Age-grading the biting midge Culicoides sonorensis using near-infrared spectroscopy.

Age-grading of insects is important in the control and monitoring of both insect populations and vector-borne diseases. Microscopy and morphological techniques exist to age-grade most blood-feeding flies, but these techniques are laborious, often destructive to the insects, and slow. Near-infrared spectroscopy (NIRS) can be automated and is a non-destructive technique for age-grading. We applied NIRS techniques to age-grade females of the biting midge, Culicoides sonorensis Wirth & Jones (Diptera: Ceratopogonidae), the vector of bluetongue and other arboviruses in North America. Female flies of five known age cohorts (1, 3, 6, 9 and 12 days post-emergence) from three laboratory colonies were used. The data indicate that NIRS can be used to differentiate age groups of C. sonorensis.

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.

Circumsporozoite protein heterogeneity in the human malaria parasite Plasmodium vivax.

Phenotypic heterogeneity in the repetitive portion of a human malaria circumsporozoite (CS) protein, a major target of candidate vaccines, has been found. Over 14% of clinical cases of uncomplicated Plasmodium vivax malaria at two sites in western Thailand produced sporozoites immunologically distinct from previously characterized examples of the species. Monoclonal antibodies to the CS protein of other P. vivax isolates and to other species of human and simian malarias did not bind to these nonreactive sporozoites, nor did antibodies from monkeys immunized with a candidate vaccine made from the repeat portion of a New World CS protein. The section of the CS protein gene between the conserved regions I and II of a nonreactive isolate contained a nonapeptide repeat, Ala-Asn-Gly-Ala-Gly-Asn-Gln-Pro-Gly, identical at only three amino acid positions with published nonapeptide sequences. This heterogeneity implies that a P. vivax vaccine based on the CS protein repeat of one isolate will not be universally protective.

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.

Immunogenicity of synthetic peptides from circumsporozoite protein of Plasmodium falciparum.

In a study of recombinant proteins that might be useful in developing a vaccine against malaria, synthetic peptides from the circumsporozoite (CS) protein of Plasmodium falciparum were found to be immunogenic for mice and rabbits. Antibody to peptides from the repeating region of the CS protein recognized native CS protein and blocked sporozoite invasion of human hepatoma cells in vitro. Antibodies to peptides from regions I and II had no biologic activity, although antibody to region I recognized processed CS protein by Western blot analysis. These data support the feasibility of developing a vaccine against the sporozoite stage of the malaria parasite by using synthetic peptides of the repeating region of the CS protein conjugated to a carrier protein.

Naturally acquired antibodies to sporozoites do not prevent malaria: vaccine development implications.

The first human vaccines against the malaria parasite have been designed to elicit antibodies to the circumsporozoite protein of Plasmodium falciparum. However, it is not known whether any level of naturally acquired antibodies to the circumsporozoite protein can predict resistance to Plasmodium falciparum malaria. In this study, 83 adults in a malaria-endemic region of Kenya were tested for circumsporozoite antibodies and then treated for malaria. They were monitored for the development of new malaria infections for 98 days. Antibody levels, as determined by four assays in vitro, were indistinguishable between the 60 individuals who did and the 23 who did not develop parasitemia during follow-up, and there was no apparent relation between day of onset of parasitemia and level of antibodies to circumsporozoite protein. Unless immunization with sporozoite vaccines induces antibodies that are quantitatively or qualitatively superior to the circumsporozoite antibodies in these adults, it is unlikely that such antibodies will prevent infection in areas with as intense malaria transmission as western Kenya.

Efficacy of murine malaria sporozoite vaccines: implications for human vaccine development.

As part of a study of potential vaccines against malaria, the protective efficacy of sporozoite subunit vaccines was determined by using the Plasmodium berghei murine malaria model. Mice were immunized with recombinant DNA-produced or synthetic peptide-carrier subunit vaccines derived from the repetitive epitopes of the Plasmodium berghei circumsporozoite gene, or with radiation-attenuated sporozoites. Immunization with subunit vaccines elicited humoral responses that were equivalent to or greater than those elicited by irradiated sporozoites, yet the protection against sporozoite challenge induced by either of the subunit vaccines was far less than that achieved by immunization with attenuated sporozoites. Passive and adoptive transfer studies demonstrated that subunit vaccines elicited predominantly antibody-mediated protection that was easily overcome whereas irradiated sporozoites induced potent cell-mediated immunity that protected against high challenge doses of sporozoites. These studies indicate that new strategies designed to induce cellular immunity will be required for efficacious sporozoite vaccines.

