Two-step chromatographic purification of recombinant Plasmodium falciparum circumsporozoite protein from Escherichia coli.

The Plasmodium falciparum circumsporozoite (PfCS) protein (aa 19-405) has been cloned and expressed in E. coli. The protein was purified in a two-step process that was rapid and reproducible. E. coli cells were grown to a high density before induction for 1 h. Cells were disrupted by high pressure microfluidization and the total bacterial protein solubilized in 6 M Gu-HCl. The protein was refolded while bound to Ni-NTA agarose by exchange of 6 M Gu-HCl for 8 M urea and then slow removal of the urea. The eluted protein was further purified on Q Sepharose Fast Flow using conditions developed to remove E. coli proteins and reduce endotoxin (to 10 EU/50 microg). Yield was 20 mg of PfCS protein from 10 g of wet cell paste. The final protein product bound to HepG2 liver cells in culture and inhibited the invasion of those cells by sporozoites in an ISI assay greater than 80% over control cultures when used at 10 microg/ml.

Enhancement of the immune response in rabbits to a malaria DNA vaccine by immunization with a needle-free jet device.

We compared the needle free jet device device Biojector with syringe/needle as a method to administer a DNA vaccine encoding the Plasmodium falciparum circumsporozoite protein (PfCSP) in albino rabbits. A group of three rabbits was injected by the intramuscular (IM) route using a syringe/needle combination, a second group IM with the Biojector device and a third group both IM and intradermal (ID) using the Biojector. When animals were immunized with the Biojector IM or IM/ID as compared to the syringe/needle IM, we observed 10- and 50-fold greater antibody titers, as measured by enzyme immunoassay (EIA) and indirect fluorescence antibody test (IFAT), respectively. We also observed that the Biojector conferred a greater ability to prime the immune system as compared with the needle. The subsequent boosting of all animals with a recombinant canary pox virus (ALVAC) expressing PfCSP induced significantly higher titers in both Biojector groups of rabbits as compared with the needle and naive animals. These results provided the foundation for a clinical trial using the same regime.

Anti-mosquito midgut antibodies block development of Plasmodium falciparum and Plasmodium vivax in multiple species of Anopheles mosquitoes and reduce vector fecundity and survivorship.

The mosquito midgut plays a central role in the sporogonic development of malaria parasites. We have found that polyclonal sera, produced against mosquito midguts, blocked the passage of Plasmodium falciparum ookinetes across the midgut, leading to a significant reduction of infections in mosquitoes. Anti-midgut mAbs were produced that display broad-spectrum activity, blocking parasite development of both P. falciparum and Plasmodium vivax parasites in five different species of mosquitoes. In addition to their parasite transmission-blocking activity, these mAbs also reduced mosquito survivorship and fecundity. These results reveal that mosquito midgut-based antibodies have the potential to reduce malaria transmission in a synergistic manner by lowering both vector competence, through transmission-blocking effects on parasite development, and vector abundance, by decreasing mosquito survivorship and egg laying capacity. Because the intervention can block transmission of different malaria parasite species in various species of mosquitoes, vaccines against such midgut receptors may block malaria transmission worldwide.

Detection of Plasmodium yoelii stage mRNA in BALB/c mice.

Relatively little information is available concerning the expression of parasite genes during the liver stage of Plasmodium infection, mostly because of low-level infection of host hepatocytes and the lack of purification techniques for the liver stage parasites. We have determined the optimal dosage of Plasmodium yoelii sporozoite inoculum and routes of inoculation, which are intravenous tail vein and the intrahepatic portal circulation. To determine which route was optimal, BALB/c mice were inoculated via 1 of these routes, and parasitemia was detected by reverse transcription polymerase chain reaction (RT-PCR) detecting both murine beta-actin and P. yoelii-specific 28S ribosomal RNA in the liver samples. Murine beta-actin was detected after 15 cycles of PCR, and its expression levels did not differ between treatment groups. However, P. yoelii-specific 28S ribosomal RNA gene product was detected after 15 cycles of PCR in animals inoculated via the tail vein but was not detected until 25 cycles in animals inoculated via the intrahepatic portal circulation. Experiments were then performed to identify the smallest inoculum required to initiate a liver stage infection that would yield sufficient parasite RNA for analysis. Inoculation with different doses of sporozoite inocula was followed by RT-PCR on the livers of the inoculated animals. The P. yoelii-specific 28S ribosomal RNA gene product was first detected in both treatment groups after 15 cycles, suggesting that both doses of sporozoite inocula provided relatively the same level of liver infection rate. We also have analyzed infected mouse liver for parasite-specific mRNA, which was detectable as early as 24 hr postinfection.

