Genetic control of the immune response in mice to a Plasmodium falciparum sporozoite vaccine. Widespread nonresponsiveness to single malaria T epitope in highly repetitive vaccine.

Different H-2 congenic strains of mice were immunized with a P. falciparum sporozoite vaccine currently being tested in humans, or with different segments of the vaccine molecule. Specific IgG production or lymph node cell proliferation in response to different antigens was then determined. Only four of seven strains (representing three of eight possible different class II restriction molecules) responded to the vaccine. Of those restriction molecules, only one, I-Ab, was associated with a response to a malaria-encoded T epitope [contained within NP(NANP)3NA], while the other two molecules (E alpha dE beta d and E alpha kE beta s) were associated with a T cell response to a nonmalarial epitope(s) carboxyterminal to the malaria sequence and encoded by a tetracycline resistance gene, read out of frame. If an analogous situation applies in humans, natural boosting by sporozoites will be very restricted. This has serious implications for the effectiveness of the vaccine, since constant high levels of antisporozoite antibodies and possibly antibody-independent T cell effector functions are required for immunity.

Proteosome-lipopeptide vaccines: enhancement of immunogenicity for malaria CS peptides.

Proteosomes are hydrophobic, membranous, multimolecular preparations of meningococcal outer membrane proteins that are also B cell mitogens. These characteristics suggested that proteosomes may serve as carrier proteins and adjuvants to enhance peptide immunogenicity. Although high titers of malaria circumsporozoite (CS) antibodies protect against malaria, vaccines thus far tested in humans have been insufficiently immunogenic to be clinically useful. Here it is shown that synthetic CS peptides hydrophobically complexed to proteosomes by way of lauroyl-cysteine become highly immunogenic in mice without other adjuvants. The high titers of antibodies produced and the safety of proteosomes in humans suggest that this novel system is widely applicable for the development of peptide vaccines to protect against many diseases.

Oral Salmonella typhimurium vaccine expressing circumsporozoite protein protects against malaria.

Immunization with radiation-attenuated malaria sporozoites induces potent cellular immune responses, but the target antigens are unknown and have not previously been elicited by subunit vaccines prepared from the circumsporozoite (CS) protein. A method is described here for inducing protective cell-mediated immunity to sporozoites by immunization with attenuated Salmonella typhimurium transformed with the Plasmodium berghei CS gene. These transformants constitutively express CS antigens and, when used to immunize mice orally, colonize the liver, induce antigen-specific cell-mediated immunity, and protect mice against sporozoite challenge in the absence of antisporozoite antibodies. These data indicate that the CS protein contains T cell epitopes capable of inducing protective cell-mediated immunity, and emphasize the importance of proper antigen presentation in generating this response. Analogous, orally administered vaccines against human malaria might be feasible.

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.

Structure of the gene encoding the immunodominant surface antigen on the sporozoite of the human malaria parasite Plasmodium falciparum.

The gene for the circumsporozoite (CS) protein of Plasmodium falciparum has been cloned and its nucleotide sequence determined. The gene encodes a protein of 412 amino acids as deduced from the nucleotide sequence. The protein contains 41 tandem repeats of a tetrapeptide, 37 of which are Asn-Ala-Asn-Pro and four of which are Asn-Val-Asp-Pro. Monoclonal antibodies against the CS protein of Plasmodium falciparum were inhibited from binding to the protein by synthetic peptides of the repeat sequence. The CS protein of Plasmodium falciparum and the CS protein of a simian malaria parasite, Plasmodium knowlesi, have two regions of homology, one of which is present on either side of the repeat. One region contains 12 of 13 identical amino acids. Within the nucleotide sequence of this region, 25 of 27 nucleotides are conserved. The conservation of these regions in parasites widely separated in evolution suggests that they may have a function such as binding to liver cells and may represent an invariant target for immunity.

Dot enzyme-linked immunosorbent assay (Dot-ELISA): a micro technique for the rapid diagnosis of visceral leishmaniasis.

