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.

Long-term persistence of sterile immunity in a volunteer immunized with X-irradiated Plasmodium falciparum sporozoites.

Three volunteers were immunized by repeated exposure to the bites of Plasmodium falciparum-infected, X-irradiated mosquitoes to characterize immunologic responses and duration of protective immunity. A primary series of immunizations had been shown previously to induce sterile immunity in these volunteers against sporozoite-induced P. falciparum malaria. In the current study, antibodies to sporozoites circulated at high levels for at least 9-12 months after the volunteers were administered booster bites from X-irradiated infective mosquitoes. One volunteer challenged a second time with P. falciparum 9 months after his last immunization was again shown to be protected, whereas all 5 control subjects developed patent infections. These results set a new standard for persistence of sterile immunity against experimental P. falciparum infection.

Effects of ingested human anti-sporozoite sera on Plasmodium falciparum sporogony in Anopheles stephensi.

We investigated the effects of human anti-sporozoite antibodies on the sporogonic development of Plasmodium falciparum in Anopheles stephensi. Equal volumes of washed human erythrocytes and human sera from 1) volunteers with protective immunity induced by immunization with irradiated P. falciparum sporozoites, 2) the same volunteers before immunization, or 3) Kenyans exposed to natural sporozoite transmission, were fed to cohorts of P. falciparum-infected A. stephensi on either day 5, 8, or 11 after infection. A fourth group of infected mosquitoes from the same cohort were not refed. In two experiments, the effects of anti-sporozoite antibodies were evaluated by determining the infection rates and parasite densities for oocysts and salivary gland sporozoites. There was no evidence that anti-sporozoite antibodies had any effect on the development or intensity of P. falciparum infection in A. stephensi. However, accelerated oocyst maturation was associated with mosquitoes taking a second blood meal, independent of serum source. Salivary gland sporozoites from mosquitoes that fed on immune human sera contained bound human IgG, which was detectable by indirect immunofluorescence assay. The infectivity and transmission potential of human IgG-coated sporozoites is unknown.

Age-specific prevalence of serum antibodies to the invasion plasmid and lipopolysaccharide antigens of Shigella species in Chilean and North American populations.

Shigella species have virulence plasmids that encode outer membrane proteins (invasion plasmid antigens, Ipa) associated with pathogenicity. Western blots were used to detect antibodies to Ipa in sera from 390 Chilean children, and these responses were compared with those of a US population of infants and adults. Antibodies to lipopolysaccharide (LPS) of Plesiomonas shigelloides and Shigella flexneri 2a were measured by ELISA. Among the Chileans, there was an age-related acquisition of Ipa antibodies, with 28% of 1-year-olds and 100% of children greater than or equal to 10 years showing positive responses. In contrast, none of the US infants and only 38% of the adults had antibodies to Ipa. Levels of LPS antibodies were also found to increase in an age-related manner among the Chileans. These results corroborate findings of previous epidemiologic studies which show that Shigella infections are endemic in Chile, as in other developing countries. The measurement of Ipa and LPS antibodies is a useful seroepidemiologic tool for investigating previous exposure to Shigella species in populations.

Safety, immunogenicity, and efficacy in monkeys and humans of invasive Escherichia coli K-12 hybrid vaccine candidates expressing Shigella flexneri 2a somatic antigen.

A live, oral Shigella vaccine, constructed by transfer of the 140-MDa invasiveness plasmid from Shigella flexneri 5 and the chromosomal genes encoding the group- and type-specific O antigen of S. flexneri 2a to Escherichia coli K-12, was tested in humans. Designated EcSf2a-1, this vaccine produced adverse reactions (fever, diarrhea, or dysentery) in 4 (31%) of 13 subjects who ingested a single dose of 1.0 x 10(9) CFU, while at better-tolerated doses (5.0 x 10(6) to 5.0 x 10(7) CFU), it provided no significant protection against challenge with S. flexneri 2a. A further-attenuated aroD mutant derivative, EcSf2a-2, was then tested. Rhesus monkeys that received EcSf2a-2 in three oral doses of ca. 1.5 x 10(11) CFU experienced no increase in gastrointestinal symptoms compared with a control group that received an E. coli K-12 placebo. Compared with controls, the vaccinated monkeys were protected against shigellosis after challenge with S. flexneri 2a (60% efficacy; P = 0.001). In humans, EcSf2a-2 was well tolerated at inocula ranging from 5.0 x 10(6) to 2.1 x 10(9) CFU. However, after a single dose of 2.5 x 10(9) CFU, 4 (17%) of 23 subjects experienced adverse reactions, including fever (3 subjects) and diarrhea (209 ml) (1 subject), and after a single dose of 1.8 x 10(10) CFU, 2 of 4 subjects developed dysentery. Recipients of three doses of 1.2 to 2.5 x 10(9) CFU had significant rises in serum antibody to lipopolysaccharide (61%) and invasiveness plasmid antigens (44%) and in gut-derived immunoglobulin A antibody-secreting cells specific for lipopolysaccharide (100%) and invasiveness plasmid antigens (60%). Despite its immunogenicity, the vaccine conferred only 36% protection against illness (fever, diarrhea, or dysentery) induced by experimental challenge (P = 0.17). These findings illustrate the use of an epithelial cell-invasive E. coli strain as a carrier for Shigella antigens. Future studies must explore dosing regimens that might optimize the protective effects of the vaccine while eliminating adverse clinical reactions.

