Pre-erythrocytic Forms Research Articles

Towards functional antibody-based vaccines to prevent pre-erythrocytic malaria infection

ABSTRACTIntroduction: An effective malaria vaccine would be considered a milestone of modern medicine, yet has so far eluded research and development efforts. This can be attributed to the extreme complexity of the malaria parasites, presenting with a multi-stage life cycle, high genome complexity and the parasite’s sophisticated immune evasion measures, particularly antigenic variation during pathogenic blood stage infection. However, the pre-erythrocytic (PE) early infection forms of the parasite exhibit relatively invariant proteomes, and are attractive vaccine targets as they offer multiple points of immune system attack.Areas covered: We cover the current state of and roadblocks to the development of an effective, antibody-based PE vaccine, including current vaccine candidates, limited biological knowledge, genetic heterogeneity, parasite complexity, and suboptimal preclinical models as well as the power of early stage clinical models.Expert commentary: PE vaccines will need to elicit broad and durable immunity to prevent infection. This could be achievable if recent innovations in studying the parasites’ infection biology, rational vaccine selection and design as well as adjuvant formulation are combined in a synergistic and multipronged approach. Improved preclinical assays as well as the iterative testing of vaccine candidates in controlled human malaria infection trials will further accelerate this effort.

Protective immune mechanisms against pre-erythrocytic forms of Plasmodium berghei depend on the target antigen

Pre-erythrocytic malaria vaccines are believed to either stop the injected sporozoites from reaching the liver or to direct cellular immune responses towards eliminating infected hepatocytes. The present study reveals for the first time the anatomical sites at which these immune mechanisms act against the malaria parasites. To determine the mechanisms leading to protection mediated by two previously characterized vaccines against either the circumsporozoite protein (CSP) or the cell traversal protein for ookinetes and sporozoites (CelTOS), mice were immunized and subsequently challenged by subcutaneous injection of salivary gland sporozoites of luciferase-transgenic Plasmodium berghei parasites. The In Vivo Imaging System (IVIS) was used to identify the anatomical site where the vaccine-induced immune response eliminates sporozoites after injection. The data demonstrate that CSP-based immunity acts at the site of infection (skin) whereas CelTOS-based immunity is only partially efficient in the skin and allows reduced levels of liver infection that can be subsequently cleared. The results of this study challenge assumptions regarding CSP-mediated immune mechanisms and call into question the validity of some commonly used assays to evaluate anti-CSP immune responses. The knowledge of the mechanism and events leading to infection or immune defense will guide supportive treatment with drugs or combination therapies and thus accelerate the development of effective antimalarial strategies.

Looking under the skin: the first steps in malarial infection and immunity

Malaria, which is caused by Plasmodium spp., starts with an asymptomatic phase, during which sporozoites, the parasite form that is injected into the skin by a mosquito, develop into merozoites, the form that infects erythrocytes. This pre-erythrocytic phase is still the most enigmatic in the parasite life cycle, but has long been recognized as an attractive vaccination target. In this Review, we present what has been learned in recent years about the natural history of the pre-erythrocytic stages, mainly using intravital imaging in rodents. We also consider how this new knowledge is in turn changing our understanding of the immune response mounted by the host against the pre-erythrocytic forms.

Expression, Purification and Characterization of a Recombinant Plasmodium Vivax Thrombospondin Related Adhesive Protein (PvTRAP)

Thrombospondin Related Adhesive Protein (TRAP) is a transmembrane parasite molecule responsible in sporozoite-host interactions. This molecule is one of the most promising vaccine candidates against the pre-erythrocytic forms of malaria. In the present study, a gene encoding the Plasmodium vivax TRAP (PvTRAP) was expressed in Escherichia coli (M15 strain) using the expression plasmid pQE30. The expressed recombinant protein PvTRAP of about 70kDa was achieved, purified and refolded according to the standardized refolding procedure. This refolded protein (PvTRAP) showed a single band monomeric form with SDS-PAGE and blot analysis. In reduced and alkylated form, PvTRAP showed less binding to hepatoma (HepG2) liver cells, when compared to the normal purified and refolded form. Purified and refolded recombinant PvTRAP bound Duffy-positive human erythrocytes, while no binding was observed with Duffy-negative erythrocytes. Our report on PvTRAP is currently documented for the first time and it has been able to provide an experimental evidence of the biochemical and binding properties of PvTRAP in the invasion of hepatocytes and interaction with Duffy-positive and Duffy-negative human erythrocytes. In conclusion, our findings have been able to demonstrate the potential of PvTRAP as a promising target for vivax malaria vaccine candidate.