A micro enzyme-linked immunosorbent assay utilizing antigen dotted onto nitrocellulose filter discs (Dot-ELISA) was developed for the rapid diagnosis of visceral leishmaniasis. Leishmania donovani promastigotes applied to filter discs in volumes of 1 microliter were placed in 96-well microtiter plates, blocked with bovine serum albumin, then incubated with 4-fold dilutions of patient sera. After incubation with peroxidase-conjugated anti-human antibody, washing and addition of precipitable substrate, positive reactions appeared as blue dots on a white background which were easily read by eye. The procedure is performed at room temperature, takes about 2 h and is economical. At a reciprocal diagnostic titer of greater than or equal to 32, 41 of 42 (98%) leishmaniasis patients were positive, and positive titers ranged from 512 to 524,288. Control sera from healthy individuals showed 1 of 50 (2%) false positive reactions. Sera from patients with African trypanosomiasis, Chagas' disease, and lupus erythematosus were cross-reactive in the Dot-ELISA. No cross-reactivity was noted with sera from patients with amebiasis, coccidioidomycosis, cutaneous leishmaniasis, viral hepatitis, hydatidosis, malaria, schistosomiasis, syphilis, toxoplasmosis or trichinosis. In replicate experiments, 90% of 167 sera tested did not vary in titer. This rapid and inexpensive test should prove to be an important field diagnostic technique for visceral leishmaniasis.

Structure of the circumsporozoite protein gene in 18 strains of Plasmodium falciparum.

Using the cloned circumsporozoite (CS) protein gene of a Brazilian strain of Plasmodium falciparum as probe, we have analyzed the structure of the CS protein gene from 17 other Asian, African, Central and South American parasite strains by nucleic acid hybridization. Each strain appears to have one CS protein gene which hybridizes readily to the Brazilian strain probe. The 5' and 3' thirds of the genes are invariant in size in all 18 strains whereas the central third containing the 12 base pair tandem repeats varies in size over a range of about 100 base pairs. Several differences were found in the locations of Sau3A sites in the genes. The Sau3A sites are significant because each of the minority Asn-Val-Asp-Pro repeats in the cloned gene has a Sau3A site. DNA melting of hybrids revealed a high degree of homology between the sequences of the cloned gene and genes from an Asian strain and an African strain. A 14 base oligodeoxynucleotide with a sequence from the central repeat region hybridized to all strains tested. We conclude that the CS protein gene is highly conserved among strains of P. falciparum and that malaria vaccine development with the CS protein is unlikely to be complicated by strain variation.

Standardization of the dot enzyme-linked immunosorbent assay (Dot-ELISA) for human visceral leishmaniasis.

The Dot-ELISA, a rapid, visually read micro enzyme immunoassay for visceral leishmaniasis utilizing minute volumes of antigen "dotted" on nitrocellulose filter discs and precipitable chromogenic substrate, was analyzed under a variety of experimental parameters. Raising assay incubation temperatures from 23 degrees C to 28 degrees C resulted in titer increases in three of five leishmaniasis patient sera; at 37 degrees C, all five patient sera and one of five normal human sera showed titer increases. The amount of antigen used could be reduced 50% by incubating patient serum overnight at 4 degrees C. Antigen discs stored at - 20 degrees C were optimally reactive with leishmaniasis sera over a 270-day period. Antigen discs stored at 4 degrees C and 23 degrees C showed reproducible titer decreases at 90 days. Aging either peroxidase-conjugated antibody or substrate for up to 28 days at 4 degrees C did not adversely affect titers of positive and negative control sera and reagent controls. Activated substrate stored at 23 degrees C was optimally reactive in the assay for at least 24 hours. No changes in titers of positive and negative control sera or nonspecific reactions in reagent controls occurred when using different brands of microtiter plates. The long shelf lives and stabilities of Dot-ELISA antigen and reagents indicate this test should prove useful both in the laboratory and in the field.