Host responses to Plasmodium yoelii hepatic stages: a paradigm in host-parasite interaction.

The liver stage of malaria, caused by the genus Plasmodium, is clinically silent, but immunologically significant. Ample evidence exists for an effective CD8(+) T cell response to this stage as well as the involvement of gammadeltaT cells and NK1.1(int) cells in immunized animal models. In contrast, there is little information concerning responses in a naive host. Here we report that several host gene expressions in the liver, spleen, and kidney of BALB/c mice are altered during the liver stage of Plasmodium yoelii infection. Really interesting new gene 3 (Ring3), semaphorin subclass 4 member G, glutamylcysteine synthetase, and p45 NF erythroid 2 were all up-regulated 24 h after infection with P. yoelii. Semaphorin subclass 4 member G expression was elevated in the kidney, whereas Ring3 was elevated in both spleen and kidney. The expression of TNF-alpha (TNF-alpha and IFN-gamma) were down-regulated in all three tissues tested except in infected spleen where IFN-gamma was elevated. P. yoelii-related host gene changes were compared with those in Toxoplasma gondii-infected livers. Ring3 expression increased 5-fold over control values, whereas expression of the other transcripts remained unchanged. TNF-alpha and IFN-gamma expressions were increased in the Toxoplasma-infected livers. The uniform increase of Ring3 expression in both Plasmodium- and Toxoplasma-infected livers suggests an innate immune response against parasitic infections, whereas the other gene expression changes are consistent with Plasmodium parasite-specific responses. Taken together, these changes suggest the immune responses to P. yoelii infection are both parasite and organ specific.

Mercury exposure and murine response to Plasmodium yoelii infection and immunization.

Malaria has re-emerged in Amazonia over the past two decades. Many factors have been proposed for this, among them changes in population distribution, failures of vector control and pharmacologic management, and local as well as global environmental changes. Among the latter factors, we have studied the potential role of increasing exposures to the immunotoxic metal mercury, which is widely used in Amazonia for artisanal extraction of alluvial gold deposits. We report here that Hg impairs host resistance to malaria infection at exo-erythrocytic stages. Hg exposed mice have higher parasitemia following infection with sporozoites, but not after transfusion of infected red cells. In mice inoculated with irradiated sporozoites, Hg blocks acquisition of immunity. In addition Hg affects immunologic parameters that are known to be involved in host response to malaria infection. These results have potential implications for the incidence and prevalence of malaria among populations exposed to mercury from artisanal goldmining and consumption of contaminated fish regions with high rates of malaria and other infectious diseases.

Retrieving parasite specific liver stage gene products in Plasmodium yoelii infected livers using differential display.

Differential display (DD) has been routinely used to identify genes whose expression pattern is altered by changes in the cellular environment and/or at different stages of development. Most reports utilizing DD contain conventional DD primers that have high guanine and cytosine content and would not be expected to be optimal for Plasmodium which has approximately 30-40% G+C. In an attempt to accommodate the high adenine and thymidine rich genome of Plasmodium yoelii, we utilized PCR primers containing 40, 50 and 60% G+C and modified the existing DD technique. Thus 40% G+C appeared to be the most suitable to amplify Plasmodium genome. Gene specific primers were generated from the sequences of selected DD bands amplified using the 40% G+C primers and were used to verify that the DD clones were of parasite origin by PCR and sequence alignment. Additional data on five of the selected DD clones, designated P2T1L5, P2T1L6, P2T6L11, P2T7L12 and P2T7L13, suggested that all are expressed during the P. yoelii liver stage infection. Interestingly, P2T1L5 is also expressed during the sporozoite stage of the life cycle and both P2T1L6 and P2T6L11 are present as blood stage antigens. The results of this study suggest that DD incorporating primers with low G+C content allows the identification of P. yoelii messages from infected mouse livers.

Improving protective immunity induced by DNA-based immunization: priming with antigen and GM-CSF-encoding plasmid DNA and boosting with antigen-expressing recombinant poxvirus.

