One hundred and ninety hepatitis B surface antigen positive (HBsAG+) sera were subtyped, belonging to : blood donors, hepatitis patients, patients and staff in a hemodialysis unit, all from Kuala Lumpur; Malaysian aborigines from three jungle locations in Peninsular Malaysia; and East Malaysians from Sarawak, East Malaysia; Three subtypes adr, adw and ayw were present in Malaysia in the following frequencies: 44%, 29%, and 27%, respectively; In Kuala Lumpur 87% had subdeterminant d and 13 per cent y, whereas in the deep jungle aborigines of Perak and Pahang, the y subdeterminant was present in 87% and the d in 13%. A similar pattern of preponderance of y prevailed in Sarawak, East Malaysia. In Kuala Lumpur the two main ethnic groups, Malays and Chinese, differed in subtype distribution, in that adr predominated in the Malays (61%), while the adw predominated in the Chinese (51%); Subtype distribution was not related to age or sex of carriers of the antigen, or to whether they had hepatitis, or asymptomatic antigenemia.
Alpha-Fetoprotein (AFP) levels of 1,335 males (15 years and older) of seven ethnic groups (Chinese, Indians, and Malays from Singapore, Caucasians from Lyon, and Blacks from Nairobi, forest, and the savanna region of the Ivory Coast) were determined by radioimmunoassay. A few elevated levels (up to 30 nanounits/ml) were detected in some normal individuals, especially in the older age-groups. In addition, there was a systematic age-dependency of AFP levels particularly evident in the groups from Singapore-Lyon, in which there was a 50% AFP increase between the ages of 20 and 40. Comparison between Africans on the one hand and people from Singapore-Lyon on the other hand revealed highly significant differences (p less than 0.001), especially in the younger groups, whereas Chinese, Malays, and Indians from Singapore had very similar AFP pattern; this suggests an important role for environmental factors in the regulation of AFP levels. The age dependency of the presumed effect of environmental factors is in keeping with experimental data showing that young animals respond more vigorously to AFP-stimulating factors. Although the incidence of hepatocellular carcinoma (HCC) differs in the three Singapore groups (the highest in Chinese and the lowest in Indians), no relationship was observed in this study between mean AFP level and HCC incidence in Singapore.
Purified schizonts (6--10 nuclei) and membranes of schizont-infected erythrocytes from the Malaysian and Philippine strain of Plasmodium knowlesi are analyzed immunochemically using immunoglobulin of rhesus monkey hyperimmune sera against schizonts and of sera from naturally immune monkeys. The anti-schizont Ig identifies less than 20 immune components in Triton X-100-solubilized schizonts and membranes of infected cells. Of these antigens, 9 (component 1, 3, 4, 5, 6, 10, 11, 18, and 20) are common to parasites and membranes of infected erythrocytes, and 12 (2A,B, 6, 8, 9, 12, 13p, 14, 16A,B, 19 A,Bp, 21, 22p, and 23) are predominantly found in the parasite; 4 components (13i, 19A,Bi, 22A, B, and 24) are unique to the membrane of infected erythrocytes. Only three parasite-specific components (1, 13, and 19) are exposed on the surface of parasitized erythrocytes as revealed by both lactoperoxidase-catalyzed radioiodination and extensive absorption of anti-schizont Ig using intact infected erythrocytes. Two plasmodium-specific antigens (1 and 13) on the surface of infected erythrocytes are recognized by sera of rhesus monkeys rendered naturally immune against P. knowlesi infections and, therefore, represent antigens in vivo. Analyses of schizonts and membranes of parasitized erythrocytes of the two different strains of P. knowlesi yields only some minor quantitative, but no qualitative differences when analyzed with both types of antisera. Importantly, components 1 and 13 appear identical in both strains.
