ABSTRACT
Hepatitis B infection causes a wide spectrum of liver diseases. Previous analyses of hepatitis B virus (HBV) genome have revealed eight HBV genotypes (A-H), with distinct geographical distribution worldwide. The epidemiology of HBV genotypes and their implications for natural history of disease progression and response to anti viral therapy have been increasingly recognized. This study was undertaken to determine the HBV genotypes in a group of Sri Lankan patients with chronic infection who presented for investigation prior to treatment. Genotypes were determined (2007-2009) in 25 patients with evidence of chronic HBV infection. A genotyping system based on multiplex-nested PCR using type-specific primers was employed in assigning genotypes A through F. Genotypes G and H were not determined. Among the 25 patients tested, genotypes B [9 (36%)], C [4 (16%)], D [3 (12%)], A [2 (8%)] and E [1 (4%)] were detected. There was a relatively high prevalence of mixed infections with genotypes B+C (3), A+D (1), and B+D (2), which overall constituted 24% of patients. Although this is a non-representative sample, HBV infections among this group of Sri Lankan patients were predominantly genotypes B, C and D.
ABSTRACT
Allelic polymorphism existing within restricted settings and time frames were evaluated in this study, for Plasmodium falciparum C-terminal merozoite surface protein-1, 42 kDa (MSP1p42), and 19 kDa polypeptides (MSP1p19), which are major blood stage vaccine candidates for malaria. The nature and significance of natural human antibody responses to P. vivax MSP1 proteins were also studied using baculovirus-expressed recombinant proteins. Potential role of relative antibody affinity in protective immunity was investigated using P.cynomolgi MSP1 vaccinated toque monkey sera. MSP1p42 sequences from 19 P. vivax isolates collected from Kataragama, in Sri Lanka, revealed classical Belem and Salvador-1 alleles with 98-100 percent homology, except for a hypervariable block of 38 amino acids with only 24 percent overall homology giving rise to 10 sequence types. These sequence types appear to have derived by re-assortment of di, tri, or quadric-morphisms at 29 variable positions within this hypervariable block, of which the amino acid composition, suggests an exposed antigenic loop. The C-terminal P.vivax MSP1 19 kDa region was highly conserved and only a single dimorphic position was detected. Acute sera of all 19 P.vivax patients reached with PvMSP1 p42 and p19 proteins indicating that both proteins are highly antigenic in natural P. vivax infections. However, a major proportion of the antibody response to PvMSP142 was primarily directed against the p19 domain of the protein. Irreversible reduction of the PvMSP19 protein almost completely abolished antibody reactivity indicating that p19 epitopes, which are naturally antigenic in humans are almost entirely conformational. All anti-sera showed reactivity to multiple epitopes in 3 Belem and/or 3 Salvado-1 overlapping hypervariable region peptides and correlated with the homology of the allele in their current infection. The humoral response to the hypervariable region includes both antibodies that can distinguish epitope fine specificity, as well as an apparent majority of lower affinity, cross-reactive species. These results are indicative of substantial natural antigenicity of polymorphic region epitopes. Sequence variation was also investigated in the P. falciparum MSP1 C-terminal 42 kDa region (spanning blocks 15-17) in 21 natural isolates from Kataragama in Sri Lanka and in 18 isolates from Ndiop in Senegal. Among the Kataragama isolates, prototypic K1 allelic type seems to predominate as it was found in 86 percent of isolates. In contrast, all Ndiop isolates represented the PNG- mad20 prototype, indicating that alleles of PNG-MAD20 origin are predominant in this region of West Africa. The 52 nucleotide and deduced amino acid sequences from Kataragama and Ndiop, encoding the PfMSP1p19 domain (block 17) were highly conserved, with amino acid variations at only four positions. Most highly represented PfMSP1p19 sequence type was the prototype Q-KNG-L (Thai-K1;81 percent. In addition, there were 15 percent E-TSR-L prototypes (PNG-MAD20). Four (4) percent of the more rare prototypes (E-TSG-L(Indian type 1) and 1 E-KNG-L (UG-Palo Alto)}. Variations in block 16 were represented by 13 and 2, PNG-MAD-20 and Thai-K1 di/tri-morphic positions respectively, whereas only a single dimorphism was found in block 15 in isolates of PNG-MAD-20 origin. It could be considered a positive feature of the recombinant baculovirus P.cynomolgi MSP1p19 (PcMSP1p19) antigen to systematically evoke high affinity antibody responses with Ka values in the range of 108-9. However, a direct correlation between relative antibody affinity and protection was not observed, in the context to primate vaccination trials with PcMSP1p19/p42 proteins, as function of the 4 adjuvants used. Although it was somewhat suggestive, the data was not adequate to validate the concept of antibody affinity maturation with parasite exposure. Results presented in this thesis, therefore have important implications for the design of a malaria vaccine based on C-terminal MSP1 proteins.