Intramuscular immunization with a naked DNA plasmid expressing the Plasmodium yoelii circumsporozoite protein (pPyCSP) protects mice against challenge with P. yoelii sporozoites. This protection can be improved either by coadministration of a plasmid expressing murine GM-CSF (pGMCSF) or by boosting with recombinant poxvirus expressing the PyCSP. We now report that combining these two strategies, by first mixing the priming dose of pPyCSP with pGMCSF and then boosting with recombinant virus, can substantially increase vaccine effectiveness. Not only were immune responses and protection improved but the pPyCSP dose could be lowered from 100 microg to 1 microg with little loss of immunogenicity after boost with recombinant poxvirus. Comparing mice primed by the 1-microg doses of pPyCSP plus 1 microg pGMCSF with mice primed by 1-microg doses of pPyCSP alone, the former were better protected (60% vs 0) and had higher concentrations of Abs (titers of 163, 840 vs 5, 120 by indirect fluorescent Ab test against sporozoites), more ex vivo CTL activity (25% vs 7% specific lysis), and more IFN-gamma-secreting cells by enzyme-linked immunospot assay (1460 vs 280 IFN-gamma spot-forming cells/106 cells). Priming with plasmid vaccine plus pGMCSF and boosting with recombinant poxviruses strongly improves the immunogenicity and protective efficacy of DNA vaccination and allows for significant reduction of dose.

gammadelta T cells are a component of early immunity against preerythrocytic malaria parasites.

We tested the hypothesis that gammadelta T cells are a component of an early immune response directed against preerythrocytic malaria parasites that are required for the induction of an effector alphabeta T-cell immune response generated by irradiated-sporozoite (irr-spz) immunization. gammadelta T-cell-deficient (TCRdelta(-/-)) mice on a C57BL/6 background were challenged with Plasmodium yoelii (17XNL strain) sporozoites, and then liver parasite burden was measured at 42 h postchallenge. Liver parasite burden was measured by quantification of parasite-specific 18S rRNA in total liver RNA by quantitative-competitive reverse transcription-PCR and by an automated 5' exonuclease PCR. Sporozoite-challenged TCRdelta(-/-) mice showed a significant (P < 0.01) increase in liver parasite burden compared to similarly challenged immunocompetent mice. In support of this result, TCRdelta(-/-) mice were also found to be more susceptible than immunocompetent mice to a sporozoite challenge when blood-stage parasitemia was used as a readout. A greater percentage of TCRdelta(-/-) mice than of immunocompetent mice progressed to a blood-stage infection when challenged with five or fewer sporozoites (odds ratio = 2.35, P = 0.06). TCRdelta(-/-) mice receiving a single irr-spz immunization showed percent inhibition of liver parasites comparable to that of immunized immunocompetent mice following a sporozoite challenge. These data support the hypothesis that gammadelta T cells are a component of early immunity directed against malaria preerythrocytic parasites and suggest that gammadelta T cells are not required for the induction of an effector alphabeta T-cell immune response generated by irr-spz immunization.

CD4(+) T-cell- and gamma interferon-dependent protection against murine malaria by immunization with linear synthetic peptides from a Plasmodium yoelii 17-kilodalton hepatocyte erythrocyte protein.

Most work on protective immunity against the pre-erythrocytic stages of malaria has focused on induction of antibodies that prevent sporozoite invasion of hepatocytes, and CD8(+) T-cell responses that eliminate infected hepatocytes. We recently reported that immunization of A/J mice with an 18-amino-acid synthetic linear peptide from Plasmodium yoelii sporozoite surface protein 2 (SSP2) in TiterMax adjuvant induces sterile protection that is dependent on CD4(+) T cells and gamma interferon (IFN-gamma). We now report that immunization of inbred A/J mice and outbred CD1 mice with each of two linear synthetic peptides from the 17-kDa P. yoelii hepatocyte erythrocyte protein (HEP17) in the same adjuvant also induces protection against sporozoite challenge that is dependent on CD4(+) T cells and IFN-gamma. The SSP2 peptide and the two HEP17 peptides are recognized by B cells as well as T cells, and the protection induced by these peptides appears to be directed against the infected hepatocytes. In contrast to the peptide-induced protection, immunization of eight different strains of mice with radiation-attenuated sporozoites induces protection that is absolutely dependent on CD8(+) T cells. Data represented here demonstrate that CD4(+) T-cell-dependent protection can be induced by immunization with linear synthetic peptides. These studies therefore provide the foundation for an approach to pre-erythrocytic-stage malaria vaccine development, based on the induction of protective CD4(+) T-cell responses, which will complement efforts to induce protective antibody and CD8(+) T-cell responses.

Pan DR binding sequence provides T-cell help for induction of protective antibodies against Plasmodium yoelii sporozoites.