AOTUS LEMURINUS GRISEIMEMBRA MONKEYS: A SUITABLE MODEL FOR PLASMODIUM VIVAX SPOROZOITE INFECTION

This study describes a successful Plasmodium vivax sporozoite infection in Aotus lemurinus griseimembra. Twenty-eight naive or previously infected monkeys, either splenectomized or spleen intact, were inoculated intravenously or subcutaneously with Plasmodium vivax sporozoites of the Salvador I strain or with two wild isolates (VCC-4 and VCC-5; Vivax-Cali-Colombia). The monkeys were successfully infected regardless of the parasite strain, spleen presence, or inoculation route and showed prepatent periods that ranged from 16 to 89 days. Only one monkey inoculated intravenously failed to develop parasitemia. Since immune protection against malaria pre-erythrocytic forms is mediated by both helper and cytolytic T cells that may home in the spleen and P. vivax cultures are not yet available; the use of spleen-intact A. lemurinus griseimembra, susceptible to both adapted and non-adapted strains of P. vivax sporozoites, is a valuable model for evaluation of pre-erythrocytic vaccine candidates.

Fluorescent Plasmodium berghei sporozoites and pre-erythrocytic stages: a new tool to study mosquito and mammalian host interactions with malaria parasites.

To track malaria parasites for biological studies within the mosquito and mammalian hosts, we constructed a stably transformed clonal line of Plasmodium berghei, PbFluspo, in which sporogonic and pre-erythrocytic liver-stage parasites are autonomously fluorescent. A cassette containing the structural gene for the FACS-adapted green fluorescent protein mutant 2 (GFPmut2), expressed from the 5' and 3' flanking sequences of the circumsporozoite (CS) protein gene, was integrated and expressed at the endogenous CS locus. Recombinant parasites, which bear a wild-type copy of CS, generated highly fluorescent oocysts and sporozoites that invaded mosquito salivary glands and were transmitted normally to rodent hosts. The parasites infected cultured hepatocytes in vitro, where they developed into fluorescent pre-erythrocytic forms. Mammalian cells infected by these parasites can be separated from non-infected cells by fluorescence activated cell sorter (FACS) analysis. These fluorescent insect and mammalian stages of P. berghei should be useful for phenotypic studies in their respective hosts, as well as for identification of new genes expressed in these parasite stages.

Identification of hypnozoites and tissue schizonts of Plasmodium vivax and P. cynomolgi by the immunoperoxidase method.

An immunoperoxidase technique was used to detect hypnozoites and liver schizonts of the primate malaria species Plasmodium vivax and P. cynomolgi bastianellii in Carnoy's-fixed sections. Anti-P. cynomolgi serum and a peroxidase-conjugated anti-monkey IgG serum rendered 7-day pre-erythrocytic forms clearly visible. The technique retains the specificity of the immunofluorescence method while having the advantage of a permanent preparation. Detection of hyponozoites by this alternative method provides further evidence for their plasmodial nature.

Antimalarial activity of floxacrine (HOE 991) II: Studies on causal prophylactic and blood schizontocidal action of floxacrine and related dihydroacridinediones against Plasmodium yoelii and P. berghei.

The causal prophylactic and blood schizontocidal action of six dihydroacridinediones were assessed in mice using the Plasmodium yoelii 17X strain in sporozoite-induced infections and the P. berghei K 173 strain in blood-induced infections respectively. A remarkable relationship was shown between the action on pre-erythrocytic forms and on blood forms of malaria parasites. A trifluoromethyl group in the para (4—) position of the 3-phenyl ring of the 7-chloro-10-hydroxy-3,4-dihydroacridine-1,9-(2H,10H)-dione (compound Hoe 991: floxacrine) proved the most favourable substituent of a group of electronegative substituents concerning antimalarial activity. The CPD50/CD50 of Hoe 991 against pre-erythrocytic/blood forms of P.yoelii/P. berghei was 0·37 mg kg−1 (X 1)/1·7 mg kg−1 (X5) after the intraperitoneal (ip)/subcutaneous (sc) route. As demonstrated by compound Hoe 990 double substitution in the 3-phenyl ring with 2-chloro-, 4-trifluoromethyl- caused a marked residual action. The causal prophylactic effect (CP...

Observations on Early and Late Post-Sporozoite Tissue Stages in Primate Malaria

Confirmation of the existence of a persistent, uninucleate, dormant pre-erythrocytic stage, the hypnozoite, of the relapsing simian malaria parasite, Plasmodium cynomolgi bastianellii, has been obtained by means of experiments involving the intravenous injection into susceptible monkeys of 48 to 85 x 10(6) sporozoites derived from mosquitoes of a different species and source than employed previously. The development of these hypnozoites was traced from 3 days until 105 days after sporozoite inoculation, employing a sensitive immunofluorescence technique followed by restaining with Giemsa. From an average mean diameter of 4 micrometers at 3 and 5 days, uninucleate hypnozoites grow to 5 micrometers at 7 days, then persist with little change until at least 105 days after infection. Strong evidence for the viability of these persistent forms was obtained by treatment of a host monkey with primaquine, which eliminated all trace of hypnozoites present 2 weeks before. Examination of hepatic tissue from a monkey injected with sporozoites 36 and 40 hours earlier revealed rare uninucleate pre-erythrocytic forms of 2.5-micrometers diameter. These early forms were present in hepatocytes in a density only approximately 1/30th of that expected on the basis of numbers of pre-erythrocytic stages found in the same animal's liver 7 days after infection. Nevertheless, subinoculation experiments appeared to rule out the circulation as a vehicle for dissemination of any putative early intermediate hepatotropic forms from another site.