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.

Phagocytosis of liposomes by macrophages: intracellular fate of liposomal malaria antigen.

Liposomes containing a synthetic recombinant protein were phagocytosed by macrophages, and the internalized protein was recycled to the cell surfaces where it was detected by enzyme-linked immunosorbent assay. The transit time of the liposome-encapsulated protein from initial phagocytosis of liposomes to appearance of protein on the surfaces of macrophages was determined by pulse-chase experiments. The macrophages were pulsed with liposomes containing protein and chased with empty liposomes, and vice versa. The amount and rate of protein antigen expression at the cell surfaces depended on the quantity of encapsulated protein ingested by the macrophages. Although liposomes were rapidly taken up by macrophages, the liposome-encapsulated protein was antigenically expressed for a prolonged period (at least 24 h) on the cell surface. Liposomes were visualized inside vacuoles in the macrophages by immunogold electron microscopy. The liposomes accumulated along the peripheries of the vacuoles and many of them apparently remained intact for a long time (greater than 6 h). However, nonliposomal free protein was also detected in the cytoplasm surrounding these vacuoles, and it was concluded that the free protein in the cytoplasm was probably en route to the macrophage surface. Exposure of the cells to ammonium chloride did not inhibit the appearance of liposomal antigenic epitopes on the cell surface, and this suggests that expression of the liposomal antigenic epitopes at the surface was not a pH-sensitive phenomenon. There was no significant effect of a liposomal adjuvant, lipid A, on the rate or extent of surface expression of the liposomal protein.

Regulation and trafficking of three distinct 18 S ribosomal RNAs during development of the malaria parasite.

The human malaria parasite Plasmodium vivax has been shown to regulate the transcription of two distinct 18 RNAs during development. Here we show a third and distinctive type of ribosome that is present shortly after zygote formation, a transcriptional pattern of ribosome types that relates closely to the developmental state of the parasite and a phenomenon that separates ribosomal types at a critical phase of maturation. The A-type ribosome is predominantly found in infected erythrocytes of the vertebrate and the mosquito blood meal. Transcripts from the A gene are replaced by transcripts from another locus, the O gene, shortly after fertilization and increase in number as the parasite develops on the mosquito midgut. Transcripts from another locus, the S gene, begins as the oocyst form of the parasite matures. RNA transcripts from the S gene are preferentially included in sporozoites that bud off from the oocyst and migrate to the salivary gland while the O gene transcripts are left within the oocyst. Although all three genes are typically eukaryotic in structure, the O gene transcript, described here, varies from the other two in core regions of the rRNA that are involved in mRNA decoding and translational termination. We now can correlate developmental progression of the parasite with changes in regions of rRNA sequence that are broadly conserved, where sequence alterations have been related to function in other systems and whose effects can be studied outside of Plasmodium. This should allow assessment of the role of translational control in parasite development.

Repellents: past, present, and future.

The use of repellents in protecting people against vector-borne diseases is predicated on the assertion that reducing human/vector contact will reduce the incidence of disease. The methods that have been used in developing countries have been simple to apply and relatively cheap. This article will discuss the use of repellents for protection against vector-borne disease in Southeast Asia and the Southwest Pacific region.

Pseudomonas aeruginosa outer membrane protein OprF as an expression vector for foreign epitopes: the effects of positioning and length on the antigenicity of the epitope.

