Evaluation of the safety and immunogenicity of Plasmodium falciparum apical membrane antigen 1, merozoite surface protein 1 or RTS,S vaccines with adjuvant system AS02A administered alone or concurrently in rhesus monkeys.

In an effort to broaden the immune response induced by the RTS,S/AS02(A),vaccine, we have evaluated the immunogenicity of the RTS,S antigen when combined with MSP1(42) and with AMA1, antigens derived from the asexual blood stage. The objectives of this study were (i) to determine whether MSP1(42) and AMA1 vaccines formulated with the AS02(A) Adjuvant System were safe and immunogenic in the rhesus monkey model; (ii) to investigate whether MSP1(42) or AMA1 induced immune interference to each other, or to RTS,S, when added singly or in combinations at a single injection site; (iii) in the event of immune interference, to determine if this could be reduced when antigens were administered at separate sites. We found that MSP1(42) and AMA1 were safe and immunogenic, eliciting antibodies, and Th1 and Th2 responses using IFN-gamma and IL-5 as markers. When malaria antigens were delivered together in one formulation, MSP1(42) and RTS,S reduced AMA1-specific antibody responses as measured by ELISA however, only MSP1(42) lowered parasite growth inhibitory activity of anti-AMA1 antibodies as measured by in vitro growth inhibition assay. Unlike RTS,S, MSP1(42) significantly reduced AMA1 IFN-gamma and IL-5 responses. MSP1(42) suppression of AMA1 IFN-gamma responses was not seen in animals receiving RTS,S+AMA1+MSP1(42) suggesting that RTS,S restored IFN-gamma responses. Conversely, AMA1 had no effect on MSP1(42) antibody and IFN-gamma and IL-5 responses. Neither AMA1 alone or combined with MSP1(42) affected RTS,S antibody or IFN-gamma and IL-5 responses. Immune interference by MSP1(42) on AMA1 antibody responses was also evident when AMA1, MSP1(42) and RTS,S were administered concurrently at separate sites. These results suggest that immune interference may be complex and should be considered for the design of multi-antigen, multi-stage vaccines against malaria.

Potentiation by a novel alkaloid glycoside adjuvant of a protective cytotoxic T cell immune response specific for a preerythrocytic malaria vaccine candidate antigen.

We have recently demonstrated that the novel glycoalkaloid tomatine, derived from leaves of the wild tomato Lycopersicon pimpinellifolium, can act as a powerful adjuvant for the elicitation of antigen-specific CD8+ T cell responses. Here, we have extended our previous investigation with the model antigen ovalbumin to an established malaria infection system in mice and evaluated the cellular immune response to a major preerythrocytic stage malaria vaccine candidate antigen when administered with tomatine. The defined MHC H-2kd class I-binding 9-mer peptide (amino acids 252-260) from Plasmodium berghei circumsporozoite (CS) protein was prepared with tomatine to form a molecular aggregate formulation and this used to immunise BALB/c (H-2kd) mice. Antigen-specific IFN-gamma secretion and cytotoxic T lymphocyte activity in vitro were both significantly enhanced compared to responses detected from similarly stimulated splenocytes from naive and tomatine-saline-immunised control mice. Moreover, when challenged with P. berghei sporozoites, mice immunised with the CS 9-mer-tomatine preparation had a significantly delayed onset of erythrocytic infection compared to controls. The data presented validate the use of tomatine to potentiate a cellular immune response to antigenic stimulus by testing in an important biologically relevant system. Specifically, the processing of the P. berghei CS 9-mer as an exogenous antigen and its presentation via MHC class I molecules to CD8+ T cells led to an immune response that is an in vitro correlate of protection against preerythrocytic malaria. This was confirmed by the protective capacity of the 9-mer-tomatine combination upon in vivo immunisation. These findings merit further work to optimise the use of tomatine as an adjuvant in malaria vaccine development.

Pre-erythrocytic immunity to Plasmodium falciparum: the case for an LSA-1 vaccine.

