines/immunology , Malaria, Falciparum/prevention & control , Protozoan Proteins/immunology , Adenoviridae/classification , Adjuvants, Immunologic , Animals , Antibodies, Protozoan/blood , Female , Humans , Immunization , Immunization, Secondary , Interferon-gamma/metabolism , Macaca mulatta , Malaria Vaccines/administration & dosage , Malaria Vaccines/genetics , Malaria, Falciparum/immunology , Malaria, Falciparum/parasitology , Male , Plasmodium falciparum/immunology , Protozoan Proteins/administration & dosage , Protozoan Proteins/genetics , Receptors, Thrombopoietin/immunology , Saponins/immunology , T-Lymphocytes/immunology , Th1 CellsABSTRACT
Liver-stage antigen 1 (LSA1) is expressed by Plasmodium falciparum only during the intrahepatic cell stage of the parasite's development. Immunoepidemiological studies in regions where malaria is endemic suggested an association between the level of LSA1-specific humoral and cell-mediated immune responses and susceptibility to clinical malaria. A recombinant LSA1 protein, FMP011, has been manufactured as a preerythrocytic vaccine to induce an immune response that would have the effect of controlling parasitemia and disease in humans. To evaluate the immunogenicity of FMP011, we analyzed the immune response of three inbred strains of mice to antigen immunization using two different adjuvant formulations, AS01B and AS02A. We report here the ability of BALB/c and A/J mice, but not C57BL/6J mice, to mount FMP011-specific humoral (antibody titer) and cellular (gamma interferon [IFN-gamma] production) responses following immunization with FMP011 formulated in AS01B or AS02A. Immunization of BALB/c and A/J mice with FMP011/AS01B induced more antigen-specific IFN-gamma-producing splenocytes than immunization with FMP011/AS02A. A slightly higher titer of antibody was induced using AS02A than AS01B in both strains. C57BL/6J mice did not respond with any detectable FMP011-specific IFN-gamma splenocytes or antibody when immunized with FMP011 in AS01B or AS02A. Intracellular staining of cells isolated from FMP011/AS01B-immunized BALB/c mice indicated that CD4(+) cells, but not CD8(+) cells, were the main IFN-gamma-producing splenocyte. However, inclusion of blocking anti-CD4(+) antibody during the in vitro restimulation ELISpot analysis failed to completely abolish IFN-gamma production, indicating that while CD4(+) T cells were the major source of IFN-gamma, other cell types also were involved.
Subject(s)
Adjuvants, Immunologic , Antigens, Protozoan/immunology , Malaria Vaccines/immunology , Plasmodium falciparum/immunology , Animals , Antibodies, Protozoan/blood , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Female , Interferon-gamma/biosynthesis , Lymphocyte Subsets/immunology , Mice , Mice, Inbred A , Mice, Inbred BALB C , Mice, Inbred C57BL , Vaccines, Subunit/immunology , Vaccines, Synthetic/immunologyABSTRACT
A successful vaccine against Plasmodium vivax malaria would significantly improve the health and quality of the lives of more than 1 billion people around the world. A subunit vaccine is the only option in the absence of long-term culture of P. vivax parasites. The circumsporozoite protein that covers the surface of Plasmodium sporozoites is one of the best-studied malarial antigens and the most promising vaccine in clinical trials. We report here the development of a novel "immunologically optimal" recombinant vaccine expressed in Escherichia coli that encodes a chimeric CS protein encompassing repeats from the two major alleles, VK210 and VK247. This molecule is widely recognized by sera from patients naturally exposed to P. vivax infection and induces a highly potent immune response in genetically disparate strains of mice. Antibodies from immunized animals recognize both VK210 and VK247 sporozoites. Furthermore, these antibodies appear to be protective in nature since they cause the agglutination of live sporozoites, an in vitro surrogate of sporozoite infectivity. These results strongly suggest that recombinant CS is biologically active and highly immunogenic across major histocompatibility complex strains and raises the prospect that in humans this vaccine may induce protective immune responses.