We have investigated the expression of a strain-specific malarial antigen on the surface of erythrocytes infected with knobless (K-) variants of knob-positive (K+) strains of Plasmodium falciparum. Aotus blood infected with K+ or K- parasites derived from two independent geographical isolates (Malayan camp and Santa Lucia) was surface iodinated by the lactoperoxidase method. Infected and uninfected erythrocytes were then separated by a new procedure involving equilibrium density sedimentation on a Percoll gradient containing sorbitol. Strain-specific antigens were readily identified on the surface of erythrocytes infected with either of the K+ strains by their characteristic size and detergent solubility. These proteins were not detected on the surface of erythrocytes infected with either of the K- variants nor on uninfected erythrocytes isolated from K+- or K- -infected blood. These results are consistent with a role for the strain-specific surface antigen in cytoadherence of P. falciparum-infected erythrocytes. Our findings represent the second biochemical difference (with the knob-associated histidine-rich protein) between K+ and K- P. falciparum.
Antigen analysis of group A streptococcus strain, "Matsuyama 2166", which had been typed as T12-28 (by the T-agglutination method), MNT (nontypeable by the M-precipitation method) using conventional typing sera was examined by the precipitation and precipitation absorption technique. The prevalence of group A streptococci, typed under the name "Matsuyama 2166" was also investigated. The results were as follows: 1) The strain "Matsuyama 2166" is OF (Opacity Factor) (+) and its serological components consist of M "Matsuyama 2166" antigen, which is unique in our collection of M-types, and T28 antigen as a major antigen along with T12 as a minor. 2) Out of the group A streptococci typed as MNT, 96.6% of the streptococci were types as T12-28 and 96.5% of T28 by conventional typing sera were strains to be typed as M "Matsuyama 2166" using the newly prepared M "Matsuyama 2166" typing serum, suggesting the great advantage to M-typing rate of T28 strains. 3) Group A streptococcus M "Matsuyama 2166" was also found in isolates from Thailand or Malaysia. It is interesting that T-types found in those isolates seems to be a little different from the T-types isolated in Japan. These results showed that there was the difficult problem when we did the speculation of M-type from the result of T-type.
The relationship between immunoglobulin allotypes and risk of developing nasopharyngeal carcinoma was examined in a comparative study of 50 Chinese cases and 140 Chinese controls and 50 Malay cases and 79 Malay controls residing in Malaysia. Although the most common Gm phenotype was elevated in both Chinese and Malay nasopharyngeal carcinoma patients compared to their controls, there were no significant differences between cases and controls in the distribution of Gm haplotypes in either population. There were no differences between cases and controls in the distribution of Km alleles in either population. Thus a previously reported association of Km(1) with increased nasopharyngeal carcinoma risk in Tunisia is not confirmed in two Mongoloid populations in Malaysia.
The Epstein-Barr virus nuclear antigen I (EBNA I) is the only latent EBV antigen consistently expressed in malignant tissues of the nasopharynx. A 20-amino-acid synthetic peptide, p107 contains a major epitope of EBNA I. We tested sera from 210 patients with nasopharyngeal carcinoma (NPC) and from 128 normal individuals (NHS) for IgA antibodies to p107 using an enzyme-linked immunosorbent assay (ELISA). Whereas 191/210 (91%) of NPC patients had IgA antibodies to p107, only 17/128 (13.3%) of NHS had such antibodies and only 6/57 (10.5%) of sera from patients with malignancies other than NPC had IgA-p107 reactivity. Thirty-nine salivary samples from 46 NPC patients (84.8%) also contained IgA-p107 antibodies whereas only 3/42 (7.1%) of normal saliva samples were IgA-p107 positive. The results suggest that IgA antibodies to EBNA I may become a useful, easily measurable, marker for NPC.
The Sarawak strain of Japanese encephalitis virus (JE-Sar) is virulent in 3-week-old mice when inoculated intraperitoneally. The nucleotide sequence for the envelope glycoprotein (E) of this virus was determined and compared with the published sequences of four other strains. There were several silent nucleotide differences and five codon changes. Monoclonal antibodies (MAbs) against the E protein of JE-Sar virus were prepared and characterized. MAb-resistant mutants of JE-Sar were selected to determine if mutations in the E protein gene could affect its virulence for mice. Eight mutants were isolated using five different MAbs that identified virus-specific or group-reactive epitopes on the E protein. The mutants lost either complete or partial reactivity with selecting MAb. Several showed decreased virulence in 3-week-old mice after intraperitoneal inoculation. Two (r27 and r30) also showed reduced virulence in 2-week-old mice. JE-Sar and the derived mutants were comparable in their virulence for mice, when inoculated intracranially. Mutant r30 but not r27 induced protective immunity in adult mice against intracranial challenge with parent virus. However, r27-2 did induce protective immunity against itself. Nucleotide sequencing of the E coding region for the mutants revealed single base changes in both r30 and r27 resulting in a predicted change from isoleucine to serine at position 270 in r30 and from glycine to aspartic acid at position 333 in r27. The altered capacity of the mutants to induce protective immunity is consistent with the immunogenicity changes predicted by computer analysis using the Protean II program.