A vaccine is urgently needed to stem the global resurgence of Plasmodium falciparum malaria. Vaccines targeting the erythrocytic stage are often viewed as an anti-disease strategy. By contrast, infection might be completely averted by a vaccine against the liver stage, a pre-erythrocytic stage during which the parasite multiplies 10000-fold within hepatocytes. Sterilizing immunity can be conferred by inoculating humans with irradiated pre-erythrocytic parasites, and a recombinant pre-erythrocytic vaccine partially protects humans from infection. Liver-stage antigen-1, one of a few proteins known to be expressed by liver-stage parasites, holds particular promise as a vaccine. Studies of naturally exposed populations have consistently related immune responses against this antigen to protection.

Plasmodium falciparum liver-stage antigen-1 peptide-specific interferon-gamma responses are not suppressed during uncomplicated malaria in African children.

Liver-stage antigen (LSA)-1 is a candidate vaccine molecule for Plasmodium falciparum malaria, but knowledge of the evolution of naturally acquired immune responses to LSA-1 in African children is lacking. We therefore assessed cellular immune responses to two defined T cell epitopes of LSA-1, during and after uncomplicated P. falciparum malaria in a group of Gabonese children. In terms of their prevalence, interferon (IFN)-gamma responses of peripheral blood mononuclear cells (PBMC) to an LSA-1 N-terminal peptide, T1, were significantly higher when measured during the acute phase compared with convalescence. IFN-gamma responses to the LSA-J (hinge region) peptide showed a similar profile, but at a lower prevalence. Depletion experiments confirmed that CD8+ T cells are a major source of peptide-driven IFN-gamma, but both lymphoproliferation and the production of IL-10 in response to either of the peptides was low in all children at all times. PBMC from 25% of the children failed to produce IFN-gamma in response to either peptide at any time-point. The results suggest that lymphocytes producing IFN-gamma in response to at least one T cell epitope of LSA-1 are most frequent in the peripheral circulation during the acute phase of P. falciparum malaria. Thus, in this case, the generalised suppression of cell-mediated responses which characterises acute malaria does not affect liver-stage antigen-specific IFN-gamma production. These findings imply that measurements of the frequency of parasite antigen-specific cellular immune responses in clinically healthy individuals may represent significant underestimations, which has important implications for the design of field-based vaccine antigen-related studies.

Influence of age and HLA type on interferon-gamma (IFN-gamma) responses to a naturally occurring polymorphic epitope of Plasmodium falciparum liver stage antigen-1 (LSA-1).

Antigenic polymorphism and HLA restriction may limit the immunogenicity of a subunit vaccine against liver-stage Plasmodium falciparum. We examined 59 clinical isolates and five laboratory clones of P. falciparum for polymorphism in the N- and C-terminal regions of LSA-1, evaluated binding of the corresponding peptides to selected HLA class I alleles, and measured IFN-gamma responses in residents of a malaria-endemic area of Papua New Guinea where HLA-A*1101, -24, -B13, and -B40 are the most common class I alleles. LSA-1 polymorphism was limited to a single non-synonymous mutation encoding serine (S), proline (P), or threonine (T) at amino acid 85. Nine-mer 84-92 peptides with S, T, or P at the primary anchor position bound differentially to HLA-A11, -A2, and -B7. IFN-gamma ELISPOT responses increased with age in malaria-exposed subjects: 14-16% and 30-36% of 2-5- and 6-54-year-olds, respectively, had > or =10 IFN-gamma-secreting cells/106 peripheral blood mononuclear cells when stimulated with at least one peptide variant (P<0.05). IFN-gamma responses to all three peptides were also greater for older than younger individuals. No children < 3 years old had lymphocytes that responded to all three 84-92 peptides, whereas 45% of adults (mean age 48 years) had aggregated IFN-gamma responses. These data support the notion that age-related cumulative exposure to P. falciparum increases the frequency of IFN-gamma responses to polymorphic epitopes of liver-stage antigens such as LSA-1.

Expression and immunogenicity of a liver stage malaria epitope presented as a foreign peptide on the surface of RNA-free MS2 bacteriophage capsids.

