David F. Clyde

Immunization of man against sporozite-induced falciparum malaria

Based on successful work with nonhuman malarias, an attempt was made to immunize man against mosquito-borne stages of Plasmodium falciparum. Strict ethical guidelines were followed. Mosquitoes carrying sporozoites of P. falciparum were X-irradiated and then fed on volunteers who had not previously been exposed to malaria. The sporozoites were inactivated at a minimum dosage of 15,000 rads, and did not produce adverse reactions in the volunteers. Three volunteers were each exposed during 84 days to 379 infected irradiated mosquitoes, and on day 98 were fed on by nonirradiated mosquitoes heavily infected with homologous strain P. falciparum. One of these men did not develop malaria, and continued to be immunized during the ensuing 217 days with 819 infected mosquitoes. On day 327, when antisporozoite antibody was first demonstrated in his serum by the circumsporozoite precipitation test, he was fed on by nonirradiated mosquitoes carrying homologous strain P. falciparum, and did not develop malaria. Possible causes of his failure to become infected were investigated, and it was concluded that he had become immunized to falciparum sporozoites of that strain. His continued susceptibility to strain-specific falciparum malaria induced by direct blood transfer was demonstrated.

Immunization of Man against Falciparum and Vivax Malaria by Use of Attenuated Sporozoites

With strict adherence to ethical guidelines, a volunteer was immunized against sporozoites of Plasmodium falciparum and P. vivax, the antigen consisting of attenuated sporozoites of each species inoculated through bites of mosquitoes X-irradiated at a minimum dosage of 15,000 rads. On one occasion this dosage did not render all P. vivax sporozoites noninfective. Species specificity of antigen and antibody was demonstrated, but within each species a wide geographical diversity of strains proved interchangeably antigenic and susceptible to the antibody. Once immunized, the volunteer was protected for not more than 3 months and 6 months, respectively, from infective P. falciparum and P. vivax sporozoites, the duration of protection being reflected by a positive species-specific circumsporozoite reaction. Studies in this volunteer, and in two others immunized with P. falciparum sporozoites, did not reveal any increase in serum levels of immunoglobulins G and M.

Specificity of protection of man immunized against sporozoite-induced falciparum malaria.

During a period of 421 days a total of 1,441 X-irradiated mosquitoes inoculated attenuated sporozoites of Burma (Thau.) strain Plasmodium falciparum into a volunteer. Strict ethical guidelines were followed. The volunteer was also exposed at various times to nonirradiated mosquitoes carrying infective sporozoites of the same strain and of strains from Malaya, Panama and the Philippines, and did not develop falciparum malaria. Following exposure to nonirradiated mosquitoes carrying Chesson strain P. vivax, he developed vivax malaria. Antisporozoite antibody, demonstrated by the circumsporozoite precipitation test, appeared in his serum and reacted equally against the Burma strain of P. falciparum and strains from Malaya, Panama and the Solomon Islands, but did not react with Chesson vivax sporozoites.

Clinical Manifestations of Plasmodium falciparum Malaria Experimentally Induced by Mosquito Challenge

To determine the characteristics of clinical illness accompanying Plasmodium falciparum infection induced by controlled exposure to infected mosquitoes, records of 118 volunteers participating in studies conducted between 1985 and 1992 were reviewed. One hundred fourteen volunteers (97%) reported at least one symptom attributable to malaria, with fatigue, myalgias or arthralgias, headache, and chills most commonly reported. The median duration of symptoms was 3 days. Fever was recorded in 61% of volunteers; 4 volunteers had temperatures >40 degrees C. Neutropenia and thrombocytopenia were present in 9% and 12% of volunteers, respectively. Despite counts as low as 658/microL (neutrophils) or 73,000/microL (platelets), no secondary infectious or hemorrhagic complications occurred. In all cases, volunteers recovered completely and laboratory values returned to baseline after specific antimalarial therapy. Recrudescence did not occur in any volunteer. In this model, mosquito inoculation of P. falciparum is a reliable, safe, and well-tolerated method of experimental challenge.

Radical cure of Chesson strain vivax malaria in man by 7, not 14, days of treatment with primaquine.

Glucose-6-phosphate dehydrogenase-normal adult volunteers infected with mosquito-bone Chesson strain vivax malaria were treated with chloroquine and primaquine during the initial attack. Administration of 60 mg (base) of primaquine daily for 7 days was as effective in preventing relapse as is the regimen customarily used for the radical cure of infections produced by this strain, namely, 30 mg daily for 14 days. However, it is stressed that because of the risk of primaquine-induced hemolysis in individuals having genetically-transmitted erythrocyte abnormalities this high dosage should not be used routinely.