Activity of pentamidine-containing human red cell ghosts against visceral leishmaniasis in the hamster.

A recent approach to the chemotherapy of visceral leishmaniasis has been the encapsulation of clinical agents within macrophage directed carriers such as liposomes. Because in mammals Leishmania are obligate intramacrophage microorganisms, injection of an encapsulated drug should deliver large quantities of drug to the organisms, thus decreasing both the number of drug administrations needed for cure and drug toxicity. Drugs contained within red cell ghosts have been used clinically to treat other macrophage disorders. We encapsulated the clinical antileishmanial agent pentamidine within human red cell ghosts and administered it to hamsters infected with Leishmania donovani. The ED50 and ED90 of single injections of this preparation were 231-240 times lower than that of the positive control drug, sodium stibogluconate (Pentostam), and essentially all parasites could be eliminated by 2.5 mg encapsulated drugs/kg. Against splenic parasites, the ED50 was 195 times lower than that of antimony, although only 80% of parasites were eliminated by the highest doses of encapsulated drug (2.5-6.4 mg/kg). The difference in liver vs. splenic parasite killing is probably related to the greater uptake of encapsulated drug by the liver (11-14 micrograms/g tissue) vs. the spleen (2-3 micrograms/g). If activity in this model is comparable to activity in humans, these results suggest that a single injection of a preparation consisting of ghosts of a patient's own red cells and the amount of pentamidine in one standard dosage (4 mg/kg) would eliminate 80%-100% of L. donovani from the spleen and liver.

Identification of pathogenic Leishmania promastigotes by DNA: DNA hybridization with kinetoplast DNA cloned into E. coli plasmids.

We report the characterization of Leishmania (L. infantum, L. donovani, and L. major) kinetoplast DNA (kDNA) by the use of restriction endonuclease digestion patterns and Southern hybridizations. Overall, the sizes and fragment patterns of MspI restriction endonuclease-produced DNA fragments vary from species to species. However, kDNA isolates from different species and strains cross-reacted to a great extent in Southern hybridization experiments. Only kDNA isolated from L. infantum and L. major had little homology during hybridization reactions. To prepare DNA probes that would differentiate between species of Leishmania, minicircle kDNA was digested with restriction enzymes and ligated to an E. coli plasmid. Several plasmids were isolated that specifically detect in hybridization experiments as few as 5 X 10(3) L. donovani or L. infantum promastigotes lysed on nitrocellulose filters.

Immune complex decomplementation of canine sera for use in a complement-fixation test for diagnosis of visceral leishmaniasis.

Canine sera frequently become anti-complementary when heat-inactivated at 56 degrees C for 30 min, and generally cannot be used in standard complement-fixation (CF) assays. Therefore, a procedure was developed for decomplementing canine sera by absorption with particulate immune complexes consisting of sheep erythrocyte stroma optimally sensitized with anti-sheep erythrocyte antibody (hemolysin). Canine sera incubated for 20 min at 30 degrees C with sensitized stroma consistently showed less than 10% residual complement and were not anti-complementary. This decomplementation procedure was applied in a complement-fixation (CF) test for detection of serum antibodies during canine visceral leishmaniasis. Two groups of German shepherd dogs were injected intravenously with Leishmania donovani or L. donovani chagasi amastigotes, and the course of infections was followed for 12 weeks. Using freeze-thaw sonicate preparations of L. donovani parasites as antigen, reciprocal CF antibody titers above 24 were detectable in sera 7 weeks after infection and gradually increased to a maximum titer of 775 at 12 weeks. Sera from control dogs had mean titers of 24. This improved methodology enhances the potential of the CF test in the serodiagnosis of canine leishmaniasis.

The response of Kenyan kala-azar to treatment with sodium stibogluconate.

