Method to map antigenic determinants recognized by monoclonal antibodies: localization of a determinant of virus neutralization on the feline leukemia virus envelope protein gp70.

Nunberg, Jack H.; Rodgers, G; Gilbert, James H.; Snead, Richard M.

doi:10.1073/pnas.81.12.3675

Cited by 63 publications

(24 citation statements)

References 55 publications

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“…By expression of subgenomic fragments in a prokaryotic expression system, 2gtll or pEX for example (Nunberg et al, 1984;Mehra et al, 1986;Luytjes et al, 1989;Lenstra et al, 1989), it is possible to localize antigenic domains on proteins. We have chosen to express relatively large subgenomic fragments in pEX to localize domains roughly and to use the Pepscan analysis to localize a number of epitopes precisely (Middeldorp & Meloen, 1988).…”

Section: Discussionmentioning

confidence: 99%

Epitope analysis of glycoprotein I of pseudorabies virus

Jacobs¹,

Meloen²,

Gielkens³

et al. 1990

Journal of General Virology

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A panel of 11 monoclonal antibodies (MAbs) raised against pseudorabies virus (PRV) was used to map epitopes on the virus glycoprotein I (gI). We employed three approaches to map epitopes on gI. By a competition binding assay, six groups of MAbs were defined as reacting with distinct antigenic domains on gI. To identify regions along the gI polypeptide chain encompassing the domains recognized by these MAbs, DNA fragments derived from the gI-coding region were cloned into pEX expression plasmids. The antigenic reactivity of the fusion proteins expressed in Escherichia coil was analysed by immunoblotting. Five antigenic domains were mapped within the first 238 amino acids of gI: domains A, B and D were mapped between amino acids 52 and 123 and domains C and E between amino acids 78 and 238. One MAb, representing domain F, did not react with the expressed fusion proteins. To assess the precise location and amino acid sequences of the epitopes, overlapping nonapeptides covering the amino acid sequence 52 to 238 were synthesized. The antibody-binding activity of these peptides was tested by an ELISA (Pepscan-method). Three antigenic domains were mapped: domain A was localized to amino acids 64 to 73 and 75 to 84, domain B to amino acids 52 to 67 and domain D to amino acids 68 to 82. Four MAbs representing antigenic domains C, E and F did not react in the Pepscan. Finally, sera from pigs infected experimentally with PRV reacted with the fusion protein ofplasmid psi (amino acids 52 to 238).

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Section: Discussionmentioning

confidence: 99%

Epitope analysis of glycoprotein I of pseudorabies virus

Jacobs¹,

Meloen²,

Gielkens³

et al. 1990

Journal of General Virology

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“…We further demonstrate the authenticity of viral genes mapped using this technique by showing that mRNA, hybrid selected on a CMV DNA fragment, is translated in vitro to yield a polypeptide of about 50 kDa which is immunologically crossreactive with ICP36. This system of mapping viral genes is generally applicable to any eukaryotic viral system and can be extended to mapping of epitopes within genes (18).…”

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confidence: 99%

Precise localization of genes on large animal virus genomes: use of lambda gt11 and monoclonal antibodies to map the gene for a cytomegalovirus protein family.

Mocarski¹,

Pereira²,

Michael³

1985

Proc. Natl. Acad. Sci. U.S.A.

