Imogene Schneider

Protection of Humans against Malaria by Immunization with Radiation-Attenuated Plasmodium falciparum Sporozoites

During 1989-1999, 11 volunteers were immunized by the bites of 1001-2927 irradiated mosquitoes harboring infectious sporozoites of Plasmodium falciparum (Pf) strain NF54 or clone 3D7/NF54. Ten volunteers were first challenged by the bites of Pf-infected mosquitoes 2-9 weeks after the last immunization, and all were protected. A volunteer challenged 10 weeks after the last immunization was not protected. Five previously protected volunteers were rechallenged 23-42 weeks after a secondary immunization, and 4 were protected. Two volunteers were protected when rechallenged with a heterologous Pf strain (7G8). In total, there was protection in 24 of 26 challenges. These results expand published findings demonstrating that immunization by exposure to thousands of mosquitoes carrying radiation-attenuated Pf sporozoites is safe and well tolerated and elicits strain-transcendent protective immunity that persists for at least 42 weeks.

SAFETY AND EFFICACY OF A RECOMBINANT DNA PLASMODIUM FALCIPARUM SPOROZOITE VACCINE

A recombinant DNA Plasmodium falciparum sporozoite vaccine produced in Escherichia coli (FSV-1) was tested in doses of 10 micrograms to 800 micrograms protein in fifteen volunteers. No serious adverse reactions occurred. Antibodies that reacted with P falciparum sporozoite antigens by enzyme-linked immunoassay developed in twelve of the volunteers. The highest antibody titres induced were similar to those resulting from lifelong natural exposure to sporozoite-infected mosquitoes. Postimmunization serum samples from a majority of volunteers mediated the circumsporozoite (CS) precipitation reaction and inhibited sporozoite invasion of hepatoma cells in vitro. Serum from the three volunteers who received 800 micrograms doses reacted with the surface of sporozoites in an immunofluorescence assay. Six immunised volunteers receiving a fourth dose of FSV-1 and two non-immunised controls were challenged by bites of mosquitoes infected from cultured P falciparum gametocytes. Parasitaemia did not develop in the volunteer with the highest titre of CS antibodies, and parasitaemia was delayed in two other immunised volunteers. This study confirms that human beings can be protected by CS protein subunit vaccines and provides a framework for the further development and testing of more immunogenic sporozoite vaccines.

An estimation of the number of malaria sporozoites ejected by a feeding mosquito

Restrained Anopheles stephensi mosquitoes infected with Plasmodium falciparum were made to produce time-dependent series of saliva droplets in mineral oil. The relative volume of each droplet and the number of sporozoites each contained were determined microscopically; gland sporozoites were estimated with an enzyme-linked immunosorbent assay. Median gland infection was 8170 and median number of sporozoites ejected was 15 (range, 0-978). Inoculum size was positively correlated to the number of sporozoites in the salivary glands. Most mosquitoes ejected sporozoites only at the beginning of salivation; this suggests that only those parasites in the common and secondary salivary ducts at the time of feeding can be ejected. The small size of inocula may explain some aspects of malaria transmission, including the often observed discrepancy between inoculation and incidence rates.

Infectivity to mosquitoes of Plasmodium falciparum clones grown in vitro from the same isolate

In an attempt to produce a line of cultured Plasmodium falciparum parasites consistently infective to mosquites, a Brazilian isolate, IMTM 22, was cloned by the limiting dilution method. Five of the resulting clones were examined in detail. The clones were found to differ in their ability to produce micro- and macrogametocytes, to exflagellate and to infect Anopheles freeborni mosquitoes. The stability of one clone in producing microgametocytes and in its ability to produce oocysts and sporozoites in mosquitoes has been documented through 15 subcultures. This clone should provide a reliable source of infectious gametocytes for genetic studies and vaccine development.

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.

Plasmodium falciparum: Immunogenicity of circumsporozoite protein constructs produced in Escherichia coli

The immunogenicity of Plasmodium falciparum recombinant circumsporozoite protein constructs R16tet32, R32tet32, and R48tet32 in mice was examined by measuring antibody responses by enzyme linked immunosorbent assay, immunofluorescence, circumsporozoite precipitation, and inhibition of sporozoite invasion. All three constructs were found to be immunogenic when administered alone, but antibody responses were greater for the larger constructs, R32tet32 and R48tet32. Increased dose, boosting, and the use of adjuvants further augmented antibody responses. R32tet32 was found to be the most immunogenic of the three constructs, and high levels of protective antibodies were found to persist for at least 44 weeks when the construct was given with alum. Clinical trials with alum adjuvanted R32tet32 have now begun.

