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.

The evolution of ultrafast electron microscope instrumentation.

Extrapolating from a brief survey of the literature, we outline a vision for the future development of time-resolved electron probe instruments that could offer levels of performance and flexibility that push the limits of physical possibility. This includes a discussion of the electron beam parameters (brightness and emittance) that limit performance, the identification of a dimensionless invariant figure of merit for pulsed electron guns (the number of electrons per lateral coherence area, per pulse), and calculations of how this figure of merit determines the trade-off of spatial against temporal resolution for different imaging modes. Modern photonics' ability to control its fundamental particles at the quantum level, while enjoying extreme flexibility and a very large variety of operating modes, is held up as an example and a goal. We argue that this goal may be approached by combining ideas already in the literature, suggesting the need for large-scale collaborative development of next-generation time-resolved instruments.

Crystallization kinetics of the phase change material GeSb6Te measured with dynamic transmission electron microscopy.

GeSb6Te is a chalcogenide-based phase change material that has shown great ptoential for use in solid-state memory devices. The crystallization kinetics of amorphous thin films of GeSb6Te during laser crystallization were followed with dynamic transmission electron microscopy, a photo-emission electron microscopy technique with nanosecond-scale time resolution. Nine-frame movies of crystal growth were taken during laser crystallization. The nucleation rate is observed to be very low and the growth rates are very high, up to 10.8 m s(-1) for amorphous as-deposited films and significantly higher for an amorphous film subject to sub-threshold laser annealing before crystallization. The measured growth rates exceed any directly measured growth rate of a phase change material. The crystallization is reminiscent of explosive crystallization of elemental semiconductors both in the magnitude of the growth rate and in the resulting crystalline microstructures.

Computer-assisted multiple categorization of absorbance values in ELISA through pictorial emulation of 96-well plates.

A simple algorithm is proposed by which multiple categorization of absorbance values from ELISA plates is performed under a microcomputer control. The printed output is a pictorial emulation of a 96-well plate with the color intensities represented for each reaction. Although the method is presented as a colorimeter computer interfaced system, a provision for manual entry of absorbance values via keyboard is also included. Simulation is based solely on the magnitude of absorbance values. Therefore, it is possible to utilize any enzyme/substrate combination within the range of filters of the colorimeter. We have tested the present system for titration of anti-malarial antibodies in human serum and for the screening of mouse hybridoma culture supernatants.

Two Plasmodium falciparum merozoite surface polypeptides share epitopes with a single Mr 185 000 parasite glycoprotein.

The malarial parasite Plasmodium falciparum synthesizes a major glycoprotein (gp) of Mr 185 000 during its asexual blood cycle. Immunoprecipitation of [35S]methionine- or [3H]glucosamine-labeled schizont antigens indicated that two groups of polypeptides were distinguished with anti-gp 185 mouse monoclonal antibodies: group A was composed of glycosylated molecules of Mr 185 000, 120 000, 90 000, 88 000, 46 000, and 40 000 while group B contained, in addition to gp 185, polypeptides of Mr 152 000, 106 000 and 83 000. The latter polypeptides lacked detectable amounts of radiolabeled saccharide. The smaller Mr polypeptides were specifically immunoprecipitated and not merely coprecipitated with gp 185. Our results suggest that gp 185 contains at least two structurally distinct domains which may be processed independently into either the group A or group B polypeptides. Although gp 185 may not be a merozoite surface protein, representative group A and group B-specific monoclonal antibodies bound to surface antigens of the merozoite as demonstrated by immunolabeling followed by electron microscopy. Therefore, at least one group A antigen and one group B antigen appeared to be on the extracellular surface of the merozoite. The proteins found in immunoprecipitates after both (1) sonication in aqueous medium and ultracentrifugation and (2) solubilization and phase separation of parasite molecules with Triton X-114 suggested that the group A and group B polypeptides and glycoproteins are either soluble or peripheral membrane proteins. Some of these, therefore, may be components of the surface coat of the merozoite.

Structure of the circumsporozoite gene of Plasmodium malariae.

The sequence of the gene encoding the circumsporozoite protein of Plasmodium malariae was determined. The central immunodominant region of the protein consists of 45 copies of the sequence Asn-Ala-Ala-Gly and 6 copies of the sequence Asn-Asp-Ala-Gly. The CSP of the monkey parasite Plasmodium brasilianum contains the same repetitive sequences. Further comparison of the two genes in regions outside the immunodominant domains reveals only three nucleotide differences and each results in an amino acid change. One is centered in a putative T-cell determinant bearing region, the second is in the putative liver binding site, and the third is part of a degenerate repeat at the start of the immunodominant region.