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.

Urinary neopterin in volunteers experimentally infected with Plasmodium falciparum.

To investigate the kinetics of monocyte/macrophage activation in falciparum malaria we determined urinary neopterin values serially in experimentally infected volunteers. Three subjects who had been immunized with irradiated sporozoites via mosquito bites served as controls. These individuals remained aparasitaemic, afebrile and without a rise in neopterin after challenge by infective mosquitoes. Four non-immune subjects developed Plasmodium falciparum parasitaemia, fever (3 of 4) and sharp rises in neopterin. Parasite densities reached 10-100 parasitized erythrocytes per microliter before elevations in temperature or neopterin levels were detected. Onset of fever preceded the rise in neopterin excretion by one day. Prompt chemotherapy was associated with the clearance of parasites from the blood and the return of temperature and neopterin levels to normal.

Safety and immunogenicity in volunteers of a recombinant Plasmodium falciparum circumsporozoite protein malaria vaccine produced in Lepidopteran cells.

A recombinant Plasmodium falciparum circumsporozoite (CS) antigen (rPfCSA) was produced in insect cells using a baculovirus expression vector containing the entire CS gene. This near full-length CS antigen was adsorbed onto aluminium phosphate for use as a malaria vaccine. In a study of safety and immunogenicity, 20 volunteers were divided into four groups of five each and inoculated intramuscularly with 10, 100, 500 or 1000 micrograms of vaccine. Primary vaccinations were followed by two booster immunizations at 2 and 6 months. Three volunteers developed prominent local reactions manifested as tenderness, redness and swelling at the injection site following the second or third vaccination. All symptoms resolved spontaneously within 72 h. Postimmunization sera from six of 20 volunteers showed seroconversions as measured by Western blot, using rPfCSA as antigen. However, specific anti-CS protein antibody could not be detected by indirect immunoflourescence against intact sporozoites or by ELISA using rPfCSA or peptide to the repeat region. In addition, 18 of 20 volunteers developed antibody to baculovirus proteins as determined by ELISA and/or Western blot. Antigen-driven replication studies using peripheral blood mononuclear cells from vaccinees failed to detect proliferative responses specific to CS protein. This recombinant CS protein vaccine, as formulated, was minimally immunogenic in humans.

Safety and immunogenicity of a recombinant sporozoite malaria vaccine against Plasmodium vivax.

A recombinant Plasmodium vivax circumsporozoite (CS) antigen representing approximately 70% of the CS protein was expressed in yeast and adsorbed onto aluminum hydroxide for use as a malaria vaccine. In a study of safety and immunogenicity, 30 volunteers were divided into four groups of 5, 5, 10, and 10 individuals, and inoculated intramuscularly with 50, 100, 200, or 400 micrograms of vaccine, respectively. Primary vaccinations were followed by two booster immunizations at six weeks and six months. Overall, the vaccine was well tolerated. Following the third vaccination, one volunteer developed acute hepatitis of uncertain etiology that resolved without sequelae. All volunteers in the 400-micrograms group, and six of 10 in the 200-micrograms group generated IgG against P. vivax CS protein, as determined by Western blot using recombinant CS protein. However, the magnitude of the antibody response measured by indirect immunofluorescence of intact sporozoites or enzyme-linked immunosorbent assay against the recombinant protein was low, and responses could not be boosted. Antigen-driven replication studies using peripheral blood lymphocytes failed to detect proliferative responses specific to peptide sequences represented in the recombinant vaccine, except in one volunteer. Minimal humoral and cell-mediated immune responses developed in most recipients who received this recombinant CS vaccine.