We have designed a novel vaccine strategy which enables display of short peptides expressed from chimeras of the gene encoding the coat protein of the RNA bacteriophage MS2 and inserted foreign DNA. MS2 coat protein has a beta-hairpin loop at the N-terminus which forms the most radially distinct feature of the mature capsid. The coat protein gene was modified to enable insertion of DNA at the central part of the beta-hairpin loop. Upon expression of the recombinant gene in E. coli, the MS2 coat protein subunits self-assemble into capsids, each comprising 180 copies of the monomer. This system was used to produce chimeras containing a putatively protective epitope, T1, from the immunodominant liver stage antigen-1 (LSA-1) of the malaria parasite Plasmodium falciparum. The immunogenicity of the native MS2 capsid and the recombinant construct was investigated in BALB/c (H-2(d)) mice. The native protein appeared to elicit both humoral and cellular immune responses, observed as a predominance of type 2 cytokines but with a mixed profile of immunoglobulin isotypes. In contrast, the LSA-1 chimera stimulated a type 1-polarised response, with significant upregulation of interferon-gamma, a finding which corroborates naturally acquired resistance to liver stage malaria. These results validate RNA phage capsid display of immunogenic determinants as a basis for the development of novel peptide vaccines and indicate that further evaluation of MS2 coat protein as a vector for malaria epitopes is merited.

Identification of heparin as a ligand for the A-domain of Plasmodium falciparum thrombospondin-related adhesion protein.

Thrombospondin-related adhesion protein (TRAP) is a Plasmodium falciparum transmembrane protein that is expressed within the micronemes of sporozoites, and is implicated in host cell invasion and motility. Contained within the extracellular region of TRAP is an A-domain, a module found in a number of membrane, plasma and matrix proteins, that is often involved in ligand recognition. In order to determine the role of the TRAP A-domain, it has been expressed as a glutathione S-transferase fusion protein and its ligand binding compared with that of other characterised glutathione S-transferase A-domain fusion proteins. Using a solid phase assay to screen for binding to known A-domain ligands, the TRAP A-domain was found to bind heparin. Binding to heparin appeared to be specific as it was saturable, and was inhibited by soluble heparin, fucoidan and dextran sulfate, but not by other negatively charged sulfated glycosaminoglycans such as chondroitin sulfates. Furthermore, unlike some A-domain ligand interactions, the A-domain of both TRAP and the leukocyte integrin, Mac-1, bound to heparin in the absence of divalent cations. It has been shown previously that another domain within TRAP, which is homologous to region II-plus of circumsporozoite protein, binds to sulfatide and to heparan sulfate on the immortalised hepatocyte line HepG2. The TRAP A-domain also bound to sulfatide and to HepG2 cells. Thus the A-domain shares certain binding properties already attributed to the region II-plus-like domain of TRAP, and may contribute to the binding of TRAP to heparan sulfate on hepatocytes.

Biology of malarial liver stages: implications for vaccine design.

The molecular events controlling sporozoite invasion and exo-erythrocytic (EE) development within hepatocytes are largely not understood, and EE parasites are probably better defined immunologically than biologically. The observation that the Plasmodium falciparum sporozoite antigen TRAP (thrombospondin-related anonymous protein) contains multiple adhesive domains that recognize endothelial and hepatocyte receptors indicates that, like leucocyte passage across capillaries, sporozoite invasion probably involves a co-ordinated interaction between sporozoite and hepatic molecules. The parallel with leucocyte extravasation is strengthened by the finding that TRAP contains a functional, integrin-like, I domain. EE parasites are an important target of immunity elicited by irradiated sporozoites, and much current effort is focused on developing malaria vaccines targeting EE parasites. Only one EE-specific antigen, liver-stage antigen 1 (LSA-1), is known to be expressed during EE development and may contribute to protective immunity elicited by irradiated P. falciparum sporozoites. In a study in Papua New Guinea, resistance to P. falciparum infection correlated with CD8+ T-cell interferon-gamma responses to an LSA-1 epitope that contains an HLA A11-restricted sequence. Since A11 is > 40% frequent in this population it is reasonable to suggest that, as with B53 responses to LSA-1 in The Gambia, P. falciparum has driven genetic selection of certain HLA haplotypes, as proposed by Haldane nearly 50 years ago. LSA-1 is thus an important vaccine candidate, and is being expressed in bacterial and phage vectors.