Immunization of mice with Trypanosoma rhodesiense exposed to ultraviolet irradiation.

Exposure time of Trypanosoma rhodesiense as short as 1 minute to ultraviolet (U.V.) light prevents the organisms from causing infection. Live trypanosome challenge of mice immunized with U.V.-irradiated trypanosomes results in sterile immunity. This allows a method for the induction of protective immunity to experimental trypanosomiasis which can be performed in most laboratories using U.V. germicidal lamps found in sterile hoods.

Is Anopheles mascarensis a new malaria vector in Madagascar?

Anopheles mascarensis De Meillon, 1947, a mosquito that is native to Madagascar, is reported for the first time to act as a vector of Plasmodium falciparum malaria. From September 1989 to March 1990, 2, 499 An. mascarensis specimens from different regions of Madagascar were tested by an enzyme-linked immunosorbent assay (ELISA), using monoclonal antibodies against the circumsporozoite (CS) protein of the four human species of Plasmodium. The salivary glands of 237 specimens were also dissected. Fourteen of 1,864 specimens obtained from Sainte Marie island on the Malagasy east coast were found by ELISA to be positive for the CS protein of P. falciparum. In addition, two of 237 specimens that were dissected were observed to have sporozoites in the salivary glands. These sporozoites were identified as P. falciparum by ELISA. In the other regions studied, no positive specimens were found. Due to observed behavioral differences between east coast and highland populations of An. mascarensis, the possible presence of a species complex is discussed.

Concentration of Plasmodium ovale- and Plasmodium vivax-infected erythrocytes from nonhuman primate blood using Percoll gradients.

Plasmodium vivax and Plasmodium ovale schizont-infected erythrocytes were separated from peripheral blood by centrifugation using discontinuous Percoll (colloidal silica) gradients. Infected Aotus monkey or chimpanzee blood was diluted and placed on a discontinuous gradient containing 30%, 40%, 45%, and 50% Percoll (v/v in media) layers before centrifugation at 1,450 X g. Parasitized erythrocytes were concentrated to greater than 95% schizont-infected cells in two bands that contained an average of one leukocyte per 500 infected cells. Mononuclear cells and trophozoites were isolated in another band and noninfected red cells, ring-infected cells, and granulocytes were pelleted to the bottom. The yield of parasitized erythrocytes ranged from 50% to close to 100% of the estimated number of infected cells in the original whole blood. Use of this Percoll procedure results in a high yield of concentrated parasitized erythrocytes and separation of these cells from host white blood cells.

Repeated immunogenic amino acid sequences of Plasmodium species share sequence homologies with proteins from humans and human viruses.

The use of recombinant peptides based upon the repeated amino acid sequences of Plasmodium has been proposed for malaria vaccines. By reducing homologies of such peptide vaccines to host proteins, the possibility of autoimmune complications may be reduced, and the effective immune response may be enhanced. The Wilbur and Lipman Wordsearch algorithm was used to identify homologous amino acid sequences between tandemly repeated Plasmodium amino acid sequences and the human and human viral sequences compiled in the National Biomedical Research Foundation database. Six published repetitive immunogenic amino acid sequences from the circumsporozoite (CS) antigen, ring-infected erythrocyte surface antigen (RESA), soluble (S) antigen, and falciparum interspersed repetitive antigen (FIRA) of P. falciparum, and the CS protein of P. vivax, were analyzed by computer. Matches of at least 4 amino acids were found for all sequences. In the database, 29 matches were found for human proteins and 26 matches were found for human viruses with the 6 antigen sequences. Most of the matched proteins, and many of the matched human viruses, are found in blood. The biological significance of these matches remains to be clarified.

Immunization of owl monkeys with a combination of Plasmodium falciparum asexual blood-stage synthetic peptide antigens.