Studies in volunteers to evaluate candidate Shigella vaccines: further experience with a bivalent Salmonella typhi-Shigella sonnei vaccine and protection conferred by previous Shigella sonnei disease.

A bivalent vaccine consisting of Salmonella typhi strain Ty21a containing the 120 MDa plasmid of Shigella sonnei and expressing both S. typhi and S. sonnei lipopolysaccharides (LPS) on its surface was previously shown to protect significantly against S. sonnei disease in experimental challenge studies. However, protective efficacy could not be reconfirmed in volunteers with five subsequent lots of vaccine. One vaccine lot which resembled the initial protective lots of vaccine in biochemical and serological tests, and by electron microscopy, was administered to 16 volunteers who ingested three doses of 10(9) organisms each. Antibody secreting cells (ASC) specific for S. sonnei LPS were detected in the blood of 100% of vaccines, but no protection of these vaccines was demonstrated during a S. sonnei challenge study. To assess the ability of the volunteer model to detect infection-derived immunity, six volunteers who had had clinical shigellosis due to S. sonnei two months earlier were rechallenged with wild-type S. sonnei, together with 12 controls. Prior infection provided 100% protection against febrile illness (p = 0.05) and diarrhea (p = 0.04), thereby validating the volunteer model for assessing Shigella vaccines.

Antigenic variation of Giardia lamblia in experimental human infections.

To determine if Giardia surface Ag vary in human infections volunteers were inoculated enterally with trophozoites of uncloned GS/M-85 and in later experiments with two clones derived from GS/M. The surface Ag of trophozoites reisolated from 6/6 volunteers differed from the inoculum. To determine if the surface Ag of trophozoites derived from clones would also change, volunteers were inoculated with two clones, B6 or H7. B6 possesses a 200-kDa surface Ag recognized by mAb 3F6 and H7 has a 72-kDa surface Ag recognized by mAb G10/4. One of thirteen B6 and four of four H7-inoculated volunteers became infected. Analysis of Giardia obtained on day 22 from the intestines of the four H7-infected volunteers and cultures derived from these trophozoites revealed loss of the initial major surface Ag as determined by surface IFA using mAb, surface radiolabeling and loss of cytotoxicity to mAb, and Western blots. Loss of the 72-kDa Ag began after day 14 and was practically complete by day 22. The 200-kDa surface Ag was almost totally absent from the surface of Giardia isolated from the single B6-infected volunteer. Serum surface-reactive antibodies, as measured by IFA and cytotoxicity to H7 and the day 22 isolates, showed high levels of antibodies to H7, primarily to the 72-kDa surface Ag, but negligible or low levels of late-appearing antibodies to the day 22 isolates. These studies document antigenic variation of Giardia in human infections and show that humoral responses are in part isolate-specific.

Specific immunoglobulin A-secreting cells in peripheral blood of humans following oral immunization with a bivalent Salmonella typhi-Shigella sonnei vaccine or infection by pathogenic S. sonnei.

The ability of bivalent Salmonella typhi-Shigella sonnei vaccine strain 5076-1C to stimulate an intestinal immunoglobulin A response in humans was evaluated by detecting gut-derived, trafficking antibody-secreting cells (ASC) in peripheral blood. Following vaccination, an immunoglobulin A-ASC response to O antigens of S. typhi and S. sonnei was observed in 10 of 13 and 13 of 13 vaccine recipients, respectively. Experimental challenge with pathogenic S. sonnei stimulated an ASC response to the S. sonnei O antigen in all subjects who developed clinical illness. The magnitude of the ASC response to challenge was significantly greater than that resulting from vaccination. Furthermore, compared with the response of the unimmunized controls, individuals previously immunized with 5076-1C demonstrated a significantly greater ASC response following challenge with S. sonnei.

Diminished immunogenicity of a recombination-deficient derivative of Vibrio cholerae vaccine strain CVD103.

To address potential concerns over the release of genetically engineered live bacterial vaccines, we constructed a recombination-deficient derivative of the Vibrio cholerae O1 vaccine strain CVD103 (CVD103RM). Oral immunization of adult volunteers with CVD103RM showed that the recA mutation significantly diminished colonization ability and immunogenicity of the vaccine strain.