Pan-DR epitope (PADRE) peptides have demonstrated the capacity to deliver help for antibody responses in vivo. They were also found, fortuitously, to be able to provide significant helper T-cell activity in vivo. This suggested that linear constructs, containing the PADRE epitope, might be as efficient at generating an immune response as large multivalent antigens. Plasmodium falciparum and P. yoelii PADRE constructs were capable of inducing a high titre IgG antibody response that recognized intact sporozoites. We now report that these antibodies can inhibit sporozoite invasion of hepatocytes in vitro and that mice immunized with the PyCSP-PADRE linear construct were protected when challenged with P. yoelii sporozoites.

Immunogenicity and in vitro protective efficacy of a recombinant multistage Plasmodium falciparum candidate vaccine.

Compared with a single-stage antigen-based vaccine, a multistage and multivalent Plasmodium falciparum vaccine would be more efficacious by inducing "multiple layers" of immunity. We have constructed a synthetic gene that encodes for 12 B cell, 6 T cell proliferative, and 3 cytotoxic T lymphocyte epitopes derived from 9 stage-specific P. falciparum antigens corresponding to the sporozoite, liver, erythrocytic asexual, and sexual stages. The gene was expressed in the baculovirus system, and a 41-kDa antigen, termed CDC/NIIMALVAC-1, was purified. Immunization in rabbits with the purified protein in the presence of different adjuvants generated antibody responses that recognized vaccine antigen, linear peptides contained in the vaccine, and all stages of P. falciparum. In vitro assays of protection revealed that the vaccine-elicited antibodies strongly inhibited sporozoite invasion of hepatoma cells and growth of blood-stage parasites in the presence of monocytes. These observations demonstrate that a multicomponent, multistage malaria vaccine can induce immune responses that inhibit parasite development at multiple stages. The rationale and approach used in the development of a multicomponent P. falciparum vaccine will be useful in the development of a multispecies human malaria vaccine and vaccines against other infectious diseases.

Plasmodium falciparum and Plasmodium yoelii: effect of the iron chelation prodrug dexrazoxane on in vitro cultures.

To determine if an iron-chelating prodrug that must undergo intracellular hydrolysis to bind iron has antimalarial activity, we examined the action of dexrazoxane on Plasmodium falciparum cultured in human erythrocytes and P. yoelii cultured in mouse hepatocytes. Dexrazoxane was recently approved to protect humans from doxorubucin-induced cardiotoxicity. Using the fluorescent marker calcein, we confirmed that the iron-chelating properties of dexrazoxane are directly related to its ability to undergo hydrolysis. As a single agent, dexrazoxane inhibited synchronized cultures of P. falciparum in human erythrocytes only at suprapharmacologic concentrations (> 200 microM). In combination with desferrioxamine B, dexrazoxane in pharmacologic concentrations (100-200 microM) moderately potentiated inhibition by approximately 20%. In contrast, pharmacologic concentrations of dexrazoxane (50-200 microM) as a single agent inhibited the progression of P. yoelli from sporozoites to schizonts in cultured mouse hepatocytes by 45 to 69% (P < 0.001). These results are consistent with the presence of a dexrazoxane-hydrolyzing enzyme in hepatocytes but not in erythrocytes or malaria parasites. Furthermore, these findings suggest that dexrazoxane must be hydrolyzed to an iron-chelating intermediate before it can inhibit the malaria parasite, and they raise the possibility that the iron chelator prodrug concept might be exploited to synthesize new antimalarial agents.

In vitro expression and in vivo immunogenicity of Plasmodium falciparum pre-erythrocytic stage DNA vaccines.

DNA vaccine plasmids were constructed that encoded four pre-erythrocytic antigens from the human malaria parasite Plasmodium falciparum: circumsporozoite protein (PfCSP); sporozoite surface protein 2 (PfSSP2); carboxyl terminus of liver stage antigen 1 (PfLSA-1 c-term); and, exported protein 1 (PfExp-1). Antigen expression was evaluated in vitro by immunoblot analysis of tissue culture cells following transient transfection with each plasmid. Clearly detectable levels of expression depended upon, or were markedly enhanced by, fusion of the antigen encoding sequences in-frame with the initiation complex and peptide leader sequence of human tissue plasminogen activator protein. Mice injected with these plasmids produced antigen specific antibody and cytotoxic T lymphocyte responses. However, the magnitudes of the responses were not always predicted by the in vitro expression assay. The results of this study provided the basis for further testing of these plasmids in primates and the formulation of multi-component pre-erythrocytic DNA vaccines for efficacy testing in human volunteers.