Azithromycin prophylaxis against a chloroquine-resistant strain of Plasmodium falciparum

Azithromycin has antimalarial activity and favourable pharmacokinetic properties for a prophylactic antimalarial agent. We investigated the ability of azithromycin to prevent malaria in volunteers infected with a chloroquine-resistant strain of Plasmodium falciparum. 4 volunteers received oral azithromycin 500 mg followed by 250 mg daily for 7 further days. Subjects were infected on the third day of azithromycin. 3 subjects were protected compared with none of 15 controls. The volunteer not protected by azithromycin had unquantifiable plasma levels of azithromycin, probably because of poor absorption. Azithromycin could be a promising prophylactic agent for P falciparum malaria.

Cultivation in vitro of Plasmodium gallinaceum oocysts.

Abstract Large numbers of viable infectious Plasmodium gallinaceum sporozoites, virtually free of mosquito tissue, were obtained by culturing somewhat younger stages of the sporogonous cycle. Twenty or more oocysts were placed in each hanging- or sitting-drop culture. Graces insect tissue culture medium, slightly modified and supplemented with either vertebrate and/or invertebrate sera, was employed. In addition, approximately one half of the cultures contained mosquito cells, organs, or whole body extracts. Nine-day-old oocysts usually completed development within 24 hours and liberated sporozoites shortly thereafter. The extent of development in 8-day-old oocysts was dependent upon whether sporoblast formation with subsequent budding of the sporozoites had or had not occurred prior to explantation. If the former, the oocysts continued their development in 60% of the cultures; if the latter, less than 1% of the oocysts were capable of producing sporozoites. Attempts to culture 6- and 7-day-old oocysts were not successful. This failure of the younger oocysts to develop in vitro was attributed to insufficiencies in the culture medium, lack of tissue bulk, suboptimal conditioning of the medium, and the relatively static environment of the present system. The suggestion was made that such factors might be largely negated if the oocysts were cultured in the presence of an actively growing mosquito cell strain.

Safety and immunogenicity of a Plasmodium falciparum sporozoite vaccine: boosting of antibody response in a population with prior natural exposure to malaria

Recombinant sporozoite vaccine or placebo were administered once to 25 volunteers from an area endemic for malaria. Antibody to R32tet32 rose in 9 of 15 receiving vaccine and remained elevated in 6 for 6 months. Mean absorbance increase was 0.43 +/- 0.40 with vaccine, 0.01 +/- 0.23 with placebo, and 0.72 +/- 0.19 in responders. Six non-responders had significantly lower pre-immunization levels (0.07 +/- 0.05) than responders (0.39 +/- 0.25). There was an association between an increase in immunofluorescence (n = 4) and an increase in absorbance (n = 9) among vaccine recipients (n = 15). Vaccine-induced increase in antibody to natural circumsporozoite antigen was indicated by increases in immunofluorescence and by increases in circumsporozoite precipitation score in 2 of the 5 responders with highest antibody increase measured by enzyme-linked immunosorbent assay. Response to subunit sporozoite vaccine paralleled response to prior natural sporozoite exposure and was significant and prolonged in a population with prior natural exposure to malaria.

Plasmodium falciparum andP. berghei: detection of sporozoites and the circumsporozoite proteins in the saliva ofAnopheles stephensi mosquitoes

Sporozoites and free circumsporozoite (CS) protein were stained immunoenzymatically in 1-min saliva samples collected fromAnopheles stephensi mosquitoes infected with eitherPlasmodium berghei orP. falciparum. The number of sporozoites in 1-min saliva-streak samples significantly increased as the salivary gland index rose from 3+ to 4+. ForP. berghei-infected mosquitoes from which saliva had been collected before 30 days postfeed, the median sporozoite counts for 3+ and 4+ gland indexes were 4.5 and 116, respectively. ForP. falciparum-infected mosquitoes, the median counts obtained in two experiments were 4.5 and 14.5 (3+) and 97 and 107 (4+), respectively. The frequency of sporozoite detection in the saliva of mosquitoes containing <100 salivary-gland sporozoites was low (0.1), whereas that in the saliva of mosquitoes with >100 sporozoites was high (0.96). In highly infected 4+P. berghei-infected mosquitoes from which saliva had been collected after 30 days postinfection, both the volume of saliva collected and the median number of sporozoites recovered decreased significantly.