OprF, the major outer membrane (OM) protein of Pseudomonas aeruginosa, has been proposed to be comprised of a series of beta-strands separated by periplasmic or surface-exposed loop regions. In this study, a simple malarial epitope was used to demonstrate that OprF can be used as an expression vector to present foreign peptide sequences, namely, the 4-amino-acid (aa) repeating epitope (Asn-Ala-Asn-Pro = NANP) of the circumsporozoite protein of the human malarial parasite Plasmodium falciparum. Eight permissive sites, that allowed the expression and surface exposure of the malarial epitope, were identified throughout OprF. Using a monoclonal antibody (mAb) specific for the malarial epitope, we investigated the effects of positioning and length of the epitope on its antigenicity in the OprF expression vector system. It was demonstrated that the malarial epitope inserted at aa26 was significantly more reactive with the epitope-specific mAb (i.e., more antigenic) when assayed in the context of whole cells whereas those at aa213 and aa290 were more antigenic when assayed in the OM. The malarial epitope inserted at aa188 and aa196 was moderately antigenic, while this epitope inserted at aa215 and aa310 showed low antigenicity with the same mAb in both whole cell and OM assays. For two insertion sites, aa26 and aa213, we demonstrated that the insertion of multiple copies of the epitope enhanced reactivity with the malarial epitope-specific mAb. These data are discussed with respect to the local OprF sequences into which the epitope was inserted.

Use of fluorodinitrobenzene to identify monoclonal antibodies which are suitable for conjugation to periodate-oxidized horseradish peroxidase.

During the recent development of a double antibody enzyme-linked immunosorbent assay for detecting malaria sporozoites in mosquitoes it was found that a large number of potentially useful monoclonal antibodies lost their capacity for binding antigen after conjugation to periodate-oxidized horseradish peroxidase (HPO). Since HPO reacts with primary amino groups, we used a simple chemical reaction with fluorodinitrobenzene (FDNB) to determine if the loss of antigen binding was due to the requirement for unmodified primary amino groups in the binding site. FDNB-treated antibodies which reacted with antigen in an indirect fluorescent antibody assay (IFA) also yielded successful HPO-antibody conjugates. Conversely, those antibodies which did not react with antigen after treatment with FDNB failed to produce useful HPO-antibody conjugates. These data suggest that conjugation of oxidized HPO to primary amines in or near the antigen-combining site yields conjugates in which the antibody is inactive. Use of FDNB to predict the suitability of antibodies for conjugation to periodate-oxidized HPO may save a considerable amount of time over randomly selecting antibodies for conjugation and testing.

Isolation of a substance from the mosquito that activates Plasmodium fertilization.

We have isolated a small, heat stabile, hydrophilic molecule from the gut lumen of unfed, female Anopheles stephensi that is a potent inducer of gametogenesis in Plasmodium falciparum and P. gallinaceum at a hydrogen ion concentration, pH 7.4, that normally suppresses activation. This gamete activation factor (GAF) was purified using reverse phase high performance liquid chromatography and determined to have a major ion m/z of 206.1 by low resolution electrospray mass spectrometry. The molecule, which was also found in the heads of both female and male A. stephensi, absorbed light in the ultraviolet region at three maxima (lambda(max) = 213, 245 and 350 nm); the 245/350 nm absorbance ratio was 7.0. The structure of the molecule and its normal function in the mosquito are not yet known, but in a sample of diverse insect species, extracts from those that feed on blood were bioactive. We propose that GAF is the previously observed malaria exflagellation factor (MEF).

Transition of Plasmodium vivax ribosome types corresponds to sporozoite differentiation in the mosquito.

Two distinct small subunit ribosomal RNA (SSUrRNA) genes were amplified from the genomic DNA of Plasmodium vivax. Comparison of the two coding sequences reveals an overall divergence of 14.5% and most differences are clustered into the regions known to diverge rapidly in all eukaryotic SSUrRNAs. Oligonucleotides complementary to unique sequences of each gene have been used to distinguish the transcripts expressed either at schizogony in human blood (A gene) or at sporogony in the mosquito (C gene). These oligonucleotides were also used to monitor turnover of ribosomes during parasite development in mosquitoes. Transcripts of the A gene were predominant in the infected human blood and engorged mosquitoes but disappeared within 24 h after feeding. Expression of the C gene in mosquitoes was not detected until day 6 after the blood meal. A period of rapid accumulation of the C type rRNA from day 6 to day 8 corresponds to differentiation of individual sporozoites within the oocyst. Possible functional implications relating to the timing of this transition are discussed.

