Primaquine-induced changes in morphology of exoerythrocytic stages of malaria.

Exposure to primaquine for 48 hours caused lesions in the exoerythrocytic stages of Plasmodium fallax grown in cultivated cells derived from embryonic turkey brain. The lesions appeared in the form of cytoplasmic vacuoles when viewed under the light microscope. The electron microscope revealed these vacuoles as swollen mitochondria readily identifiable by their typical protozoan cristae. Mitochondria of the host cell were unaffected.

In vitro cultivation of the exoerythrocytic stage of Plasmodium berghei from sporozoites.

When inoculated with sporozoites of Plasmodium berghei, the human embryonic lung cell line WI38 supports the complete asexual developmental cycle of the exoerythrocytic stage. Cultured parasites were sensitive to primaquine, were shown to resemble parasites in living hosts by immunofluorescent antibody tests, and on subinoculation into mice induced a red blood cell infection, the gametocytes of which produced sporozoites in anopheline mosquitoes.

Protection against malaria by vaccination with sporozoite surface protein 2 plus CS protein.

The circumsporozoite (CS) protein has been the target for development of malaria sporozoite vaccines for a decade. However, immunization with subunit vaccines based on the CS protein has never given the complete protection found after immunization with irradiated sporozoites. BALB/c mice immunized with irradiated Plasmodium yoelii sporozoites produced antibodies and cytotoxic T cells against a 140-kilodalton protein, sporozoite surface protein 2 (SSP2). Mice immunized with P815 cells that had been transfected with either SSP2 or CS genes were partially protected, and those immunized with a mixture of SSP2 and CS transfectants were completely protected against malaria. These studies emphasize the importance of vaccine delivery systems in achieving protection and define a multi-antigen sporozoite vaccine.

Effect of antibodies to recombinant and synthetic peptides on P. falciparum sporozoites in vitro.

Antibodies were raised in mice immunized with several recombinant and synthetic peptides of the circumsporozoite protein of Plasmodium falciparum. The antibodies were evaluated for protective activity in a human hepatocyte culture system. They exerted their protective effect against the parasite at three points: sporozoite attachment to the hepatocyte surface, entry, and subsequent intracellular development. Inhibition of attachment and entry were found to be related to the antibody titer against the authentic circumsporozoite protein on the sporozoite surface, especially when peptides were administered with alum or complete Freund's adjuvant. Even when invasion was not totally inhibited, the presence of abnormal trophozoites and a frequent inhibition of schizont development in long-term cultures suggested continued activity of antibodies at the intracellular level after sporozoite penetration had been completed.

Expression of Plasmodium falciparum circumsporozoite proteins in Escherichia coli for potential use in a human malaria vaccine.

The circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum may be the most promising target for the development of a malaria vaccine. In this study, proteins composed of 16, 32, or 48 tandem copies of a tetrapeptide repeating sequence found in the CS protein were efficiently expressed in the bacterium Escherichia coli. When injected into mice, these recombinant products resulted in the production of high titers of antibodies that reacted with the authentic CS protein on live sporozoites and blocked sporozoite invasion of human hepatoma cells in vitro. These CS protein derivatives are therefore candidates for a human malaria vaccine.

Immunogenicity of synthetic peptides from circumsporozoite protein of Plasmodium falciparum.

In a study of recombinant proteins that might be useful in developing a vaccine against malaria, synthetic peptides from the circumsporozoite (CS) protein of Plasmodium falciparum were found to be immunogenic for mice and rabbits. Antibody to peptides from the repeating region of the CS protein recognized native CS protein and blocked sporozoite invasion of human hepatoma cells in vitro. Antibodies to peptides from regions I and II had no biologic activity, although antibody to region I recognized processed CS protein by Western blot analysis. These data support the feasibility of developing a vaccine against the sporozoite stage of the malaria parasite by using synthetic peptides of the repeating region of the CS protein conjugated to a carrier protein.

Sequence of the immunodominant epitope for the surface protein on sporozoites of Plasmodium vivax.

Plasmodium vivax is one of the four malaria parasites that cause disease in humans. The structure of the immunodominant repeating peptide of the circumsporozoite (CS) protein of P. vivax was determined. A fragment of P. vivax DNA that encodes this tandemly repeating epitope was isolated by use of an oligonucleotide probe whose sequence is thought to be conserved in CS protein genes. DNA sequence analysis of the P. vivax clone indicates that the CS repeat is nine amino acids in length (Gly-Asp-Arg-Ala-Asp-Gly-Gln-Pro-Ala). The structure of the repeating region was confirmed with synthetic peptides and monoclonal antibodies directed against P. vivax sporozoites. This information should allow synthesis of a vaccine for P. vivax that is similar to the one being tested for P. falciparum.

Inability of malaria vaccine to induce antibodies to a protective epitope within its sequence.