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We describe an efficient procedure, which uses monoclonal antibodies directed against specific viral proteins, for the precise mapping of genes on large DNA virus genomes. We have used the technique to locate the gene encoding a family of antigenicaily related DNA-binding proteins on the 240-kilobase-pair human cytomegalovirus (CMV) genome. A random library of CMV DNA fragments was generated using the prokaryotic vector Xgtll, which expresses open reading frames as ,-galactosidase fusion proteins in infected Escherichia coli. The library was screened with a mixture of monoclonal antibodies directed against the gene products of interest. The coding sequence for infected cell protein 36 (ICP36) was localized to a 2800-base-pair EcoRI fragment (map coordinates 0.228-0.240) on the CMV(Towne) and CMV(AD169) genomes by using DNA from immunoreactive Xgtll as probe. A 5000-nucleotide transcript from this region was detected during the early and late phases of the CMV growth cycle. This transcript directed the synthesis of the predominant member of the ICP36 family when hybrid-selected and translated in vitro. Immunoprecipitation of the in vitro translation product with the same monoclonal antibodies used in the initial mapping confirmed the location of the ICP36 gene. These studies establish the utility of the Agtll expression system for rapid and precise mapping of CMV genes (or other large animal virus genes) that encode proteins for which serological reagents exist.Human cytomegalovirus (CMV), a herpesvirus carried by most of the population, causes disease in infants and immunocompromised adults. CMV has a large DNA genome of -240 kilobase pairs (kbp) on which few viral genes have been precisely localized (1-4). Even less is known concerning the functional organization of the viral genome. Studies on the temporal expression of viral proteins and mRNA transcripts (4-11) have suggested that, as for herpes simplex virus (12), CMV gene expression is coordinately regulated and sequentially ordered; however, viral regulatory functions have not been identified. Major obstacles to locating viral genes have been (i) the large size of the CMV genome, (ii) the slow growth of the virus in cell culture, (iii) the continued synthesis of host proteins during infection, (iv) the lack of viral mutants, and (v) a host range limited to human fibroblast cells. We have modified the recently described procedures for cloning in the prokaryotic expression vector Xgtll (13,14), thereby establishing a system using serological reagents for rapid and precise mapping of viral gene products.We constructed a Xgtll library of randomly generated 400-to 500-base-pair (bp) CMV DNA fragments and probed this library with monoclonal antibodies to isolate clones expressing portions of viral proteins as f3-galactosidase fusion proteins. DNAs from immunoreactive phage were used as hybridization probes for precise localization of the coding sequences on the CMV genome.We used two monoclonal antibodies isolated by Pereira et al. (15) to map a major DN...

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“…This leads to a marrow-origin, cellfree viraemia and further tertiary replication in mucosal sites from where it is shed in high concentrations (Francis et al, 1977). Antigenic targets for the virus are less well defined although sites involved in complementmediated lysis, antibody-dependent cellular cytotoxicity of virus-infected cells (McCarty & Grant, 1983;Grant et al, 1980a) and neutralization (Osterhaus et al, 1989;deNoronha et al, 1983;Grant et al, 1980b) have been mapped to the gp70 and pI5E envelope proteins Nunberg et al, 1984;Nick et al, 1990). Studies with analogous viruses would also suggest that both the core gag and env protein can act as important immunological targets for cytotoxic T cells (Tc) (Holt et al, 1986;van der Hoorn et al, t985;Flyer et al, 1983;Manjunath et al, 1986;Martin & Rouse, 1990).…”

Section: Discussionmentioning

confidence: 99%

The use of feline herpesvirus and baculovirus as vaccine vectors for the gag and env genes of feline leukaemia virus

Wardley¹,

Berlinski²,

Thomsen³

et al. 1992

Journal of General Virology

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The env and gag genes from feline leukaemia virus were expressed in a thymidine kinase-negative feline herpesvirus and a baculovirus. Cats were vaccinated with various combinations of these recombinant viruses and 100% protection against feline leukaemia virus challenge was achieved using an immunization schedule which utilized both env and gag products delivered at both a mucosal and systemic site.

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Method to map antigenic determinants recognized by monoclonal antibodies: localization of a determinant of virus neutralization on the feline leukemia virus envelope protein gp70.

Cited by 63 publications

References 55 publications

Epitope analysis of glycoprotein I of pseudorabies virus

Epitope analysis of glycoprotein I of pseudorabies virus

Precise localization of genes on large animal virus genomes: use of lambda gt11 and monoclonal antibodies to map the gene for a cytomegalovirus protein family.

The use of feline herpesvirus and baculovirus as vaccine vectors for the gag and env genes of feline leukaemia virus

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