Browsing by Author "Holm, I."

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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.