Saimiri monkeys immunized with a recombinant protein containing 20 copies of the nine amino acid repeat of the Plasmodium vivax circumsporozoite (CS) protein developed high concentrations of antibodies to the repeat sequence and to sporozoites, but were not protected against challenge. After intravenous injection of an immunoglobulin G3 monoclonal antibody (NVS3) against irradiated P. vivax sporozoites, four of six monkeys were protected against sporozoite-induced malaria, and the remaining two animals took significantly longer to become parasitemic. Epitope mapping demonstrated that NVS3 recognizes only four (AGDR) of the nine amino acids within the repeat region of the P. vivax CS protein. The monkeys immunized with (DRAADGQPAG)20 did not produce antibodies to the protective epitope AGDR. Thus, determination of the fine specificity of protective immune responses may be critical to the construction of successful subunit vaccines.

Complete development of hepatic stages of Plasmodium falciparum in vitro.

An in vitro model was developed to study the hepatic phase of Plasmodium falciparum, the only malaria parasite lethal to man. Primary cultures of human hepatocytes were inoculated with sporozoites of Brazilian and African strains of P. falciparum. On days 1 through 7 after inoculation examination of fluorescence-labeled and Giemsa-stained preparations demonstrated the presence of many intracellular parasites. In three separate sets of experiments all cultures were found to be infected with as many as 650 liver schizonts measuring up to 40 micrometers. After the addition of red blood cells, intraerythrocytic forms of P. falciparum were detected on days 12 and 13 by an immunofluorescence assay, indicating that the hepatic cycle had been completed in vitro.

Field trial in Chiapas, Mexico, of a rapid detection method for malaria in anopheline vectors with low infection rates.

A method consisting of filtration of up to 100 macerated mosquitoes in a batch, followed by fixation with glutaraldehyde and concentration of filtrate by centrifugation has been developed to rapidly assess malaria infection in anopheline mosquitoes. Determination of the presence of sporozoites is made by observation of a sample of the final filtrate with a phase microscope. The method is simple and field adaptable, essential factors for the application of any technique to large scale field operations. Application of the technique in El Gancho, Chiapas, Mexico, in February 1984 yielded an infection rate for Anopheles albimanus of 0.9% for intradomicile-collected human bait mosquitoes and 0.1% for peridomicile-collected human bait mosquitoes.

Freeze-fracture studies on the sporoblast and sporozoite development in the early oocyst.

Freeze-fracturing has been used to study the formation of the triple layer pellicular complex of budding sporozoites of Plasmodium falciparum in the early oocyst. Sporozoites are formed from sporoblasts within the oocyst. The outer membrane of the sporozoites is derived from the single plasma membrane of the sporoblast while the inner 2 membranes are formed anew at the base of the differentiating sporozoites. A dense collar of intramembranous particles located on the P face of the outer membrane encircles the base of each budding sporozoite. The fact that this collar of intramembranous particles is located in the same region where the inner membranes of the sporozoites first make their appearance strongly suggests that the 2 are related, and that the collar may be related to either membrane synthesis or to membrane organization and assembly.

Serological reactivity of in vitro cultured exoerythrocytic stages of Plasmodium berghei in indirect immunofluorescent or immunoperoxidase antibody tests.

Exoerythrocytic (EE) stages of Plasmodium berghei were cultivated in vitro in WI38 cells inoculated with sporozoites, and examined for serological reactivity by indirect immunofluorescent or immunoperoxidase tests. At 24 hours post-inoculation, sporozoite and red blood cell (RBC) stage antigens were equally distributed, but by 48 hours, RBC stage antigens predominated. Merozoites, produced by 72 hours, reacted strongly with anti-RBC stage sera, but were weakly reactive with anti-sporozoite sera. Hybridoma-produced monoclonal antibodies to the surface protective antigen of P. berghei sporozoites also reacted with 24- and 48-hour EE parasites, and with RR merozoites, suggesting that the sporozoite-protective antigen may also be synthesized by the EE stage. Extra-EE parasite antigens associated with the host cell nucleus were detected as early as 24 hours using anti-sporozoite and anti-RBC stage sera, but did not contain the sporozoite-protective antigen.

Iron chelators: in vitro inhibitory effect on the liver stage of rodent and human malaria.

The activity of desferrioxamine (Desferal) and desferrithiocin (a newly developed oral iron chelator) was evaluated against the liver stage of Plasmodium yoelii and P. falciparum in the rodent and the human hepatocyte in vitro culture system. The two iron chelators were found to inhibit the liver schizogony of both the rodent and the human Plasmodium species at concentrations achievable in vivo. P. falciparum proved to be more sensitive (ic 95% below 20 micromol/l than P. yoelii (ic 95% 50-100 micromol/l). As assessed by electron microscopy, drug administration was associated with focal clarification of the cytoplasm thought to be reversible. As desferrioxamine and desferrithiocin are known to be equally active on the blood stage of rodent and human plasmodia, iron chelators are deserving of further investigation as potential alternative candidates to existing drugs for radical cure of malaria.