NYVAC-Pf7: a poxvirus-vectored, multiantigen, multistage vaccine candidate for Plasmodium falciparum malaria.

The highly attenuated NYVAC vaccinia virus strain has been utilized to develop a multiantigen, multistage vaccine candidate for malaria, a disease that remains a serious global health problem and for which no highly effective vaccine exists. Genes encoding seven Plasmodium falciparum antigens derived from the sporozoite (circumsporozoite protein and sporozoite surface protein 2), liver (liver stage antigen 1), blood (merozoite surface protein 1, serine repeat antigen, and apical membrane antigen 1), and sexual (25-kDa sexual-stage antigen) stages of the parasite life cycle were inserted into a single NYVAC genome to generate NYVAC-Pf7. Each of the seven antigens was expressed in NYVAC-Pf7-infected culture cells, and the genotypic and phenotypic stability of the recombinant virus was demonstrated. When inoculated into rhesus monkeys, NYVAC-Pf7 was safe and well tolerated. Antibodies that recognize sporozoites, liver, blood, and sexual stages of P. falciparum were elicited. Specific antibody responses against four of the P.falciparum antigens (circumsporozoite protein, sporozoite surface protein 2, merozoite surface protein 1, and 25-kDa sexual-stage antigen) were characterized. The results demonstrate that NYVAC-Pf7 is an appropriate candidate vaccine for further evaluation in human clinical trials.

T lymphocytes from volunteers immunized with irradiated Plasmodium falciparum sporozoites recognize liver and blood stage malaria antigens.

The model of protective immunity induced by immunization with irradiated plasmodia sporozoites (SPZ) has become the prototype for a promising vaccine strategy based on Ab and CTL responses directed against pre-erythrocytic stage Ags, in particular the circumsporozoite protein (CSP) and sporozoite surface protein 2 (SSP2). However, results from recently conducted vaccine studies suggest that T cell responses directed against additional specificities might also be required for protection. We have tested this hypothesis by examining human T lymphocytes from irradiated Plasmodium falciparum SPZ-immune volunteers for proliferative reactivities to parasitized red blood cells (pRBC) and recombinant proteins and synthetic peptides representing certain liver and blood stage Ags. In this work, we report that although SPZ-induced protective immunity is stage-specific, SPZ-immune lymphocytes recognized determinants associated with erythrocytic and liver stage parasites. Thus, protective immunity induced by irradiated SPZ may depend upon responses against pre-erythrocytic Ags in addition to CSP and SSP2.

Induction of Plasmodium falciparum sporozoite-neutralizing antibodies upon vaccination with recombinant Pfs16 vaccinia virus and/or recombinant Pfs16 protein produced in yeast.

Pfs16 is a sexual stage/sporozoite-specific antigen of Plasmodium falciparum and is a potential candidate for a sporozoite-neutralizing vaccine. To obtain more information on the function of Pfs16 and to investigate its role during transmission and hepatocyte invasion, immunization experiments were performed with both a Pfs16-specific recombinant vaccinia virus and virus-like particles produced in yeast composed of the hepatitis B surface antigen (HBsAg) and antigen Pfs16 fused to HBsAg. Upon transformation of yeast cells, harbouring a genomic copy of the HBsAg gene, with a plasmid carrying the fusion gene Pfs16-HBsAg (Pfs16-S) virus-like hybrid particles composed of HBsAg and Pfs16-S were formed of a size similar to those present in human sera after infection with the hepatitis B virus. Cells infected with recombinant Pfs16 vaccinia virus synthesized a polypeptide of approx. 16 kDa that reacted with a Pfs16-specific polyclonal antibody. Animals vaccinated with the yeast hybrid particles and/or recombinant vaccinia virus both produced Pfs16-specific antibodies. These antibodies showed no transmission-blocking activity, but they efficiently diminished or abolished in vitro invasion of sporozoites into human hepatoma cells (HepG2-A16) and primary human hepatocytes.