The distribution of circumsporozoite protein (CS) in Anopheles stephensi mosquitoes infected with Plasmodium falciparum malaria.

We monitored the distribution of Plasmodium falciparum circumsporozoite protein (CS) in Anopheles stephensi using an immunohistochemical method. An alkaline phosphatase-labeled monoclonal antibody, specific for the CS protein of P. falciparum, was incubated with tissue sections from infected and non-infected mosquitoes. Sections were stained for phosphatase activity using a new fuchsin/naphthol AS-BI phosphate capture system. Distribution of the CS protein in mosquitoes was dependent on the time after post-infective blood meal. CS protein was first detected in immature oocysts on the mosquito midgut. As oocysts differentiated to mature sporoblasts, detectable CS protein increased. Between 11-16 days post infective blood meal, CS protein was detected on the surface of sporozoites that were released into the hemolymph from oocysts. Although sporozoites were found throughout the hemocoel, they were most frequently associated with the salivary glands and flight muscle. Once in the salivary glands, sporozoites massed into bundles. The amount of CS protein associated with bundles of sporozoites was highly variable.

Analysis of malaria parasite RNA from decade-old Giemsa-stained blood smears and dried mosquitoes.

We have analyzed RNA isolated from recently prepared and historically preserved slides containing smears of Plasmodium-infected blood. We found that slides preserved as long as 20 years can yield RNA that is a suitable template for polymerase chain reaction (PCR) amplification. Mosquitoes that have been stored for years under ambient temperature can also be used as an RNA source. The RNA amplification from slide-derived material is shown to be dependent upon the addition of reverse transcriptase and the resultant products are specific to the developmental state of the parasite. Amplification of ribosomal RNA with primers conserved for Plasmodium and hybridization with species-specific probes provide a general, unbiased method for species determination. Messenger RNA transcripts from slides also appear to serve as templates. The procedure may add complementary information to that derived from microscopic examination of Giemsa-stained blood smears including species identification, variant antigen identification and drug resistance status.

The epidemiology of Plasmodium vivax circumsporozoite protein polymorphs in Thailand.

Enzyme-linked immunosorbent assays (ELISAs) highly specific for the characteristic repeat units of the circumsporozoite proteins of the VK 247 and VK 210 polymorphs of Plasmodium vivax were used to test sporozoites produced by feeding mosquitoes on 1,711 human volunteers presenting at four locations in Thailand over five years. There was no evidence for the existence of any polymorph other than the two already described. Based on the ELISAs, the overall prevalence of the VK 247 type was 29.5%, including those found mixed with VK 210. Relative proportions of VK 210 and VK 247 differed between collection sites. At all places, the ratio of VK 210 to VK 247 was significantly higher at the end of the nontransmission season than it was later during the annual monsoon, suggesting that there may be intrinsic biological differences between the polymorphs that affect their survival.

Field evaluation of an enzyme-linked immunosorbent assay for estimating the sporozoite rate in Anopheles albimanus.

We have verified for specimens of Anopheles albimanus that an enzyme-linked immunosorbent assay (ELISA) used to assess Plasmodium vivax and P. falciparum sporozoite antigen rates gives results comparable to the salivary gland dissection method for estimating sporozoite rates. For 14,150 adults of An. albimanus, captured at five locations in Guatemala, we report sporozoite antigen rates of 0.03-0.57%, which correlate with the malaria prevalences at the study sites. We also present data that suggest that specimens of An. albimanus for the ELISA can be obtained more efficiently by cattle corral collections than by the human bait capture method.

Short report: prevention of Schistosoma mansoni infections in mice by the insect repellents AI3-37220 and N,N-diethyl-3-methylbenzamide.

N,N-diethyl-3-methylbenzamide (DEET) has recently been reported to kill cercariae of Schistosoma mansoni in vitro. In addition, it blocked cercarial entry into mouse tail skin. We confirmed these results and compared the efficacy of DEET to a second insect repellent, 1-(3-Cyclohexen-1-yl-carbonyl)-2-methylpiperidine (AI3-37220), in preventing S. mansoni infections in mice. Both AI3-37220 and DEET conferred 100% protection against S. mansoni infection via percutaneous exposure to cercariae.