Immunogenicity in mice of human metapneumovirus with a truncated SH glycoprotein.

The SH glycoprotein of human metapneumovirus (HMPV) is twice the size of that of human respiratory syncytial virus and possesses a large, hydrophilic luminal domain. The glycoprotein is located on the surface of the virion and of virus infected cells and, if immunogenic, might be expected to play a role in anti-viral immunity. Initial attempts to study anti-SH antibody immunogenicity were thwarted by the instability of the SH gene on passage both in human bronchial epithelial cells and in mice. Repeated passage of virus isolates in human bronchial epithelial cells in culture resulted in the appearance and eventual predominance of HMPV mutants lacking all or most of the luminal domain of SH coincidental with the loss of productive infection in mouse lungs. Where infection was established in mice with an early cell culture passage, the virus recovered from mouse lung differed markedly from the inoculum, carrying 19 coding mutations in the SH luminal domain. Immunization of mice with a mutant virus variant expressing only 14 amino acids of the luminal domain of SH induced a cross-reactive antibody response to both the F glycoprotein and the SH glycoprotein but a largely sub-group specific response to the G glycoprotein. Similar patterns of response were achieved by immunization with individual HMPV glycoproteins expressed from recombinant vaccinia viruses. Recombinant truncated SH glycoprotein induced sub-group cross-reactive antibodies capable of neutralizing wild-type virus. Recombinant F glycoprotein also induced cross-reactive neutralizing antibodies whilst recombinant G glycoprotein induced largely strain-specific, non-neutralizing antibodies.

Diagnosis of human metapneumovirus by immunofluorescence staining with monoclonal antibodies in the North-East of England.

BACKGROUND: Since its discovery in 2001 human metapneumovirus (hMPV) has been shown to be a significant cause of human respiratory disease, responsible for 5-8% of respiratory infections in hospitalised children. Diagnosis hitherto has been largely carried out by reverse tanscriptase polymerase chain reaction (RT-PCR) but immunofluorescence staining of cells from nasopharyngeal secretions (IF) offers advantages for some laboratories and may produce a more rapid result in urgent cases. We have recently demonstrated that IF with a rabbit antiserum gave sensitivity equal to that of RT-PCR. However, monoclonal antibodies offer a more plentiful, uniform IF reagent. OBJECTIVES: Here we have evaluated a pool of anti-hMPV monoclonal antibodies in the routine diagnosis of respiratory infections in hospitalised infants and children. STUDY DESIGN: Eight hundred and fifty-seven routine respiratory specimens were tested by IF with rabbit polyclonal antiserum and monoclonal antibody pool in parallel. A further 1003 specimens were tested with the monoclonal antibody pool alone. All specimens were also tested for a panel of other respiratory viruses by IF. RESULTS: Both rabbit polyclonal antiserum and monoclonal antibody pool gave positive results in 56 and negative results in 797 specimens. The rabbit polyclonal antibody detected virus in a further two specimens which were negative when tested with the monoclonal pool giving a concordance of 96.6% and a specificity of 100% for the monoclonal antibody pool. Overall hMPV was detected in 5% of specimens whilst 18.4% were positive for hRSV. CONCLUSIONS: The monoclonal antibody pool-based IF is a robust assay suitable for routine use with a sensitivity only slightly less than that of the other major diagnostic methodologies available.

Detection of human metapneumovirus in respiratory secretions by reverse-transcriptase polymerase chain reaction, indirect immunofluorescence, and virus isolation in human bronchial epithelial cells.

Over two winters in Newcastle upon Tyne, respiratory secretions, negative by immunofluorescence staining for other respiratory viruses, were tested for the presence of human metapneumovirus (HMPV) by RT-PCR. In the second winter, specimens were also tested by immunofluorescence staining with an anti-HMPV polyclonal rabbit antiserum and immunofluorescence positive specimens were inoculated into a line of human bronchiolar cells, 16HBE140. Overall, 55 of 549 (10%) specimens tested were positive for HMPV by RT-PCR. Of 162 specimens tested by both RT/PCR and immunofluorescence staining, 23 were positive by both techniques. Of five specimens positive by RT-PCR alone, only one was confirmed with a second set of primers. Of three specimens positive by immunofluorescence alone, only one was confirmed by virus culture. All four previously recognized sub-genotypes of the virus were identified by both RT-PCR and immunofluorescence staining. Sub-genotype A1 was prevalent in the first winter and B1 prevalent in the second. HMPV replication and virus isolation rates were higher in 16HBE140 cells than in monkey kidney cells and did not require exogenous trypsin. Low passage isolates of both sub-genotypes A2 and B1 replicated slowly reaching peak titers only 12 days after inoculation. In summary, single round RT/PCR and immunofluorescence staining with a polyclonal rabbit antiserum proved of equal sensitivity in the diagnosis of HMPV infection in respiratory secretions both detecting 96% of confirmed positive specimens. 16HBE40 cells provided a significant improvement on monkey kidney cells for the isolation and propagation of the virus.

Immunohistochemical assessment of hepatitis C virus antigen in cholestatic hepatitis after liver transplantation.