A mixture of 3 synthetic peptides (35.1, 55.1, and 83.1) corresponding to portions of the 35 kDa, 55 kDa, and 83 kDa proteins from the asexual blood stages of Plasmodium falciparum and a polymer of a syntheic peptide incorporating the 3 individual peptides (SPf66) were tested as candidate malaria vaccine antigens in Aotus nancymai. Monkeys were immunized with combinations of the 3 peptides from 2 separate sources (Centers for Disease Control [CDC], Atlanta, GA or Colombia) or with the synthetic polymer. Animals immunized with a combination of the 3 peptides from CDC had higher antibody titers to the 35.1 and 55.1 peptides than to the 83.1 peptide. Monkeys immunized with a combination of the 3 peptides produced in Colombia developed higher levels of antibody to the 55.1 than to the 83.1 and 35.1 peptides. Animals immunized with the polymer produced detectable antibodies to the 55.1 peptide alone. Following challenge with P. falciparum, no differences were observed between the 3 vaccine groups and 2 control groups with respect to the number of animals with parasitemias greater than or equal to 10%. The inconsistency of serologic response to all 3 peptides in these animals contrasted with previous trials performed in Colombia where the monkeys developed high antibody titers against the 3 peptides and were protected against the experimental infection.

Isolation and characterization of a new serodeme of Trypanosoma rhodesiense.

The isolation and characterization of a new serodeme of Trypanosoma brucei rhodesiense is described. A clone of organisms derived from a human infection produced chronic infections in mice. Additional clones of differing antigenic specificities were isolated from peaks of parasitemia which occurred in these mice. The variable antigen types (VATs) of these clones were determined by agglutination, immunofluorescence, and protection of actively immunized mice. Thirteen distinct VATs were isolated and designated Walter Reed Army Trypanozoon antigenic types. The described methodology and reagents, together with the chronicity of the infection produced in mice by this serodeme, provide a model for further study of immunopathology and antigenic variation in African trypanosomiasis. The use of these reagents in determining the incidence of VATs in an endemic area may allow an assessment of the feasibility of immunoprophylaxis.

Detection of Plasmodium falciparum using a synthetic DNA probe.

A labeled synthetic polynucleotide representing a repetitive sequence from Plasmodium falciparum was hybridized with genomic DNA spotted on nitrocellulose. After an overnight exposure, 0.1 ng of P. falciparum DNA was specifically detected and 0.01 ng was detected after an exposure of 1 week. The synthetic probe showed no cross-hybridization with host DNA or with DNA isolated from other species in the phylum Apicomplexa, P. vivax and Babesia species. Since synthetic DNA is easily prepared, the observed sensitivity and specificity suggests that synthetic DNA probes would be generally useful in diagnosis.

Age-specific prevalence of antibody to a synthetic peptide of the circumsporozoite protein of Plasmodium falciparum in children from three villages in Kenya.

The presence of antibody to the repeating epitope of the circumsporozoite protein of Plasmodium falciparum was determined in children 1 month to 10 years old from three villages in western Kenya using the synthetic peptide (PNAN)5 in an enzyme-linked immunosorbent assay. The percentage of antibody-positive children increased with age and differed in the three villages. The village with the lowest percentage of antibody-positive children had the lowest percentage of infections as determined by detection of blood stage parasites. The villages also differed in the age at which antibody first appeared. In one village, only 12% of the children had antibody by the age of 5; while in the other two villages, 60% and 73% had antibody by 4 years of age.

Malaria transmission and vector biology in Manarintsoa, high plateaux of Madagascar.

To evaluate the factors which determine the transmission level of falciparum malaria, entomological and parasitological surveys were conducted from October 1988 to February 1990 in Manarintsoa in the central highland plateaux of Madagascar. Mosquitoes were collected for 928 man-nights in pit shelters and indoor resting sites. Malaria vectors were Anopheles arabiensis and An. funestus, with no evidence of the presence of An. gambiae sensu stricto. Vectors were mainly exophilic and zoophilic. The index of stability was less than 1.5. The sporozoite rate was 0.11 for An. gambiae sensu lato and 0.47 for An. funestus. The transmission level was low, with an inoculation rate of 0.91 infective bites/person/year and an infection risk of 0.62. Malaria transmission occurs 7 months of the year in this area, from November to May. Human parasite rates fluctuated from 29% in October to 53% in May.

Monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for detection of Plasmodium malariae sporozoites in mosquitoes.

A monoclonal antibody specific for a repeated epitope of the circumsporozoite protein of Plasmodium malariae sporozoites has been used to develop a two-site, single antibody-based enzyme-linked immunosorbent assay that can detect P. malariae sporozoites in mosquitoes. The assay uses a purified monoclonal antibody produced against sporozoites of the Uganda I/CDC strain of P. malariae to capture the antigen and the same monoclonal antibody labeled with horseradish peroxidase as the detector. Sporozoites have been detected in laboratory-infected mosquitoes stored at room temperature in the presence of a desiccant for as long as 18 months. The detection limit of the assay is approximately 50 P. malariae sporozoites per test well. Cross-reaction has not been observed with mosquitoes infected with P. falciparum, P. vivax, or P. ovale sporozoites.

