Generation of an antibody that recognizes Plasmodium chabaudi cysteine protease (chabaupain-1) in both sexual and asexual parasite life cycle and evaluation of chabaupain-1 vaccine potential.

Malaria cysteine proteases have been shown to be immunogenic and are being exploited as serodiagnostic markers, drug and vaccine targets. Several Plasmodium spp. cysteine proteases have been described and the best characterized of these are the falcipains, a family of papain-family enzymes. Falcipain-2 and falcipain-3 act in concert with other proteases to hydrolyze host erythrocyte hemoglobin in the parasite food vacuole. Falcipain-1 has less similarity to the other falcipains and its physiological role in parasite asexual blood stage still remains uncertain. Immunolocalization studies using an antibody developed against the Plasmodium chabaudi recombinant chabaupain-1, the falcipain-1 ortholog, were performed confirming its cellular localization in both erythrocyte and mosquito ookinete stage. Immunostaining of chabaupain-1 preferentially in apical portion of parasite ookinete suggests that this protease may be related with parasite egression from mosquito midgut. Immune responses to chabaupain-1 were evaluated using two different adjuvants, chitosan nanoparticles and hydroxide aluminum. Mice immunized with the recombinant protein alone or in association with nanoparticles were challenged with P. chabaudi showing that immunization with the recombinant protein confers partial protection to blood stage infection in BALB/c animal model.

Bis-alkylamine quindolone derivatives as new antimalarial leads.

Quindolone derivatives, designed to target the malaria parasite digestive vacuole and heme detoxification pathway, have been synthesized by reaction with 2-chloro-N,N-diethylethanamine. This reaction gave N,O-, N,N- and O-alkylated products containing one or two basic side-chains. The compounds were evaluated for antiplasmodial activity against the chloroquine-resistant Plasmodium falciparum W2 strain and for cytotoxicity in HepG2 A16 hepatic cells. By incorporating alkylamine side chains and chlorine atoms in the quindolone nucleus we transformed the inactive tetracyclic parent quindolones into moderate or highly active and selective antimalarial compounds. The most active and selective compound, 5c, showed an IC(50)=51 nM for P. falciparum and a selectivity ratio of 98.

Plasmodium chabaudi: expression of active recombinant chabaupain-1 and localization studies in Anopheles sp.

Plasmodium cysteine proteases have been shown to be immunogenic and are being used as malaria potential serodiagnostic markers and vaccine targets. Genes encoding two Plasmodium chabaudi cysteine proteases chabaupain-1 (CP-1) and chabaupain-2 (CP-2) were identified and further expressed in Escherichia coli. Solubilisation of recombinant CP-1 and CP-2 was achieved by decreasing the temperature of induction. Anopheles gambiae tissues infected with Plasmodium were analyzed by Western blotting using the anti-CP-1 antibody showing that CP-1 is only present in the A. gambiae midguts being absent from other infected mosquito biological material. Anti-CP-1 anti-serum recognized a 30 kDa band in P. chabaudi, Plasmodium berghei and Plasmodium yoelii lysates but does not recognize the recombinant CP-2 extracts suggesting high antibody specificity.

'A mate or a meal'--pre-gravid behaviour of female Anopheles gambiae from the islands of São Tomé and Príncipe, West Africa.

BACKGROUND: Malaria prevalence differs between the two islands that comprise the archipelago of São Tomé and Príncipe. This may be due to differences in the biology of local Anopheles gambiae, the only vector on the islands. Survival rate and feeding frequency are two factors influencing vectorial capacity. Anophelines generally feed just once per gonotrophic (oviposition) cycle. Newly emerged insects, however, may feed two or more times during their first oviposition cycle thus increasing the likelihood of becoming infected. The reasons for multiple feeding are not clearly understood and it is still uncertain whether the behaviour is facultative or obligatory. We, therefore, determined survival and sporozoite rates, and examined the behaviour of An. gambiae from the two islands during their first gonotrophic cycle. METHODS: The wing size of 1,410, abdominal condition of 687, gonotrophic age and mated status of 7,264 female M form An. gambiae collected by light-trap, landing catch, resting outdoors or in copula, was determined from four sites in the archipelago. Sporozoite rates assessed by ELISA in 15,533 females from São Tomé and 2,111 from Príncipe were determined. RESULTS: Estimated survival rates ranged between 0.834-0.849 per day in São Tomé and 0.801-0.818 per day in Príncipe. Sporozoite rates of 0.63% in São Tomé were significantly higher than the 0.24% from Príncipe. Overall 49% of females mated on the second night after emergence before feeding, and 51% on the third night and thus fed before mating. The likelihood of mating before feeding increased with wing size. None of the 3,776 parous insects collected showed evidence of recent mating. All but two of the 198 females collected in copula had undeveloped ovaries. Mean wing sizes and the number of insects collected in a sentinel light-trap varied but the proportion of newly emerged insects in the collection did not. The estimated survival rate of the smallest insects was lower than other size groups, but the overall size distribution of each age group was normal. Parous insects were gonotrophically concordant. CONCLUSION: Differences in mosquito survival contributed to the lower sporozoite rates and endemicity of malaria on Príncipe compared to São Tomé. On both islands all newly emerged insects blood fed on the second night following emergence but only became gonotrophically active on the third night after emergence. Smaller insects had a higher 'mortality/emigration' rate than larger ones. We suggest that insufficiency of Juvenile Hormone until the third day of adult life is responsible for gonotrophic inactivity and that by partitioning mating between the second or third day after emergence females maximise their chances of out-crossing.