Plasmodium vivax: correlation of circumsporozoite precipitation (CSP) reaction with sporozoite-induced protective immunity in man.

Abstract A volunteer was immunized with X-irradiated sporozoites of Plasmodium vivax against a nonirradiated, infective sporozoite challenge of the same species and strain. Shortly before his first successful challenge, his serum was found to contain anti-sporozoite antibodies. Subsequent immunizations and challenges with another strain demonstrated that his protective immunity lasted 3–5 months and could be restored or boosted by challenge with a small number of nonirradiated, infective sporozoites. His CSP serum reaction paralleled his level of protective immunity.

Suppressive Activity of Mefloquine in Sporozoite-Induced Human Malaria

Mefloquine hydrochloride [WR 142,490; α-(2-piperidyl)-2,8-bis(trifluoromethyl)-4-quinolinemethanol hydrochloride] was tested for suppressive effect on sporozoite-induced malaria in nonimmune volunteers living in an area where malaria is not naturally transmitted. Single doses of 250 mg were given at weekly intervals, 500 mg at intervals of 2 weeks and 1,000 mg at intervals of 4 weeks, to men bitten by 10 to 15 mosquitoes heavily infected with a chloroquine- and pyrimethamine-resistant strain of Plasmodium falciparum. None of the individuals so treated developed infections during the period of drug delivery or during the follow-up period of 60 days. Doses of 250 or 500 mg produced no adverse reactions; mild epigastric discomfort occurred in all three men given 1,000 mg. Sporozoite-induced P. vivax infections were suppressed by single doses of 250 mg of mefloquine given at weekly intervals, but malaria developed after completion of the course. At treatment intervals longer than 1 week, vivax malaria was not suppressed.

Plasmodium falciparum: in vitro characterization and human infectivity of a cloned line.

The culture-adapted NF54 isolate of Plasmodium falciparum was subjected in vitro to three sequential limiting dilution titrations and the resulting clone was given the designation CVD1. DNA sequence analysis of the gene encoding the circumsporozoite (CS) protein revealed differences between CVD1 and the published NF54 CS gene. CVD1 had 1191 bp, 397 amino acids, and 42 repeat units while NF54 had 1218 bp, 405 amino acids, and 44 repeat units. The CVD1 clone was more sensitive to chloroquine than was the parental line, in vitro. Anopheles stephensi mosquitoes were infected equally by the cloned and uncloned parasites. Volunteers were readily infected by NF54 and CVD1 following infectious mosquito bites. The availability of a well-characterized, chloroquine-sensitive clone which safety infects humans should facilitate performance of experimental challenge studies to assess vaccine efficacy.

Estimate of anti-plasmodium falciparum sporozoite activity in humans vaccinated with synthetic circumsporozoite protein (NANP)3

A mathematical model was defined to estimate the degree of in vivo activity against Plasmodium falciparum sporozoites expressed by volunteers vaccinated with a synthetic peptide comprising the immunodominant epitope of the circumsporozoite protein. Relative to the course of infection in non-immunized controls, infections in vaccinated volunteers corresponded to the neutralization or delay of development of greater than 99% of challenge sporozoites.

Prophylactic Activity of a Phenanthrene Methanol (WR 33063) and a Quinoline Methanol (WR 30090) in Human Malaria

WR 33063 (3-bromo-10-[α-hydroxy-β-(n, n-diheptylamino)ethyl]-phenanthrene hydrochloride) and WR 30090 (6,8-dichloro-2,3,4-dichlorphenyl-di-n-butylaminoethyl-4- quinolinemethanol hydrochloride) were tested for suppressive prophylactic effect on induced malaria in nonimmune volunteers living in an area where malaria is not naturally transmitted. Doses of 800 mg of WR 33063 and 690 or 460 mg of WR 30090 were given at weekly intervals to men exposed on the day of the first dose to mosquitoes heavily infected with chloroquine- and pyrimethamine-resistant strains of Plasmodium falciparum. WR 33063 did not interfere with early development of infection, but WR 30090 given for 8 weeks provided suppressive cures in 20 of 26 men. P. vivax infections similarly induced broke through WR 30090 treatment in 4 of 15 men, and most of the remainder experienced malaria after completion of the prophylactic course. No side effects of treatment were observed.