BACKGROUND: Patients with common variable immunodeficiency may exhibit rapidly progressive hepatitis when infected with hepatitis C virus (HCV), leading to cirrhosis and liver failure. Liver transplantation in these patients may result in a cholestatic form of HCV reinfection with exceptionally high virus loads. AIMS: To report an immunohistochemical investigation of the pretransplant and post-transplant liver of one such patient. METHODS/RESULTS: On immunohistochemical staining of frozen sections with anti-HCV core monoclonal antibody or fluorescein labelled human polyclonal anti-HCV IgG, no HCV antigens were demonstrated in the native cirrhotic liver removed at transplant, despite a viral load of 10(6.4) genomes/g. The transplanted liver, collected six weeks post-transplant, exhibited cholestatic recurrent hepatitis, had an HCV virus load of 10(10) genomes/g of liver, and revealed HCV antigen in the cytoplasm of most hepatocytes, with a pronounced periportal distribution. No virus antigen was demonstrable in other cell types. The core antigen was also detected in paraffin wax embedded, formaldehyde fixed tissue of this liver after high temperature antigen retrieval, but not in the native cirrhotic liver or a selection of HCV positive livers collected pretransplant from immunocompetent patients. Attempts to delineate the distribution of E1, NS3, and NS4 antigens were unsuccessful because monoclonal antibodies to these antigens produced "false positive" staining of foci of hepatocytes in the post-transplant livers of HCV seronegative patients with cholestasis. CONCLUSION: This case provided an opportunity to study the natural development of HCV during acute infection in the absence of an immune response, and may help to elucidate the pathogenesis of HCV recurrence in liver allografts.

Measurement of antibody against contemporary virus lineages of human respiratory syncytial virus sub-group A in infants and their mothers.

BACKGROUND: Human respiratory syncytial virus (hRSV) infects the majority of infants in their first year of life. Maternal antibodies offer some protection although a small proportion of infected infants develop bronchiolitis and require admission to hospital. A number of lineages of the virus co-circulate in the population and the prevalent virus lineage changes from epidemic to epidemic. The effect of antigenic variation between virus lineages upon the protection offered by maternal antibodies has not been assessed. OBJECTIVES: To explore the possibility that infants may develop bronchiolitis because of a virus lineage-specific deficiency in their maternal antibodies. STUDY DESIGN: Virus isolates from infants admitted to hospital in Newcastle upon Tyne with hRSV infection during two consecutive winter epidemics were classified into lineages by genotypic analysis. Antibodies to the surface glycoproteins of contemporary sub-group A lineages and to the A2 virus strain were assayed in the acute sera of infected infants, in a group of uninfected infants and in the mothers of both groups. RESULTS: Four lineages of sub-group A hRSV were found circulating during the study period. Antibody titres measured against all virus lineages in the acute serum of infants with hRSV bronchiolitis were similar. In the uninfected infants and in the mothers of both infected and uninfected groups antibody titres to all four contemporary virus lineages were also similar. However, in these groups antibodies to the A2 virus strain were four-fold lower than those to contemporary isolates. CONCLUSIONS: Infants admitted to hospital with hRSV bronchiolitis exhibited no apparent selective deficiency in maternal antibodies to the viral glycoproteins of the infecting virus strain or lineage.

Expression of topoisomerase IIIalpha in normal and neoplastic tissues determined by immunohistochemistry using a novel monoclonal antibody.

Topoisomerases are nuclear enzymes that modulate the topological structure of DNA in order to facilitate cellular events such as replication and transcription. These enzymes are also the cellular targets of certain classes of chemotherapeutic agents termed topoisomerase poisons. A new human topoisomerase isoform, IIIa, was discovered in 1996, which is thought to have roles in genome stability and possibly chromosome separation during mitosis. It is possible that novel or existing anti-topoisomerase agents target topoisomerase IIIa, suggesting that this enzyme may have potential as a prognostic marker and chemotherapeutic target. In order to study expression patterns of topoisomerase IIIa we have produced a novel monoclonal antibody to human topoisomerase IIIa (TOPO3a-1A4), and used it to assess topoisomerase IIIa expression in a wide range of normal and neoplastic tissues. We have found that topoisomerase IIIa is expressed in a wide range of tissue types, with especially high concentrations in endothelial cells and stromal fibroblasts. No general relationship was observed between expression of topoisomerase IIIa and proliferation. Expression in neoplastic tissues often paralleled their normal counterparts, although certain tumours showed either increased (e.g. colonic adenoma) or reduced (e.g. gastric carcinoma, small cell carcinoma of bronchus) expression. If topoisomerase IIIa is found to be a target for chemotherapeutic agents, clinical response in different tumour types may be related to topoisomerase IIIa expression, which may be assessed using TOPO3a-1A4.

A monoclonal antibody pool for routine immunohistochemical detection of human respiratory syncytial virus antigens in formalin-fixed, paraffin-embedded tissue.

Four monoclonal antibodies (MAbs) with specificities for epitopes on human respiratory syncytial virus (RSV) proteins preserved after formalin fixation and paraffin embedding were identified in fixed and embedded virus-infected HEp-2 cell pellets. The MAbs bound epitopes on the fusion protein, the nucleoprotein, the phosphoprotein, and the M2 protein of the virus. Following high-temperature antigen unmasking, immunohistochemical staining revealed RSV antigens in the lungs of five of seven children who died with confirmed RSV infection and in none of nine children who died for other reasons, with no evidence of RSV infection. Staining was cytoplasmic, granular, and confined to epithelial cells. Intense staining was seen at the apex of ciliated bronchial and bronchiolar epithelial cells in all five positive cases. In one case, of pneumonitis, infected pneumocytes were present in the alveoli and in several cases, CD68-positive, cytokeratin-negative alveolar macrophages stained for viral antigens. These antibodies may prove useful in studies of the pathogenesis of RSV infection.

Candidiasis and atrophic tongue lesions.

In an attempt to shed more light on the role of Candida albicans in the genesis of median rhomboid glossitis, a three-part study of normal, atrophic, and cadaver tongues was undertaken. Occasional fungal hyphae were found in 36 percent of the clinically normal tongues. Hyphae were far more numerous in cytologic smears from atrophic tongue lesions than in those from normal tongues. More than 40 percent of the cadaver tongues showed fungal hyphae. The possible role of C. albicans as a cause of median rhomboid glossitis is discussed in the light of these findings.