Observations on Early and Late Post-Sporozoite Tissue Stages in Primate Malaria

Previous work in this laboratory has demonstrated the ability of the immunofluorescence technique to detect pre-erythrocytic stages of the primate malaria parasite, Plasmodium cynomolgi bastianellii, in hepatic tissue obtained as early as 48 hours after sporozoite inoculation. In an attempt to visualize still earlier post-sporozoite stages, hepatic tissue obtained from a rhesus monkey infected with 12,000,000 sporozoites was examined at 2, 12, 24, and 48 hours after inoculation, employing antisera reactive with both invertebrate and vertebrate stages of the parasite. Tissue was also obtained at 7, 50, 102, and 105 days after sporozoite inoculation, and was examined for adequacy of the hepatic infection and for the presence of late exoerythrocytic schizonts. Although a new, previously unrecognized, uninucleate latent stage of 5 micrometer diameter (the "hypnozoite") was detected among large maturing schizonts in the 7-day and later biopsies, no intrahepatic parasites were found in tissue taken at 24 hours or earlier, despite the presence of up to 61 7-day schizonts and eight hypnozoites per 5 X 8 mm section. Pre-erythrocytic forms again were detected at 48 hours, although in far smaller numbers than expected on the basis of the density of parasites at 7 days after infection. The significance of these observations is discussed in the context of previous negative findings.

ChemInform Abstract: SYNTHESIS AND EVALUATION OF 6‐ARYLACETAMIDO‐2,4‐DIAMINOQUINAZOLINES AND RELATED COMPOUNDS AS FOLIC ACID ANTAGONISTS

A series of 2,4-diaminoquinazolines bearing an aryl function attached to the 6 position through an acetamido or related linkage was synthesized. Each compound was evaluated as an inhibitor of rat liver dihydrofolate reductase as well as for suppressive antimalarial effects against Plasmodium berghei in mice. Significant in vivo activity was found to reside primarily with 5-chloro-6-arylacetamido derivatives. Most of these compounds were also tested for prophylactic activity against sporozoite-induced Plasmodium gallinaceum in chicks. Thirteen compounds, each of which possesses a 5-Cl or 5-CH3 group, displayed curative activity in this test system. Since several of these showed markedly greater potency against the avian infection, selective inhibitory action upon preerythrocytic forms of the malaria parasite is thus implied.

Synthesis and evaluation of 6-arylactamido-2,4-diaminoquinazolines and related compounds as folic acid antagonists.

A series of 2,4-diaminoquinazolines bearing an aryl function attached to the 6 position through an acetamido or related linkage was synthesized. Each compound was evaluated as an inhibitor of rat liver dihydrofolate reductase as well as for suppressive antimalarial effects against Plasmodium berghei in mice. Significant in vivo activity was found to reside primarily with 5-chloro-6-arylacetamido derivatives. Most of these compounds were also tested for prophylactic activity against sporozoite-induced Plasmodium gallinaceum in chicks. Thirteen compounds, each of which possesses a 5-Cl or 5-CH3 group, displayed curative activity in this test system. Since several of these showed markedly greater potency against the avian infection, selective inhibitory action upon preerythrocytic forms of the malaria parasite is thus implied.

Plasmodium gallinaceum: Selective immunosuppression by cyclophosphamide in preerythrocytic malaria

When chicks are injected with the immunosuppressant cyclophosphamide (Cy) on days 1 and 2 after hatching and then injected with sporozoites from infected mosquitoes on day 4, the normal susceptibility of only one host cell type to the sequential invasive stages of the preerythrocytic forms of avian malaria ( Plasmodium gallinaceum) is increased. Thus, only endothelial cells lining capillaries showed an increased susceptibility to invasion or development of second generation preerythrocytic parasites. There is some indication that such an increased susceptibility also occurs after X-irradiation of chicks but not after treatment with endotoxin. Neither the infectivity or development of sporozoites within macrophages nor the invasion of erythrocytes by parasites released from the tissues was apparently affected by Cy-treatment of chicks. Neither suppression of natural anti-sporozoite humoral antibody nor the possibility of suppression of acquired immunity to preerythrocytic stages of the parasite was shown to be responsible for the observed increased parasitemia of Cy-treated chicks. The apparent specificity of the immunosuppression of a natural immunity was ascertained by inoculation of a selected preerythrocytic stage into Cy-treated and control birds, and, in addition, by observing the increased tissue parasite levels of spleens and brains of similarly treated birds after sporozoite inoculation when compared to controls.