Ultrastructural localization of CD36 in human hepatic sinusoidal lining cells, hepatocytes, human hepatoma (HepG2-A16) cells, and C32 amelanotic melanoma cells.

Ultrastructural localization of CD36 in human hepatic sinusoidal lining cells, hepatocytes, human hepatoma (HepG2-A16) cells, and C32 amelanotic melanoma cells. Experimental Parasitology 79, 383-390. CD36 is expressed in the endothelial cells of some human organs, but the ultrastructural localization of this molecule in the sinusoidal lining cells of human liver is not well established. We report the ultrastructural localization of CD36 in the liver using a novel murine monoclonal antibody against CD36, namely MO30, as a primary antibody. Immunocytochemistry by the postembedding method showed that CD36 was localized in endothelial cells of sinusoids and in hepatocyte microvilli protruding into the space of Disse. Moreover, in cultured human hepatoma (HepG2-A16) cells and C32 amelanotic melanoma cells, MO30 reacted with microvilli. Hence, CD36 expressed on these cells may be involved in recognition and/or entry of these cells by malaria sporozoites.

Effects of hormones and cysteine protease modulators on infection of HepG2 cells by Plasmodium berghei sporozoites in vitro determined by ELISA immunoassay.

An enzyme-linked immunosorbent assay (ELISA) detecting a Plasmodium berghei liver-stage-specific protein Pbl-1 is described. The quantitative detection limits ranged from 0.01 to 0.05 microgram of parasite protein. Qualitatively the assay detected as little as 0.001 microgram Pbl-1 per well. Using the ELISA dexamethasone and insulin together was shown to promote higher parasite infections in HepG2 cells compared to unsupplemented medium. Anti-cowpea-protease cysteine inhibitor significantly increased hepatocyte invasion as compared to controls, whereas a significant decrease was recorded in the presence of the protease inhibitor E64. Partial involvement of cysteine proteases in HepG2 invasion by P. berghei sporozoites is therefore suggested.

Long-term persistence of sterile immunity in a volunteer immunized with X-irradiated Plasmodium falciparum sporozoites.

Three volunteers were immunized by repeated exposure to the bites of Plasmodium falciparum-infected, X-irradiated mosquitoes to characterize immunologic responses and duration of protective immunity. A primary series of immunizations had been shown previously to induce sterile immunity in these volunteers against sporozoite-induced P. falciparum malaria. In the current study, antibodies to sporozoites circulated at high levels for at least 9-12 months after the volunteers were administered booster bites from X-irradiated infective mosquitoes. One volunteer challenged a second time with P. falciparum 9 months after his last immunization was again shown to be protected, whereas all 5 control subjects developed patent infections. These results set a new standard for persistence of sterile immunity against experimental P. falciparum infection.

Plasmodium falciparum sporozoite immunization protects against Plasmodium berghei sporozoite infection.

Irradiated sporozoites are generally thought to elicit protective immune responses that are parasite stage and species specific. But immunization with Plasmodium falciparum sporozoites delivered by the bite of infected mosquitoes protects an average of 60% mice from Plasmodium berghei sporozoite infection. Protection appears to be specific as P. falciparum sporozoite-immunized mice protected against P. berghei remain susceptible to Plasmodium yoelii sporozoite infection. Passively transferred immunoglobulin from P. falciparum sporozoite-immunized mouse serum protected naive mice against challenge with P. berghei sporozoites, indicating that cross-protection is mediated, at least in part, by anti-sporozoite antibody. Antibody-mediated protection may not be due to cross-reaction between P. falciparum and P. berghei CS proteins because mice immunized with the recombinant P. falciparum CS protein repeat vaccine candidate, R32tet32, remain susceptible to P. berghei sporozoite infection.