Monoclonal antibody characterization of Plasmodium falciparum antigens.

We characterized a set of eight monoclonal antibodies produced against Plasmodium falciparum. In an indirect fluorescent antibody assay the antibodies produced small dots of fluorescence in schizonts and individual merozoites. This merozoite-associated dot reactivity occurred with 21 different strains of P. falciparum, but there was no reactivity with other human, nonhuman primate, or rodent Plasmodium species. Three of the monoclonal antibodies precipitated proteins of Mr 145,000, 135,000, and 104,000. Five of the monoclonal antibodies precipitated proteins of Mr 78,000, 63,000, 42,000, and 40,000.

Proteins responsible for a punctate fluorescence pattern in Plasmodium falciparum merozoites.

Mouse monoclonal antibodies (McAbs) have been used to characterize the proteins of the asexual erythrocytic cycle of Plasmodium falciparum. Three different McAbs react with antigens of the schizont and extracellular merozoite to give a punctate fluorescence pattern. In many cases, such areas of fluorescence were composed of two adjacent, fluorescent bodies; these were distinct from the nuclei. In contrast, McAbs which bound to the ring-stage parasite were not localized, but were diffusely distributed within or around the ring-stage parasite. These McAbs immunoprecipitated five prominent, 35S-methionine-labeled schizont proteins (p) of Mr 82K, 70K, 67K, 39K, and 37K. Only p82, p39, and p37 were immunoprecipitated from schizont-labeled ring-stage parasites; thus, it appears that p70 and p67 are modified, degraded, or secreted some time between intracellular merozoite maturation and erythrocyte invasion.

Significance of circumsporozoite-specific antibody in the natural transmission of Plasmodium falciparum, Plasmodium vivax, and Plasmodium malariae in an aboriginal (Orang Asli) population of central peninsula Malaysia.

Two hundred and seventy-five Orang Asli volunteers living in nine villages in the Pos Legap Valley of Perak State, peninsular Malaysia, participated in a prospective study designed to characterize the epidemiological, parasitological, and entomological characteristics of Plasmodium falciparum, P. vivax, and P. malariae malaria transmission. Prevalence rates for the three plasmodial species at initiation of the study ranged from 56% in the 0-4-year-old age group to 0% in individuals over the age of 40. Entomological surveys were conducted, enabling us to determine mosquito salivary gland-positive rates and entomological inoculation rates of 1.2 infectious mosquito bites per person per month for P. falciparum, 2.4 for P. vivax, and 0.3 for P. malariae. Cumulative incidence rates over the 16 weeks of the study, following radical cure of all volunteers, were 22.5% for P. falciparum, 12.7% for P. vivax, and 1.5% for P. malariae. The median baseline antibody titer against the immunodominant repetitive B cell epitope of P. falciparum or P. vivax circumsporozoite protein was significantly higher for volunteers who did not become parasitemic. Volunteers were selected for further study if they had evidence of being challenged with P. falciparum sporozoites during the study, based on a two-fold or greater increase in antibody titer against the immunodominant repetitive B cell epitope of the circumsporozoite protein. Resistance to infection was seen in six of 10 individuals who had high (greater than 25 OD units) baseline ELISA titers, compared with only three of 24 individuals who had low baseline ELISA titers (chi 2 P less than 0.02). A similar analysis for P. vivax did not show a significant correlation.

Detection of antibodies in human sera to the repeating epitope of the circumsporozoite protein of Plasmodium falciparum using the synthetic peptide (NANP)3 in an enzyme-linked immunosorbent assay (ELISA).

An enzyme-linked immunosorbent assay (ELISA) was developed to detect antibody in human sera to a synthetic peptide, Asn-Ala-Asn-Pro (NANP)3, derived from the repeating amino acid sequence found in the surface circumsporozoite protein of Plasmodium falciparum sporozoites. One hundred four sera from U.S. residents were used to determine a cut-off value for reactivity. Test sera were considered reactive when the absorbance was greater than that at the 95th percentile of the control sera. Sera from 112 Kenyans living in an area of holoendemic malaria transmission were tested. Of the total number of sera, 65% had detectable antibody to (NANP)3. The percentage of reactive sera increased from 41% in sera from children under 4 years of age to 85% in sera from adults 20 to 39 years of age. The high exposure to malaria parasites of the Kenyans was reflected in indirect fluorescent antibody assay titers to blood stage P. falciparum parasites. All of the Kenyan sera had antibody present at titers greater than 1:256.