Of 16 patients with kala-azar treated with sodium stibogluconate (0.1 ml/kg body weight a day), one died on the 12th day of treatment and nine were cured by a 30-day course, although two subsequently relapsed. Extending the course cured a further five patients, and in one patient allopurinol was used in addition before a cure was achieved. Clinical and hematological recovery began within a few days of the start of treatment, but parasites continued to be seen in splenic aspirates for 3 weeks or more.

Lymphocyte responsiveness to a candidate malaria sporozoite vaccine (R32tet32) of individuals with naturally acquired Plasmodium falciparum malaria.

Lymphocyte proliferative responses to the candidate malaria sporozoite vaccine antigen R32tet32 were evaluated in 29 patients with acute Plasmodium falciparum malaria, 20 convalescent patients, 11 nonimmune individuals, and 22 healthy residents of two endemic malarious areas in Thailand. The results indicate that 14 of 20 (70%) convalescent patients and 14 of 22 (64%) residents of endemic areas responded to the R32tet32 antigen. However, only 8 of 29 (28%) patients with acute P. falciparum malaria responded. When 4 of the convalescent patients who remained in a malaria-free area were restudied 5-10 months after the acute infection, they were either not responsive or their responses had greatly diminished. These findings show that sensitization to R32tet32 occurs following a natural P. falciparum infection, but the cellular immune response to sporozoite antigens may be short-lived and may be suppressed during acute P. falciparum malaria.

Rapid serodiagnosis of leptospirosis using the IgM-specific Dot-ELISA: comparison with the microscopic agglutination test.

The dot enzyme-linked immunosorbent assay (Dot-ELISA) was compared to the microscopic agglutination test (MA test) for the diagnosis of human leptospirosis. Of 177 sera from 68 soldiers who trained in the Republic of Panama, 102 sera were positive in the MA test and 93 of these sera were positive in the IgM-specific Dot-ELISA. Incidence of infection was 50 of 68 patients with the MA test and 48 of 68 in the IgM Dot-ELISA. Five MA test-positive sera were reactive only in the IgG-specific Dot-ELISA, suggesting previous exposure. All 21 infecting serovars of Leptospira interrogans, as determined by positive reactions in the MA test or culture of blood and urine specimens, were reactive in the Dot-ELISA. Of 75 sera negative in the MA test, 61 were nonreactive in the Dot-ELISA. However, 9 of these 14 Dot-ELISA-positive/MA test-negative sera were acute samples from patients whose later sera were MA test-positive. Positive reactions in the IgM Dot-ELISA occurred in 2 of 30 control, 4 of 10 Lyme disease, 1 of 11 relapsing fever, and 1 of 8 yaws sera; 10 syphilis patient sera were nonreactive. The IgM-specific Dot-ELISA appears to be sensitive and specific for the serodiagnosis of acute leptospirosis. In addition, this rapid test is inexpensive, simple to perform, utilizes minute volumes of killed leptospiral antigen and is easily adaptable to field use.

Field evaluation of an enzyme-linked immunosorbent assay (ELISA) for Plasmodium falciparum sporozoite detection in anopheline mosquitoes from Kenya.

An enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody that recognizes a repetitive epitope on the circumsporozoite protein of Plasmodium falciparum was used in Kenya to assess malaria infections in Anopheles gambiae s.l. and An. funestus. The ELISA confirmed that 88% of 44 sporozoite-positive gland dissections were P. falciparum. The ELISA infection rate of 18.6% (n = 736) for individually tested mosquitoes for both species was significantly higher than the 10.4% (n = 537) salivary gland sporozoite rate determined by dissection. This difference was due to ELISA detection of medium and large sized oocysts on the midguts of infected mosquitoes which did not contain salivary gland sporozoites. From a series of 379 Anopheles that were cut at the thorax, ELISA tests on "head" and "body" portions showed that 29.5% of 95 positive mosquitoes contained circumsporozoite antigen in the body portion in the absence of salivary gland infections. This field evaluation demonstrates that the ELISA can most accurately be used to estimate sporozoite rates by cutting mosquitoes at the thorax and testing anterior portions.