Monoclonal antibodies (MAbs) directed against different epitope regions on three sexual stage-specific gamete surface proteins of Plasmodium falciparum, Pfs 25, Pfs 230, and Pfs 48/45, were used to study the genetic diversity of these epitopes among fresh isolates of P. falciparum from Malaysia, using immunofluorescence microscopy (IFA). Among 45 Malaysian isolates, one epitope of Pfs 25, designated region I, showed evidence of variable reactivity with MAbs among different isolates; the Pfs 25 epitope, region II, was universally recognized by MAbs in all isolates. Two apparently distinct epitope regions of Pfs 230 were defined by MAbs, one of which was universally recognized by MAbs among the 45 isolates; the other was conserved in all but three isolates. The epitope regions of gamete-surface protein Pfs 48/45, designated regions I, IIa, IIb, IIc, III, and IV, were examined for reactivity by IFA in 33 isolates. Epitope regions I, IIb, III, and IV were conserved in all isolates; regions IIa and IIc existed in variant forms.
An ELISA using the Epstein-Barr virus nuclear antigen 1 (EBNA 1) was found to detect selectively specific IgA in sera from patients with nasopharyngeal carcinoma (NPC). The antigen, p107, was a 20-amino acid synthetic peptide, representing a major epitope of EBNA 1.267/294 (90.8%) of NPC patients had IgA antibodies to p107 but in normal individuals, only 41/577 (7.1%) had IgA/p107. In sera from patients with other cancers, 11/77 (14.3%) had IgA/p107 reactivity. 124 IgA/VCA positive and 86 IgA/VCA negative NPC sera were also tested for IgA/p107 binding in ELISA. The majority of IgA/VCA positive sera (117) also contained IgA/p107 antibodies. Of interest was the detection of 74/86 IgA/p107 reactive sera in the IgA/VCA negative group. The results suggest that the IgA/p107 ELISA could become a useful, complementary screening assay to the IgA/VCA immunofluorescence test for detection of NPC.
Binding studies of 160 overlapping, synthetic octapeptides from the hydrophilic regions of the Sta58 major outer membrane protein of Rickettsia tsutsugamushi with sera from patients with scrub typhus revealed 15 immunodominant peptides which are recognized by all the sera tested. Further analysis of the specificity of peptide binding with five of these peptides indicated that the peptides showed significantly stronger binding to scrub typhus patients' sera than they did to sera from patients with other febrile illnesses common in the region, i.e., malaria, dengue fever, typhoid fever, and leptospirosis. The main antibody class binding to these peptides appears to be immunoglobulin M, and there appears to be little correlation between reactivity with peptides and antibody titers measured by the indirect immunoperoxidase test.
Previous studies have found Kunjin (KUN) virus isolates from within Australia to be genetically homogenous and that the envelope protein of the type strain (MRM61C) was unglycosylated and lacked a potential glycosylation site. We investigated the extent of antigenic variation between KUN virus isolates from Australia and Sarawak using an immunoperoxidase assay and a panel of six monoclonal antibodies. The glycosylation status of the E protein of each virus was also determined by N glycosidase F (PNGase F) digestion and limited sequence analysis. The results showed that KUN viruses isolated within Australia oscillated between three antigenic types defined by two epitopes whose expression was influenced by passage history and host cell type. In contrast an isolate from Sarawak formed a stable antigenic type that was not influenced by passage history and was distinct from all Australian isolates. PNGase F digestions of KUN isolates indicated that 19 of the 33 viruses possessed a glycosylated E protein. Nucleotide sequence of the 5' third of the E gene of selected KUN isolates revealed that a single base change in PNGase F sensitive strains changed the tripeptide N-Y-F (amino acids 154-156 of the published sequence) to the potential glycosylation site N-Y-S. Further analysis revealed that passage history also had a significant influence on glycosylation.