Subject(s)
Antibodies, Protozoan/genetics , Malaria Vaccines/immunology , Malaria, Vivax/prevention & control , Plasmodium vivax/immunology , Protozoan Proteins/immunology , Recombinant Fusion Proteins/immunology , Sporozoites/immunology , Animals , Antibodies, Protozoan/metabolism , Antibodies, Protozoan/physiology , Female , Humans , Malaria Vaccines/genetics , Malaria, Vivax/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Plasmodium vivax/genetics , Protozoan Proteins/genetics , Recombinant Fusion Proteins/genetics , Sporozoites/metabolism , Vaccines, Subunit/genetics , Vaccines, Subunit/immunologyABSTRACT
BACKGROUND: RTS,S/AS02A, a pre-erythrocytic Plasmodium falciparum vaccine based upon the circumsporozoite protein, is the only vaccine demonstrated in field trials to confer partial protection against a range of malaria disease manifestations. Pre-clinical studies are on-going to identify new RTS,S formulations with improved magnitude and duration of specific immunity. METHODS: Rhesus macaques were immunized with saline or one of four "RTS,S/adjuvant" formulations at 0, 4, and 12 weeks: RTS,S/AS01B, RTS,S/AS02A-standard (current formulation), RTS,S/AS05 or RTS,S/AS06. An RTS,S/AS02A-accelerated group was immunized at 0, 1, and 4 weeks. Outcomes were safety, RTS,S-specific antibody, and IFN-gamma and IL-5 ELISpots (weeks 14 and 34). FINDINGS: All regimens were safe and, except for RTS,S/AS06, generated equivalent high titer antibody levels. For IFN-gamma ELISpots, RTS,S/AS01B had the highest geometric mean (GM) values at weeks 14 and 34, and was the only group with an overall GM mean (weeks 14+34) higher than RTS,S/AS02A-standard (p<0.015). For IFN-gamma to IL-5 ELISpot response ratios, RTS,S/AS01B had the highest values at weeks 14 and 34, and was the only group higher than RTS,S/AS02A-standard at each individual time point and overall (weeks 14+34) (p<0.015). INTERPRETATION: RTS,S/AS01B is a safe and immunogenically superior formulation for cellular responses, in comparison with the RTS,S/AS02A-standard. Phase 1, 2a, and 2b clinical trials are underway to determine if RTS,S/AS01B demonstrates improved immunogenicity and protective efficacy against experimental challenge and natural mosquito-borne malaria.
Subject(s)
Adjuvants, Immunologic/pharmacology , Malaria Vaccines/immunology , Malaria, Falciparum/immunology , Plasmodium falciparum/immunology , Adjuvants, Immunologic/administration & dosage , Adjuvants, Immunologic/chemistry , Animals , Antibodies, Protozoan/analysis , Antibodies, Protozoan/biosynthesis , Antibody Specificity , Blood Chemical Analysis , Chemistry, Pharmaceutical , Cytokines/biosynthesis , Data Interpretation, Statistical , Erythrocyte Count , Immunity, Cellular/physiology , Immunization, Secondary , Interferon-gamma/pharmacology , Interleukin-5/pharmacology , Leukocyte Count , Macaca mulatta , Malaria Vaccines/administration & dosage , Malaria Vaccines/adverse effects , Monocytes/immunology , Quality ControlABSTRACT