Simultaneous induction of multiple antigen-specific cytotoxic T lymphocytes in nonhuman primates by immunization with a mixture of four Plasmodium falciparum DNA plasmids.

CD8(+) T cells have been implicated as critical effector cells in protective immunity against malaria parasites developing within hepatocytes. A vaccine that protects against malaria by inducing CD8(+) T cells will probably have to include multiple epitopes on the same protein or different proteins, because of parasite polymorphism and genetic restriction of T-cell responses. To determine if CD8(+) T-cell responses against multiple P. falciparum proteins can be induced in primates by immunization with plasmid DNA, rhesus monkeys were immunized intramuscularly with a mixture of DNA plasmids encoding four P. falciparum proteins or with individual plasmids. All six monkeys immunized with PfCSP DNA, seven of nine immunized with PfSSP2 DNA, and five of six immunized with PfExp-1 or PfLSA-1 DNA had detectable antigen-specific cytotoxic T lymphocytes (CTL) after in vitro restimulation of peripheral blood mononuclear cells. CTL activity was genetically restricted and dependent on CD8(+) T cells. By providing the first evidence for primates that immunization with a mixture of DNA plasmids induces CD8(+) T-cell responses against all the components of the mixture, these studies provide the foundation for multigene immunization of humans.

Development of two monoclonal antibodies against Plasmodium falciparum sporozoite surface protein 2 and mapping of B-cell epitopes.

The Plasmodium yoelii sporozoite surface protein 2 (PySSP2) is the target of protective cellular immunity. Cytotoxic T cells specific for the Plasmodium falciparum analog PfSSP2, also known as thrombospondin-related anonymous protein (TRAP), are induced in human volunteers immunized with irradiated sporozoites. PfSSP2 is an important candidate antigen for a multicomponent malaria vaccine. We generated and characterized three monoclonal antibodies (MAbs) specific for PfSSP2/TRAP. The MAbs PfSSP2.1 (immunoglobulin G1 [IgG1]), PfSSP2.2 (IgG2a), and PfSSP2.3 (IgM) were species specific and identified three distinct B-cell epitopes containing sequences DRYI, CHPSDGKC, and TRPHGR, respectively. PfSSP2.1 partially inhibited P. falciparum liver-stage parasite development in human hepatocyte cultures (42 and 86% in two experiments at 100 microg/ml). Mice immunized with vaccinia virus expressing full-length PfSSP2 protein produced antibodies to (DRYIPYSP)3, and humans living in malaria-endemic areas (Indonesia and Kenya), who have lifelong exposure and partial clinical immunity to malaria, had antibodies to both (DRYIPYSP)3 and (CHPSDGKCN)2. Mice immunized with multiple antigen peptides MAP4 (DRYIPYSP)3P2P30 and MAP4 (CHPSDGKCN)3P2P30 in TiterMax developed antibodies to sporozoites that partially inhibited sporozoite invasion of human hepatoma cells (39 to 71% at a serum dilution of 1:50 in three different experiments). The modest inhibitory activities of the MAbs and the polyclonal antibodies to PfSSP2/TRAP epitopes do not suggest that a single-component vaccine designed to induce antibodies against PfSSP2/TRAP will be protective. Nonetheless, the MAbs directed against PfSSP2, and the peptides recognized by these MAbs, will be essential reagents in the development of PfSSP2/TRAP as a component of a multivalent P. falciparum human malaria vaccine.

Murine typhus: updated roles of multiple urban components and a second typhuslike rickettsia.

Studies using serologic and polymerase chain reaction-(PCR) facilitated analysis of field samples from southern Texas indicate the presence of Rickettsia typhi and ELB agent infected cat fleas, Ctenocephalides felis (Bouché), and the first observation of ELB infected vertebrates (opossums). The ELB agent is a recently described typhus-like rickettsia that is not distinguished from R. typhi or R. prowazekii by currently available serologic reagents. Restriction digests of PCR products from 399 fleas revealed an ELB agent infection rate of 3.8% and a R. typhi infection rate of 0.8%. Three of nine tested opossums (Didelphis virginiana) were shown to harbor ELB agent infections. No R. typhi infected rats, Rattus norvegicus, or rat-fleas, Xenopsylla cheopis Rothschild, were detected among surveyed samples. The persistence of this murine typhus disease focus appears to be better accounted for by the presence of infected cat fleas, opossums, and other non-rat hosts found in close association with human populations. Involvement of the ELB agent in the biology of murine typhus is suggested by its prevalence among suspected vectors and reservoir hosts.