The linear antigenic epitopes of the Epstein-Barr virus replication activator protein (ZEBRA), recognised by specific serum IgG in nasopharyngeal carcinoma (NPC), were determined. This was achieved by synthesizing the entire amino acid sequence of ZEBRA as a set of 29, 22-residue peptides with an overlap of 14 amino acids. The ZEBRA peptides were tested in enzyme-linked immunosorbent assay (ELISA) for IgG binding in sera from 37 selected NPC patients who had IgG antibodies to the native ZEBRA protein. The most immunogenic epitope was peptide 1 at the amino-terminal end with 36 of the sera reactive against it. Further analysis of peptide 1, using the multipin peptide-scanning technique, defined a 10-amino-acid sequence FTPDPYQVPF, which was strongly bound by IgG. Two other regions of ZEBRA were also identified as immunodominant IgG epitopes, namely peptide 11 (amino acids 82-103) and peptide 19/20 (amino acids 146-175) with 8-13 of the NPC sera reactive against the peptides. The number of peptides reactive with individual NPC serum varies from 1 to 6 or more and there is some correlation between a greater number of peptide (at least 4) bound and a higher (at least 1:40) titre of serum IgA to viral capsid antigen. The immunodominant ZEBRA peptide 1 could be utilised in IgG ELISA for the detection of NPC.
Rice tungro spherical virus (RTSV) has an RNA genome of more than 12 kb with various features which classify it as a plant picornavirus. The capsid comprises three coat protein (CP) species, CP1, CP2 and CP3, with predicted molecular masses of 22.5, 22.0 and 33 kDa, respectively, which are cleaved from a polyprotein. In order to obtain information on the properties of these proteins, each was expressed in E. coli, purified as a fusion to the maltose-binding protein and used for raising a polyclonal antiserum. CP1, CP2 and CP3 with the expected molecular masses were detected specifically in virus preparations. CP3 is probably the major antigenic determinant on the surface of RTSV particles, as was shown by ELISA, Western blotting and immunogold electron microscopy using antisera obtained against whole virus particles and to each CP separately. In some cases, especially in crude extracts, CP3 antiserum detected several other proteins (40-42 kDa), which could be products of CP3 post-translational modification. No serological differences were detected between the three CPs from isolates from the Philippines, Thailand, Malaysia and India. The CP3-related 40-42 kDa proteins of the Indian RTSV isolate have a slightly higher electrophoretic mobility (42-44 kDa) and a different response to cellulolytic enzyme preparations, which allows them to be differentiated from south-east Asian isolates.
The present study was undertaken to investigate the antibody responses of dengue fever (DF) patients to specific dengue virus proteins. Partially purified dengue 2 New Guinea C (NGC) strain virus was used as antigen. Under the present experimental protocols, it was observed that almost all DF patients' sera had detectable presence of antibodies which recognize the dengue 2 envelope (E) protein. The convalescent-phase sera especially had significant detectable IgG, IgM and IgE against the protein. In addition, IgGs specific against the NS1 dimer and PrM were also detected. Antibody against the core (C) protein, however, was not detectable in any of the DF patients' sera. The substantial presence of IgG against the PrM in the convalescent-phase sera, and the presence of IgE specific for the E, reflect the potential importance of these antibody responses in the pathogenesis of dengue.
Random 15-mer peptides displayed on filamentous phages were screened in binding studies using a Chlamydia pneumoniae-specific monoclonal antibody (RR-402) and affinity-purified, polyclonal sera from patients seropositive for C. pneumoniae infections by the microimmunofluorescence (MIF) test. One 15-mer epitope, epitope Cpnl5A (LASLCNPKPSDAPVT) was identified in both the monoclonal and polyclonal screenings, and showed higher ELISA reactivity with C. pneumoniae MIF-positive sera compared to patients with other chlamydial infections, non-chlamydial respiratory infections and normal healthy sera (MIF-negative). Interestingly, epitope Cpnl5A also showed significant (52%) amino acid sequence homology to the 56 kDa type-specific antigen of Rickettsia tsutsugamushi, a protein implicated in the virulence of this organism.