The 42-kDa fragment of the merozoite surface protein 1 (MSP-1(42)) is a leading candidate for the development of a vaccine to control malaria. We previously reported a method for the production of Plasmodium vivax MSP-1(42) (PvMSP-1(42)) as a soluble protein (S. Dutta, L. W. Ware, A. Barbosa, C. F. Ockenhouse, and D. E. Lanar, Infect. Immun. 69:5464-5470, 2001). We report here a process to manufacture the same PvMSP-1(42) protein but as an insoluble inclusion body-derived protein which was then refolded in vitro. We compared the immunogenicity and protective efficacy of the soluble and refolded forms of PvMSP-1(42) protein by using a heterologous but closely related P. cynomolgi-rhesus monkey challenge model. As comparative controls we also expressed, purified, and immunized rhesus with the soluble and refolded forms of the P. cynomolgi MSP-1(42) (PcMSP-1(42)) proteins. All proteins induced equally high-titer, cross-reacting antibodies. Upon challenge with P. cynomolgi, none of the MSP-1(42)-vaccinated groups demonstrated sterile protection or a delay in the prepatent period. However, following an initial rise in parasitemia, all MSP-1-vaccinated animals had significantly lower parasite burdens as indicated by lower cumulative parasitemia, lower peak parasitemia, lower secondary peak parasitemia, and lower average daily parasitemia compared to the adjuvant control group (P < 0.05). Except the soluble PcMSP-1(42) group, monkeys in all other groups had fewer numbers of days with parasitemia of >10,000 parasites mm(-3). Interestingly, there was no significant difference in the level of partial protection observed in the homologous and heterologous groups in this challenge model. The soluble and refolded forms of PcMSP-1(42) and PvMSP-1(42) proteins also appeared to have a similar partially protective effect.
Subject(s)
Malaria Vaccines/immunology , Malaria/prevention & control , Merozoite Surface Protein 1/immunology , Plasmodium cynomolgi/immunology , Plasmodium vivax/immunology , Animals , Macaca mulatta , Malaria/blood , Malaria/immunology , Merozoite Surface Protein 1/genetics , Merozoite Surface Protein 1/isolation & purification , Molecular Sequence Data , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Sequence Analysis, DNAABSTRACT
Plasmodium falciparum liver-stage antigen 1 (LSA-1) is expressed solely in infected hepatocytes and is thought to have a role in liver schizogony and merozoite release. Specific humoral, cellular, and cytokine immune responses to LSA-1 are well documented, with epitopes identified that correlate with antibody production, proliferative T-cell responses, or cytokine induction. With the goal of developing a vaccine against this preerythrocyte-stage protein, we undertook the good manufacturing practices (GMP) manufacture of a recombinant LSA-1 construct, LSA-NRC, incorporating the N- and C-terminal regions of the protein and two of the centrally placed 17-amino-acid repeats. To improve the protein yield, a method of codon harmonization was employed to reengineer the gene sequence for expression in Escherichia coli. A 300-liter GMP fermentation produced 8 kg of bacterial cell paste, and a three-step column chromatographic method yielded 8 mg of purified antigen per g of paste. The final bulk protein was >98% pure, demonstrated long-term stability, and contained <0.005 endotoxin units per 50 microg of protein. To accomplish the initial stages of evaluation of this protein as a human-use vaccine against malaria, we immunized rabbits and mice with LSA-NRC in Montanide ISA 720. New Zealand White rabbits and A/J (H-2K) mice produced high-titer antibodies that recognized liver-stage parasites in infected cultured human hepatocytes. Gamma interferon-producing cells, which have been associated with LSA-1-mediated protection, were detected in splenocytes harvested from immunized mice. Finally, sera taken from people living in a region where malaria is holoendemic recognized LSA-NRC by Western blotting.
