Date of Award

1-1-2000

Thesis Type

Masters

Document Type

Thesis

Divisions

Institute for Advanced Studies

Department

-

Institution

Universiti Malaya

Abstract

Human herpes virus 6 (HHV6) is an ubiquitous, lymphoproliferative P-herpesvirus, which infects almost all individuals during infancy and persists lifelong. Two variants of HHV6 (A and B) have been described. The present study is on HHV6B, which causes exanthem subitum, also known as roseola infantum, a common febrile childhood illness. Two methods of detection for anti-HHV6B in body fluids were used, being indirect immunofluorescence assay (IFA) and peptidylated pin-ELISA. For IFA, the target cells were HHV68-infected human cord blood mononuclear cells (HCBMC). Ninety eight infant sera were tested by IFA and all were found to be IgG-HHV6 positive. Seventeen breast milk samples were also analyzed and 9 were IgA-HHV6 positive against HHV6-infected HCBMC. Eight saliva samples tested were all IgA-HHV6 negative in IFA. Serum, breast milk and saliva samples were collected from different individuals. No two sample was from the same individual. Anti-HHV6 IFA was carried out on 10 cord blood samples and all were found IgG positive but IgA negative, thus eliminating the possibility that IgA is transferred through cord blood. Age prevalence study was carried out on 240 subjects between 0-20 years old. Distinctly different serum lgA and IgG profiles were obtained. However, a correlation study carried out showed that there is correlation between prevalence of IgG and IgA. For the IgG anti-HHV6, the proportion with antibody was high at birth, about 80% and the antibody levels remained high till about 2 years. It then decreases slowly and increases again before reaching a plateau at adulthood. As for HHV6 anti-lgA antibodies were low during childhood till about 4 years of age and then increased gradually. However, the increase did not follow a consistent pattern with age. For the peptidylated pin-ELISA, HHV6 peptides were synthesized onto polyethylene pins and served as target antigens in ELISA. A IOlkDa (pI0I) protein of the HHV6 variant B strain Z29 (858 amino acids) has been found to be highly imrnunoreactive (Pellet et al, 1993). In the present study, 334 amino acids of the carboxyl terminal was synthesized as a set of 46, 14 merpeptides, overlapping by 7-mers, since antigenicity of a protein is frequently localized at the carboxyl terminal (Neipel et al, 1992). The sequence selected was also based on antigenic predictions using hydrophilicity based on Hopp and Woods (I 982), accessibility and secondary structure based on the NNPREDICT method. All three methods were obtained through Protscale Analysis from SWISSPROT (http:/expasy.hcuge. ch/cgi-bin/protscale. pl.) The antigenic profiles for IgG for 25 HHV6 IFA positive sera to HHV6 peptides were studied and 6 regions were identified. Of the 6, peptide 44 (IRQDGETDEETVP) showed the highest reactivity, recognized by IgG in 24 of the 25 sera tested. A percentage reactivity of 96% was obtained. In the saliva samples tested, 3 samples were lgA positive when reacted with 5 major HHV6 peptides. The reason for the HHV6 IF A-negative saliva showing reactivity in ELISA may be because the peptide regions recognized by salivary IgA were not exposed in the virus- infected HCBMC. This may be due to the conformational structure of the protein. When synthesized the protein structure is linearized and thus may have been possible to recognized. But as a whole antigen in IFA, it exists in its native conformation and could not be dletected. Six of the HHV6 !FA-positive and 3 HHV6 !FA-negative breast milk samples were tested against the HHV6 peptides. The IF A-positive and IF A-negative both reacted with the similar HHV6 peptides in ELISA for lgA, except for peptide 13 (KGNSRDL YSGGNAE). This peptide was predominantly recognized by IgA in HHV6 IF A-positive breast milk. This suggests the presence of a shared lgA antigenic epitope of IgA ex pressed in both the virally-infected HCBMC and the HHV6 peptide. Since serum IgG but not lgA to HHV6 was detected by IFA in cord blood, the presence of lgA antiHHV6 in breast milk may serve to provide another means of passive immunity besides the transplancental IgG. The HHV6 antigenic regions from the ELISA studies were also analyzed for their predicted hydrophilicity, accessibility and secondary structure by methods from Protscale, Swissprot. Besides these prediction methods, mobility as well as coil structured parameters were used to confirm our findings. The major immunodominant regions for IgG and IgA, being peptides 44 and peptide 13 respectively were found to be hydrophilic as well as surface accessible. They were also highly mobile and showed to be made up of coiled structures. The secondary structure of both these peptides were made up of mostly coiled structures. Peptide 44 had a helix-coil-helix structure and peptide 13 was a coil-helix-coil structure. As coils frequently exist as protrusions from the main body, they are preferentially recognized by antibodies.

Initial

snms

Additional Information

Dissertation (M.A) -- Institute for Advanced Studies, Universiti Malaya, 2000.

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