Activity of human volunteer sera to candidate Plasmodium falciparum circumsporozoite protein vaccines in the inhibition of sporozoite invasion assay of human hepatoma cells and hepatocytes.

Sera from human volunteers immunized with either synthetic peptide (NANP)3-TT or recombinant protein R32tet32 Plasmodium falciparum CS vaccines were tested in the inhibition of sporozoite invasion (ISI) assays using human hepatoma (HepG2-A16) cells or primary human hepatocytes. Sera or purified immunoglobulin (Ig) from volunteers who were completely protected against P. falciparum sporozoite challenge had higher ISI activity than sera from non-protected volunteers, or the highest titre endemic serum. However, Ig from protected and non-protected volunteers did not block sporozoite invasion of human hepatocytes, suggesting that P. falciparum sporozoites invade hepatocytes by mechanisms which differ from those concerned with invasion of HepG2-A16 cells.

Continuation of chloroquine-susceptible Plasmodium falciparum parasitemia in volunteers receiving chloroquine therapy.

Volunteers infected with a chloroquine-susceptible line of Plasmodium falciparum were administered standard oral chloroquine therapy at the first detection of parasites in the blood. Parasitemias progressed in the face of therapy for up to 5 days and to levels up to 100-fold greater than those at the initiation of treatment. Thereafter, infections cleared without a requirement for additional chemotherapy. This course of infection and response to treatment has not been previously reported and may have been detected because volunteers were exposed to an unusually large number of sporozoites. The observations are consistent with the hypothesis that prolonged parasitemia resulted from the continued release of merozoites from liver.

Human studies with synthetic peptide sporozoite vaccine (NANP)3-TT and immunization with irradiated sporozoites.

The synthetic peptide Plasmodium falciparum circumsporozoite (CS) protein conjugate vaccine (NANP)3-TT was safe when given parenterally to 202 volunteers. However, with a few notable exceptions, antibody responses were low and could not be boosted. Vaccinees' lymphocytes did not proliferate when exposed in vitro to (NANP)3. The tetanus toxoid (TT) carrier immunomodulated the response to the CS peptide in that both epitopic suppression and immune enhancement were demonstrated during the course of the clinical trials. During efficacy challenge studies, 1 of 7 vaccinees was protected against sporozoite challenge and in other vaccinees the prepatent period was significantly delayed. P. falciparum-infected mosquitos were irradiated with 20,000 rad (200 Gy). Five volunteers were immunized with 54, 55, 224, 663, and 715 total infective bites of irradiated mosquitos in an attempt to immunize with attenuated sporozoites. Four of these volunteers had significant humoral and cellular immune responses. Two volunteers (who received the largest immunizing doses) were challenged by the bites of infective mosquitos and both developed parasitaemia. In the volunteer with the highest antibody titre there was a marked delay in patency as determined by serial plasmodial cultures. T-cell clones are being obtained and characterized.

Effect of priming with carrier on response to conjugate vaccine.

To examine whether prior immunity against a carrier protein modulates the serological response to injected peptide haptens attached to the same carrier in man, baseline tetanus antitoxin levels in volunteers who received a malaria sporozoite peptide-tetanus toxoid conjugate vaccine were compared with post-vaccination IgM and IgG antibody titres against the sporozoite antigen. In tetanus-vaccinated North American recipients of low doses of conjugate vaccine there were significant dose-dependent negative correlations between these variables, which suggests that epitopic suppression may occur in man. In contrast, Venezuelans living in non-malarious areas and mostly naive to tetanus toxoid showed a notable IgM response to the sporozoite antigen. The findings indicate that epitopic suppression and immune enhancement occur in man, and that the specific immunological responses to conjugate peptide vaccines may be difficult to predict.

Conserved repetitive epitope recognized by CD4+ clones from a malaria-immunized volunteer.

T cell clones obtained from a human volunteer immunized with Plasmodium falciparum sporozoites specifically recognized the native circumsporozoite (CS) antigen expressed on P. falciparum sporozoites, as well as bacteria- and yeast-derived recombinant falciparum CS proteins. The response of these CD4+ CD8- cells was species-specific, since the clones did not proliferate or secrete gamma interferon when challenged with sporozoites or recombinant CS proteins of other human, simian, or rodent malarias. The epitope recognized by the sporozoite-specific human T cell clones mapped to the 5' repeat region of the CS protein and was contained in the NANPNVDPNANP sequence.