Subject(s)
Antigens, Protozoan/immunology , Malaria Vaccines/immunology , Plasmodium falciparum/immunology , Vaccines, Synthetic/immunology , Amino Acid Sequence , Animals , Antibodies, Protozoan/blood , Antigens, Protozoan/biosynthesis , Escherichia coli/genetics , Female , Interferon-gamma/biosynthesis , Mice , Molecular Sequence Data , Recombinant Proteins/biosynthesisABSTRACT
Antibodies against apical membrane antigen 1 (AMA-1) of Plasmodium falciparum inhibit merozoite invasion into erythrocytes. Invasion-inhibitory polyclonal AMA-1 antibodies inhibit secondary proteolytic processing and surface redistribution of AMA-1 on merozoites. We present evidence supporting inhibition of processing and redistribution as probable causes of inhibition of invasion by polyclonal antibodies. Polyclonal anti-AMA-1 was much more inhibitory than monoclonal antibody (MAb) 4G2dc1 in an invasion assay. Although both polyclonal and monoclonal immunoglobulin G (IgG) inhibited secondary processing of the 66-kDa form of AMA-1, only polyclonal IgG caused its anomalous processing, inhibited its redistribution, and cross-linked soluble forms of AMA-1 on merozoites. Moreover, Fab fragments of polyclonal IgG that fail to cross-link did not show the enhancement of inhibitory effect over intact IgG, as observed in the case of Fab fragments of MAb 4G2dc1. We propose that although blocking of biologically important sites is a common direct mode of action of anti-AMA-1 antibodies, blocking of AMA-1 secondary processing and redistribution are additional indirect inhibitory mechanisms by which polyclonal IgG inhibits invasion. We also report a processing inhibition assay that uses a C-terminal AMA-1-specific MAb, 28G2dc1, to detect merozoite-bound remnants of processing (approximately 20 kDa from normal processing to 48 and 44 kDa and approximately 10 kDa from anomalous processing to a 52-kDa soluble form of AMA-1). The ratio of intensity of 10-kDa bands to the sum of 10- and 20-kDa bands was positively correlated with inhibition of invasion by polyclonal antibodies. This assay may serve as an important immunochemical correlate for inhibition of invasion.
Subject(s)
Antibodies, Protozoan/pharmacology , Antigens, Protozoan/immunology , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/immunology , Plasmodium falciparum/immunology , Protozoan Proteins/antagonists & inhibitors , Protozoan Proteins/immunology , Animals , Antibodies, Monoclonal/pharmacology , Antigens, Protozoan/metabolism , Erythrocytes/parasitology , Immune Sera/pharmacology , Immunoglobulin Fab Fragments/pharmacology , Immunoglobulin G/pharmacology , Membrane Proteins/metabolism , Mice , Protozoan Proteins/metabolism , RabbitsABSTRACT
The goal of the Malaria Vaccine Program at the Walter Reed Army Institute of Research (WRAIR) is to develop a licensed multi-antigen, multi-stage vaccine against Plasmodium falciparum able to prevent all symptomatic manifestations of malaria by preventing parasitemia. A secondary goal is to limit disease in vaccinees that do develop malaria. Malaria prevention will be achieved by inducing humoral and cellular immunity against the pre-erythrocytic circumsporozoite protein (CSP) and the liver stage antigen-1 (LSA-1). The strategy to limit disease will target immune responses against one or more blood stage antigens, merozoite surface protein-1 (MSP-1) and apical merozoite antigen-1 (AMA-1). The induction of T- and B-cell memory to achieve a sustained vaccine response may additionally require immunization with an adenovirus vector such as adenovirus serotype 35. RTS,S, a CSP-derived antigen developed by GlaxoSmithKline Biologicals in collaboration with the Walter Reed Army Institute of Research over the past 17 years, is the cornerstone of our program. RTS,S formulated in AS02A (a GSK proprietary formulation) is the only vaccine candidate shown in field trials to prevent malaria and, in one instance, to limit disease severity. Our vaccine development plan requires proof of an individual antigen's efficacy in a Phase 2 laboratory challenge or field trial prior to its integration into an RTS,S-based, multi-antigen vaccine. Progress has been accelerated through extensive partnerships with industrial, academic, governmental, and non-governmental organizations. Recent safety, immunogenicity, and efficacy trials in the US and Africa are presented, as well as plans for the development of a multi-antigen vaccine.