Plasmodium falciparum-infected Anopheles stephensi inconsistently transmit malaria to humans.

Malaria was transmitted to only 5 of 10 volunteers bitten by 1-2 Anopheles stephensi carrying sporozoites of the 3D7 clone of the NF54 strain of Plasmodium falciparum in their salivary glands. Parasites were detectable by culture in blood taken 7-10 days following exposure and by thick blood film 14-16.5 days after exposure. Infectivity did not correlate with the numbers of sporozoites in the salivary glands.

Specific identification of Plasmodium sporozoites using an indirect fluorescent antibody method.

The specific identification of plasmodial sporozoites is not possible on morphological grounds. This study presents a serological method for the identification of sporozoite species, indicating the suitability of this approach for detection and determination of sporozoites in wild vectors collected from malaria endemic areas. Specific antisera and monoclonal antibodies prepared against each of two species of rodent malaria (Plasmodium berghei or P. yoelii) were evaluated for their ability to discriminate among sporozoites of different malaria species (P. vivax, P. gallinaceum, P. berghei, P. yoelii) from varied host types. Antisera produced by intravenous immunization of rabbits or mice and hybridoma-produced monoclonal antibodies reacted only with homologous sporozoites in an immunofluorescent antibody test. Antisera produced by intravenous challenge showed no significant difference in reactivity with fresh sporozoites as compared with sporozoites frozen at -80 degrees C for up to six months, whereas antisera produced by infective mosquito bites showed reduced sensitivity to frozen-stored homologous sporozoites and some cross-reactions with heterologous sporozoites. Antisera prepared against the erythrocytic stages of P. berghei or P. yoelii also cross-reacted with sporozoites of all four plasmodia tested, indicating that it is advantageous to use anti-sporozoite antibodies for the identification of malaria sporozoite species by means of serological tests.

Retention of Plasmodium berghei sporozoites within perfused mouse livers.

A mouse liver perfusion model was adapted to evaluate the efficiency of the liver in retaining Plasmodium berghei sporozoites. Specific numbers of sporozoites were perfused into each liver via a portal vein cannula. The numbers of sporozoites in the perfusate effluent were counted and the percent sporozoite retention calculated. Over 95% of sporozoites suspended in medium with plasma were retained in a normal liver following a single passage. Sporozoites were seen in sinusoids of perfused livers using scanning electron microscopy. This liver perfusion model offers a valuable method to help clarify sporozoite interactions with elements of the liver.

The use of dextran as a blocking agent on nitrocellulose membrane in the analysis of sporozoite antigens of Plasmodium vivax.

Dextran (molecular weight, 71,200) has been found to block the unbound sites of the nitrocellulose membrane, to which antigens have been electroblotted from acrylamide gel, for use in assaying monoclonal antibodies. The use of polysaccharide as a blocking agent allows the antigens on the nitrocellulose membrane to be digested with pronase and subsequently reacted with monoclonal antibodies. Sporozoite antigens of Plasmodium vivax, after being digested with pronase, completely lost their antigenicity to bind to the sporozoite-specific monoclonal antibodies, thus suggesting that they are proteins or protein conjugates in nature. The method described here for qualitative determination of protein antigens requires as little as 2000 sporozoites for each assay.

The influence of cell type and culture medium on the in vitro cultivation of exoerythrocytic stages of Plasmodium berghei.

Plasmodium berghei sporozoites successfully entered and developed into exoerythrocytic schizonts in a variety of cell types cultured in vitro, but segmentation and release of merozoites was only observed in human embryonic lung cells. Exoerythrocytic development was generally not influenced by the culture medium, and NCTC-135 was used routinely. In vitro infectivity of P. berghei sporozoites was unaffected by the serum type used for isolation.

Effects of transmission-blocking immunity on Plasmodium vivax infections in Anopheles albimanus populations.

Two colonized populations of Anopheles albimanus isolated from the Suchiate region, Chiapas State, Mexico, were compared for their susceptibility to coindigenous Plasmodium vivax. Groups of mosquitoes were fed in vitro with either autologous donor blood or the same blood cells substituted with serum negative for anti-gametocyte antibody. Significant differences in susceptibility between the 2 colonies were encountered if the autologous blood from a patient was fed to mosquitoes: mean infection rates of AnA2-positive groups was double that in AnA1 mosquitoes. Consistent for both colonies, only 23.6% of samples positive from malaria-negative serum-substituted blood were infected with an autologous blood feed. Vector competence in these mosquito populations was partially linked to the human populations's immune response to the parasite.