Malaria transmitted to humans by mosquitoes infected from cultured Plasmodium falciparum.

Malaria was transmitted to six normal human volunteers by mosquitoes infected from cultured gametocytes of Plasmodium falciparum. This method, which offers advantages over other methods of infecting volunteers, will be useful for evaluating the efficacy of human malaria vaccines.

Evaluation of promastigote and amastigote antigens in the indirect fluorescent antibody test for American cutaneous leishmaniasis.

Leishmania braziliensis panamensis promastigotes, temperature-induced in vitro-cultivated amastigotes, Vero cell-derived amastigotes, and rodent lesion-derived amastigotes were evaluated as antigens in the indirect immunofluorescent antibody (IFA) test for American cutaneous leishmaniasis. Test sensitivity was determined using sera from 34 U.S. soldiers with leishmaniasis diagnosed by demonstrating parasites in their skin lesions. Sera were collected from 3 to 24 months after exposure to Leishmania. Positive IFA reactions among patient sera were 82% with promastigotes or lesion amastigotes, 79% with in vitro amastigotes, and 76% with Vero cell amastigotes (P = N.S.). Positive titers ranged from 1:8 to 1:128 using all antigens. Test specificity was determined with 30 sera from healthy individuals. False positive reactions ranged from 0-5% depending on the antigen and all titers were less than or equal to 1:8. Test cross-reactivity was assessed with 47 sera from patients with other diseases. Depending on the antigen, cross-reactions occurred with sera from patients with Chagas' disease, lupus erythematosus, malaria, toxoplasmosis and amebiasis. None of the antigens cross-reacted with sera from patients with viral hepatitis, coccidioidomycosis, syphilis, schistosomiasis, and trichinosis. In replicate experiments, 99-100% of the sera varied no more than +/- 1 titer dilution. As sensitivity, specificity, cross-reactivity, and reproducibility of the four antigens were statistically similar, promastigotes, which can be easily and economically cultured in large numbers in vitro are recommended for use in the IFA test for American cutaneous leishmaniasis.

Restriction endonuclease analysis of Leishmania kinetoplast DNA characterizes parasites responsible for visceral and cutaneous disease.

The kinetoplast DNA (kDNA) from promastigotes of Leishmania responsible for Old and New World cutaneous and visceral disease was characterized to determine if species and strains causing similar or different diseases could be identified. Restriction enzymes were used to digest kDNA into fragments that were separated into characteristic banding patterns after electrophoresis in agarose or linear gradient polyacrylamide gels. Hybridization was conducted with a 32P-kDNA probe and kDNA fragments transferred from agarose gels to nitrocellulose paper. Leishmania species causing cutaneous diseases in the New and Old Worlds all had different kDNA digest patterns. Visceralizing Leishmania from the New and Old Worlds also had different kDNA restriction fragment patterns although Leishmania donovani parasites with similar fragment patterns were isolated from several humans from central Kenya. Nucleotide sequences were shared among kDNA networks from L. donovani, Leishmania d. chagasi, Leishmania d. infantum, Leishmania tropica, and Leishmania major as determined by hybridization with a 32P-kDNA probe from L. donovani. However, no hybridization was detected between the L. donovani 32P-kDNA probe and kDNA from Leishmania aethiopica or Leishmania braziliensis panamensis. Leishmania characterization results for the same isolates from the published literature were compared and kinetoplast DNA analysis was found to be one of the most sensitive procedures for species and strain identification.

Proteosome-hydrophobic 'foot' malaria peptide vaccines for Plasmodium falciparum and P. vivax.

The immunogenicity of synthetic peptides representing the repeating portions of circumsporozoite proteins of Plasmodium sporozoites was greatly increased by complexing them to proteosomes via hydrophobic moieties added to their amino termini. Proteosomes have been used safely in people in the development of meningococcal vaccines and therefore proteosome-peptide vaccines are prime candidates for use against malaria.