Subject(s)
Malaria Vaccines/isolation & purification , Plasmodium falciparum/immunology , Academies and Institutes , Adenoviridae/genetics , Animals , Antigens, Protozoan/genetics , Antigens, Protozoan/immunology , Antigens, Protozoan/isolation & purification , Clinical Trials as Topic , Genetic Vectors , Humans , Malaria Vaccines/genetics , Malaria Vaccines/immunology , Malaria Vaccines/pharmacology , Malaria, Falciparum/prevention & control , Membrane Proteins/genetics , Membrane Proteins/immunology , Merozoite Surface Protein 1/genetics , Merozoite Surface Protein 1/immunology , Plasmodium falciparum/genetics , Protozoan Proteins/genetics , Protozoan Proteins/immunology , United StatesABSTRACT
The apical membrane antigen 1 of Plasmodium falciparum is one of the leading candidate antigens being developed as a vaccine to prevent malaria. This merozoite transmembrane protein has an ectodomain that can be divided into three subdomains (D I, D II, and D III). We have previously expressed a major portion of this ectodomain and have shown that it can induce antibodies that prevent merozoite invasion into red blood cells in an in vitro growth and invasion assay. To analyze the antibody responses directed against the individual subdomains, we constructed six different genes that express each of the domains separately (D I, D II, or D III) or in combination with another domain (D I+II, D II+III, or D I+III). These proteins were purified and used to immunize rabbits to raise construct-specific antibodies. We demonstrated that D I+II induced a significant amount of the growth-inhibitory antibodies active in the growth and invasion assay.
Subject(s)
Antibodies, Protozoan/blood , Antigens, Protozoan/chemistry , Antigens, Protozoan/immunology , Membrane Proteins/chemistry , Membrane Proteins/immunology , Plasmodium falciparum/immunology , Plasmodium falciparum/pathogenicity , Protozoan Proteins/chemistry , Protozoan Proteins/immunology , Animals , Antibodies, Protozoan/immunology , Antigens, Protozoan/genetics , Antigens, Protozoan/metabolism , Erythrocytes/parasitology , Escherichia coli/genetics , Escherichia coli/metabolism , Immunization , Malaria Vaccines/administration & dosage , Malaria Vaccines/immunology , Malaria, Falciparum/prevention & control , Membrane Proteins/genetics , Membrane Proteins/metabolism , Plasmodium falciparum/chemistry , Plasmodium falciparum/growth & development , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Rabbits , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolismABSTRACT
The apical membrane antigen 1 (AMA1) has emerged as a promising vaccine candidate against malaria. Advanced evaluation of its protective efficacy in humans requires the production of highly purified and correctly folded protein. We describe here a process for the expression, fermentation, refolding, and purification of the recombinant ectodomain of AMA1 (amino acids 83(Gly) to 531(Glu)) of Plasmodium falciparum (3D7) produced in Escherichia coli. A synthetic gene containing an E. coli codon bias was cloned into a modified pET32 plasmid, and the recombinant protein was produced by using a redox-modified E. coli strain, Origami (DE3). A purification process was developed that included Sarkosyl extraction followed by affinity purification on a Ni-nitrilotriacetic acid column. The recombinant AMA1 was refolded in the presence of reduced and oxidized glutathione and further purified by using two ion-exchange chromatographic steps. The final product, designated AMA1/E, was homogeneous, monomeric, and >99% pure and had low endotoxin content and low host cell contamination. Analysis of AMA1/E showed that it had the predicted primary sequence, and tertiary structure analysis confirmed its compact disulfide-bonded nature. Rabbit antibodies made to the protein recognized the native parasite AMA1 and inhibited the growth of the P. falciparum homologous 3D7 clone in an in vitro assay. Reduction-sensitive epitopes on AMA1/E were shown to be necessary for the production of inhibitory anti-AMA1 antibodies. AMA1/E was recognized by a conformation-dependent, growth-inhibitory monoclonal antibody, 4G2dc1. The process described here was successfully scaled up to produce AMA1/E protein under GMP conditions, and the product was found to induce highly inhibitory antibodies in rabbits.