Browsing by Author "Longacre, S."

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A Double antibody sandwich ELISA for the diagnosis of vivax malaria: a tool for further research
(University of Colombo, 2000) Seneviratna, G.D.C.N.; Manamperi, A.A.P.S.; Kapilananda, G.M.G.; Longacre, S.; Handunnetti, S.M.; Udagama-Randeniya, P.V.
A diagnostic assay for Plasmodium vivax based on a double antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA) was established to detect the merozoite surface protein 1 (PvMSP1) in wild isolates. This assay was based on the recombinant protein p19, a C-terminal processing product of PvMSP1. Of the two anti-P. vivax monoclonal antibodies (MAbs) selected, A21 was used as the capture antibody while horse radish peroxidase labelled A8 served as the probing second antibody. Optimized conditions established for p19 based DAS ELISA with the exception of a lower concentration of Tween-20 in buffers were suitable to screen lysed whole blood of malaria patients. This assay had a specificity of 100 percent for P. vivax and all the isolates of P. falciparum tested negative. Of the P. vivax-infected blood samples, only those containing both compact and schizont stages were diagnosed positive. The rest of the isolates tested negative either due to stage specificity of this assay or to the antigenic diversity of PvMSP1 in wild isolates. This test demonstrated a sensitivity of 27.58 percent and an accuracy of 63.15 percent. The positive and negative predicted values of this ELISA were 100 percent and 57.14 percent, respectively. Therefore, the P. vivax specific DAS ELISA developed and tested in the present study is not sufficiently sensitive to be used as a diagnostic tool for vivax malaria. Nevertheless, a baseline was established for development of diagnostic ELISA for future use with a more appropriate antigen.
Hypervariability in a leading P.vivax malaria vaccine candidate, C-terminal merozoite surface protein 1
(Sri Lanka Association for the Advancement of Science, 2000) Manamperi, A.; Holm, I.; Perera, L.; Handunnetti, S.M.; Longacre, S.
It is widely accepted that the C-terminal 42 kDa (p42) and 19 kDa (p19) processing fragments of plasmodium Merozoite Surface Protein-1 (MSP-1) are targets of immune protection. To begin to assess the degree of polymorphism in these MSP-1 vaccine candidates, we have investigated the sequence diversity in the p.vivax MSP-1 p42 processing fragment, in 19 natural isolates, from p.vivax infected patients in Kataragama. Sequence analysis of PvMSP-1 p42 in the 19 PCR positive isolates reveald 11 sequences of Belem origin and 8 sequences of Salvador-1 (Sal-1) origin. Among the isolates, these two stains are 98-100% homologous across this region, with one notable exception. This corresponds to a highly polymorphic block of 38 amino acids (24% amino acid homology among isolates). However, this polymorphism appears to be derived largely by re-assorting a dimorphism at each variable position. This type of restricte variability suggests that in spite of its diversity, there may nevertheless be a defined structure for this region of the molecule and that the diversity may be functionally important. Alternatively, it may be specifically designed for maximal effect in immune evasion, as a highly exposed immunogenic loop structure. In striking contrast, a single nucleotide substitution was detected in the cysteine rich C-terminal 19 kDa region, resulting in a lysine to glutamate substitution. This was detected in only one isolate among the 19 isolates investigated for sequence diversity. Since the PvMSP-1 C-terminal antigen is clearly hypervariable in the context of natural infections, a vaccine based on a single version of this antigen, might not induce an effective immunity against the multiple forms. In contrast, the PvMSP-1 p19 domain appears to be well conserved and thus appears to be a considerably more promising vaccine candidate.
Protection against malaria in toque mokeys immunized with p.cynomolgi MSP1inv in alum
(Sri Lanka Association for the Advancement of Science, 2000) Amaratunga, C.; Nandasiri, K.; Weerasinghe, S.; Manamperi, A.; Holm, I.; Longacre, S.; Handunnetti, S.
Immunization of toque monkeys with baculovirus-expressed, His-tagged recombinant plasmodium cynomoigi ceylonensis (Pcc) C-terminal 19 kDa proteins of the major merozoite surface protein 1 (MSP1inv) with Freunds adjuvant, mediates long-term protection against homologous and heterologous p. cynomolgi blood-stage challenge infection. However, Freunds adjuvant is unsuitable for use in humans, which necessitates the testing of alternative adjuvants. Antigen-alum formulation and binding was optimized for the new preparation of matalloaffinity-purified MSP1inv antigen, under conditions acceptable for human trails, including 800ug aluminium per dose, in accordance with the permitted FDA maximum. This formulation was used in an immunization trial using the p. cynomolgi-toque monkeys system, which is analogous to the p.vivax-human system. Group 1 comprising 4 animals were immunized with alum+MSP1inv , and group 2 comprising 3 monkeys received alum alone. Four doses of immunization were given intramuscularly at 0,1,3 and 4 month intervals. After immunization, the anti-MSP1inv antibody titres of immunized animals reached 2.8x104 . all animals were given a homologous Pcc challenge infection one month after the last dose of immunization. One of the four immunized animals was completely protected while the other 3 animals showed low patent parasitaemia, resulting In an overall partially protective effect (p=0.02). immunization with alum did not result in sterile immunity, as seen with Freunds, where antibody titres range from 106 - 107 . Following treatment, the animals were given a second heterologous, blood-stage challenge infection of p.cynomolgi Gombak, (PcG) four months after the first challenge infection. Sequence analysis of PcG DNA reveald a single amino acid differing from that of Pcc. The substitution which occurs at the nt 207 position in the C-terminal 19-kDa sequence changes the amino acid glutamate into lysine. Statistically significant partial protection was observed in the immunized animals (p=0.04), despite having a lower titre of antibodies (3.3x103) at the time of re-challenge. Together with sequence data, this documents the ability of recombinant MSG1inv to protect against a heterologous infection.
Protection against malaria in toque monkeys immunized with P. cynomolgi MSPI-19 in alum
(The royal society of tropical medicine and hygiene, British society for parasitology, The American society of tropical medicine and hygiene, 2000) Amaratunga, C.; Nandasiri, K.; Weerasinghe, S.; Manamperi, A.; Longacre, S.; Holm, I.; Handunnetti, S.
In a pre-clinical trail of a vaccine against P.vivax malaria, toque monkeys were immunized with matalloaffinity-purified baculovirus-expressed. His-tagged recombinant Plasmodium cynomolgy ceylonensis (Pcc) C-terminal 19 kDa protiens of the major merozoite surface protein 1 (MSP1p19) with alum, under conditions acceptable for clinical trials. The P. cynomolgi-toque monkey system is highly analogous to P.vivax in humans. Eight animals were immunized with alum+MSP1p19 and 3 monkeys received alum alone. After four doses, the anti-MSP1p19 antibody titres of immunized animals reached 2.8x104. all animals were challenged with Pcc asexual blood stage parasites. The immunized animals showed significant, partial protection (p=0.0024). Four of these animals were given a second heterologous challenge infection of p.cynomolgi Gombak, (PcG) four months after the first challenge infection. Sequence analysis of PcG DNA revealed a single amino acid challenge differing from that of Pcc. The substitution which occurs at the nt 207 position in the C-terminal 19-kDa sequence changes the amino acid glutamate into lysine. Statistically significant protection was observed in the immunized animals (p=0.04), despite having a lower titre of antibodies at the time of re-challenge. This documents the ability of recombinant MSP1p19 with alum to protect against Pcc and PcG infections.