Humoral immune responses in volunteers immunized with irradiated Plasmodium falciparum sporozoites.

Volunteers immunized with gamma-irradiated Plasmodium falciparum sporozoites serve as the gold standard for protective immunity against mosquito-borne malaria transmission and provide a relevant model for studying protective immune effector mechanisms. During a 7-12 month period, we immunized four volunteers via the bites of irradiated, infected mosquitoes. Following these exposures to attenuated sporozoites, all four volunteers developed antibodies to sporozoites as measured by an immunofluorescence assay and by an enzyme-linked immunosorbent assay using the circumsporozoite (CS) protein repeat-based molecule R32LR as capture antigen. Three volunteers also developed antibodies against the nonrepeating (flanking) regions of the CS protein; the level of these antibodies paralleled the serum activity to inhibit sporozoite invasion of hepatoma cells in vitro. These three volunteers were protected against malaria transmitted by the bites of five infected mosquitoes. Two of these protected volunteers received additional immunizing doses of irradiated sporozoites and were subsequently protected against challenge with a heterologous P. falciparum clone. No detectable fluctuations were observed in circulating levels of tumor necrosis factor, interferon-gamma, or interleukin-6 during the course of this study. Analysis of the humoral and cellular immune responses of these protected volunteers is expected to yield important clues to additional targets of immunity against the pre-erythrocytic stages of malaria parasites.

Thrombospondin related anonymous protein (TRAP) of Plasmodium falciparum binds specifically to sulfated glycoconjugates and to HepG2 hepatoma cells suggesting a role for this molecule in sporozoite invasion of hepatocytes.

Thrombospondin related anonymous protein (TRAP) of Plasmodium falciparum contains an amino acid motif based around the sequence WSPCSVTCG which is also found in region II of the circumsporozoite (CS) proteins of different species of Plasmodium. This amino acid motif confers on the CS protein the ability to bind specifically to sulfated glycoconjugates and to hepatocytes. This suggests that the interaction of CS protein with sulfated glycoconjugates on the surface of the hepatocytes may represent the first molecular event of sporozoite invasion of liver cells. Experimental evidence indicates that TRAP is localized both on the micronemes and on the surface of P. falciparum sporozoites implying that TRAP with its putative sulfated glycoconjugate binding motif may also be involved in recognition and/or entry of hepatocytes by the sporozoite. We show here that different TRAP constructs expressed in Escherichia coli bind to sulfogalactosyl-cerebrosides (sulfatides) and to the surface of HepG2 cells. These interactions are dependent on the presence of the conserved amino acid motif WSPCSVTCG within the sequences of the constructs and are completely inhibited by several sulfated glycoconjugates as well as by suramin, a polysulfonated drug with anti-protozoan activity. Moreover, sporozoite invasion of HepG2 cells is inhibited by antisera raised against these different TRAP constructs and by the presence of low concentrations of suramin. We concluded that TRAP may be one of the parasite encoded molecules in the host-parasite interaction that results in sporozoite invasion of hepatocytes.

Plasmodium falciparum: stage-specific ribosomal RNA as a potential target for monitoring parasite development in Anopheles stephensi.

The transcriptional switch of Plasmodium falciparum ribosomal RNA A gene to the C gene was demonstrated during the developmental transition from the vertebrate blood stage to the invertebrate sporozoite stage. Expression of the sporozoite specific C gene in infected mosquitoes was not detected until Day 10 postinfectious blood meal, the time of mature oocyst formation on the midgut. As a potential target for monitoring malaria parasite development in mosquitoes, oligonucleotide probes based on sequences of small subunit ribosomal RNA were evaluated for specificity and sensitivity by filter blot hybridization against different species and stages of malaria parasites. Probes PfC02 and PfA02 were selected as the most sensitive for sporozoite and blood stage parasites, respectively. Filter blot hybridization using probe PfC02 resulted in sensitivity comparable with microscopic dissection in single mosquitoes, detecting mosquitoes with an average of 1.2 oocysts per gut or as few as 800 salivary gland sporozoites.