The localization of infective pre-erythrocytic forms of Plasmodium cynomolgi

1. 1) This report deals with preliminary efforts to localize the site(s) of development of the pre-erythrocytic forms of P. cynomolgi by use of tissue implantation techniques. Twelve infant rhesus monkeys killed 4, 5, 6, and 7 days after inoculation with massive numbers of sporozoites served as donors of liver, spleen, lymph nodes, lung, and bone marrow. Fragments of these organs were implanted into the abdominal cavities of 115 subjects of the same species. 2. 2) Positive results (development of parasitaemia) were obtained with 26 of 61 liver implants carried out 4–6 days after sporozoite inoculation. The incidence of takes was largest at 6 days, at which time the peripheral blood was still negative. None of 44 implants of spleen, lymph node, lung, or bone marrow induced infections. 3. 3) These observations add strength to earlier morphological studies which have focussed attention on the liver as the site of development of pre-erythrocytic forms of primate plasmodia. Because of technical problems not yet resolved, they should not be interpreted as excluding the possibility that such development occurs in other organs.

Studies on Malaria in Chimpanzees

SummaryThe 2-, 2.5-, 3-, 3.5-, and 4-day-old pre-erythrocytic forms of Laverania falcipara are described from the liver of the chimpanzee. These ranged with age from 4 µ to 24 µ in diameter, and from 2 to 250 in number of nuclei. All lay in parenchyma cells of the liver.

Studies on Malaria in Chimpanzees

SummaryThe distribution of the pre-erythrocytic forms of Laverania falcipara in the volume of the liver (as a whole) of the chimpanzee was found to be random.The area distribution of pre-erythrocytic forms within the lobules was found to be random.No pre-erythrocytic forms were found in sections of the lung, spleen or thyroid gland.

Chemotherapeutic and other studies on the pre-erythrocytic forms of simian malaria ( Plasmodium cynomolgi)

1. (1) A study was made of the chemotherapeutic reactions of the pre-erythrocytic forms of Plasmodium cynomolgi. 2. (2) Quinine and mepacrine did not destroy these forms; neither did chloroquin, although it possibly diminished the number of pre-erythrocytic forms developing, and it caused vacuolation in them. 3. (3) Proguanil given daily during the prepatent period suppressed the pre-erythrocytic forms but did not prevent the development of a latent infection; if given towards the end of the prepatent period it caused vacuolation of the parasites. Sulphadiazine, given throughout the prepatent period, incompletely suppressed the parasites. 4. (4) Pamaquin given daily during the prepatent period completely prevented the infection. 5. (5) Sporozoites were injected intraperitoneally into two immune monkeys and the pre-erythrocytic forms developed apparently normally; but the resultant parasitaemia of the blood was very small and brief. This confirms the view that in malaria the immune mechanisms are directed against the forms in the blood and not against those in the tissues. 6. (6) Prolonged search for pre-erythrocytic parasites less than 4 days old was unsuccessful. 7. (7) Sporozoites of Plasmodium vivax were injected into the liver of a rhesus monkeys but not infection could be detected.

A strain of monkey malaria ( Plasmodium cynomolgi) made resistant to proguanil (paludrine)

1. (1) A high degree of resistance to proguanil (paludrine, chlorguanide) has been produced in P. cynomolgi by repeated subeffective treatment. Significant resistance was observed after 6 months and resistance to maximum tolerated doses after 16 months. The resistance was a thousand times as great as that of the original normal strain. 2. (2) Resistance was not diminished by passage through mosquitoes, or by preservation of the blood by freezing. 3. (3) Resistance was shown by the pre-erythrocytic forms and by the gametocytes and early forms in the mosquito as well as by the endo-erythrocytic forms. 4. (4) The proguanil-resistant strain was normally sensitive to sulphadiazine

The pre-erythrocytic development of Plasmodium cynomolgi and Plasmodium viv ax

A description is given of experiments resulting in the discovery of the pre-erythrocytic stages in the life cycles of P. cynomolgi and P. vivax In the case of P. cynomolgi, daily development of the forms commencing from the 5th day of the incubation period to the establishment of the blood infection on the 9th and 10th days is described. In the case of P. vivax, the forms described are those present on the 6th and 7th days of the incubation period. Certain questions connected with immunity, the mechanism of relapses, the earliest forms of the pre-erythrocytic cycle, the non-infectivity of the preerythrocytic forms on sub-inoculation and the possible modifications in the taxonomic status of the plasmodia are discussed.