Natural variation of poliovirus neutralization epitopes.

Poliovirus-neutralizing monoclonal antibodies were prepared against type 1, type 2, and type 3 wild laboratory (Mahoney, MEF1, and Saukett) and Sabin vaccine strains. Fifty-five poliovirus laboratory strains and field isolates were assayed by neutralization index test with a panel of homotypic monoclonal antibodies. A total of 27 monoclonal antibodies were used. Two categories of neutralization epitopes were found, i.e., cross-reacting (K), which is present on almost all strains of the same serotype, and strain-specific (V, variable), either wild (VW) or Sabin (VS). Several distinct neutralization epitopes were defined for each of the three poliovirus serotypes in almost every category. The study of antigenic variation of the Sabin type 1 vaccine virus during replication in human intestine showed that the VS neutralization epitope may be lost and even replaced by the VW epitopes of the parental Mahoney virus. A late isolate from a vaccine-fed infant recovered the complete neutralization epitope pattern of the Mahoney strain. Upon in vivo virus replication, a different kind of antigenic variation was also detected in which an epitope lost its function in virus neutralization but kept its antigenic conformation unaltered. Neutralization epitope analysis demonstrated that the presence of VS epitopes on a field isolate suggests the Sabin origin of the strain when the isolate displays the same epitope pattern as the original Sabin virus, or confirms it when the VS epitope(s) is mutually exclusive of VW epitopes. The lack of VS epitopes on a field isolate does not rule out its being of Sabin origin.

Localization of a poliovirus type 1 neutralization epitope in viral capsid polypeptide VP1.

Poliovirus type 1 cDNA sequences coding for viral capsid polypeptide VP1 were inserted into the beta-lactamase sequence of Escherichia coli plasmid pBR322. Resulting recombinant plasmid pSW119 expressed in Escherichia coli a VP1-beta-lactamase fusion protein that reacted with antibodies raised against poliovirus capsid polypeptide VP1 and with a monoclonal poliovirus type 1 neutralizing antibody, C3. Deletions of various lengths were generated within the VP1 sequence. The hybrid proteins expressed by the deleted plasmids did not react any more with C3 when the region of VP1 amino acids 95-110 (poliovirus nucleotides 2,754-2,806) was deleted. Therefore, the C3 epitope responsible for virus neutralization is most probably located in this region of the capsid polypeptide.

Detection by monoclonal antibodies of an antigenic determinant critical for poliovirus neutralization present on VP1 and on heat-inactivated virions.

Hybridoma cell lines were established against poliovirus type 1 (Mahoney) heat-denatured virions (C particles). Each anti-C monoclonal antibody (McAb) immunoprecipitated specifically one of the individualized poliovirus capsid polypeptides VP1, VP2, or VP3. One of the anti-C McAb (C-3), reacting with VP1, neutralized homologous virus and immunoprecipitated infectious D particles. Its properties have been compared to those of a neutralizing anti-D McAb (D-Ic). In contrast with the C-3 antigenic site, the D-Ic epitope was not present on C particles nor on individualized structural polypeptide. This demonstrates that C-3 and D-Ic epitopes represent two independent antigenic determinants, both critical for poliovirus neutralization.

Late transient expression of human hepatitis B virus genes in monkey cells.

The expression of human hepatitis B virus (HBV) surface (HBS) and e (HBe) antigens has been studied comparatively in monkey and mouse cell lines co-transfected with HBV DNA and the dominant selective marker aminoglycoside 3'-phosphotransferase gene. We have found that the kinetics and stability of expression of the HBS gene varies with the cell lines used. Only a late transient expression of both HBS and HBe is observed between 1 and 5 weeks after transfection in monkey kidney Vero cells transfected with the complete HBV genome, while a permanent expression of HBS and HBe is obtained in mouse cells. HBS and HBe are excreted into the cell culture medium. HBe is expressed in cells transfected with the complete HBV genome, but not with isolated HBS gene. In clones of Vero cells transformed with the HBS gene, HBV sequences were rearranged or lost.

[Structural polypeptide VP1 in poliovirus type 1 induced by neutralizing antibodies]. / Le polypeptide structural VP1 du poliovirus type 1 induit des anticorps neutralisants.

Anti-type 1 (Mahoney) poliovirus neutralizing antibodies were obtained by immunizing rabbits with structural polypeptide VP1. This polypeptide was isolated from the virion by PAGE-SDS electrophoresis and extracted from the gel by electroelution. Anti VP2 and anti VP3 sera prepared in the same was did not display any significant antipolio neutralizing activity.

Freeze-dried erythrocytes for an indirect hemagglutination test for detection of cytomegalovirus antibodies.

An indirect hemagglutination test with lyophilized, fixed, tanned, and cytomegalovirus (CMV)-sensitized sheep erythrocytes for the detection of CMV antibodies is reported. To avoid nonspecific hemagglutination, cells were fixed with glutaraldehyde or Formalin directly in whole blood. The lyophilized, CMV-sensitized erythrocytes obtained by this technique were stable up to 9 months at 37 degrees C and retained the same reactivity at fresh, CMV-sensitized cells. Indirect hemagglutination performed with lyophilized, sensitized cells was highly efficient in detecting CMV-antibodies as compared with complement fixation and enzyme immunoassay.

Expression of herpes simplex virus type I thymidine kinase gene in Escherichia coli.

A herpes simplex virus type I DNA fragment containing the sequence coding for thymidine kinase was fused to the very beginning of the Escherichia coli lac Z gene in the three possible reading frames. When the thymidine kinase sequence was in the orientation fit to be transcribed from the lac promoter, functional thymidine kinase was made under lac control in all three cases. Sequences data indicate that translation reinitiation occurs at the 5' end of the thymidine kinase gene after stop signals. Two T+A-rich sequences, which may be part of eukaryotic promoters, are found in the same region.

Construction of a dominant selective marker useful for gene transfer studies in animal cells.

Biochemically transformed clones of murine, simian and human cells were obtained after transfection with a new dominant selective marker. This marker is a hybrid gene which was constructed with a bacterial neomycin-kanamycin resistance gene coding region and the transcription signals of the herpes simplex virus thymidine kinase gene.

Determination of type 1 poliovirus subtype classes with neutralizing monoclonal antibodies.

Hybridomas secreting neutralizing monoclonal antibodies (MA) against type 1 poliovirus were developed by fusion of mouse myeloma cells with splenocytes of immunized mice. Strain-specific MA selected for their capacity to neutralize wild (Mahoney) or attenuated (Sabin, LSc 2ab) poliovirus strains were used for identification of field isolates. A simple and reliable neutralization-index-assay was developed and used to test for intratypic serodifferentiation of poliovirus with the selected MA. Three subtype classes of type 1 poliovirus have so far been identified by testing 4 reference strains and 96 field isolates: Mahoney-like, LSc-like, and strains not recognized by either anti-Mahoney or anti-LSc MA. Neutralizing MA proved an excellent tool for unequivocal antigenic characterization of different poliovirus strains.

Cell substrate and risk in killed poliomyelitis vaccine.

By using 3H-T labeled HeLa cell DNA it was possible to evaluate the quantity and the state of substrate DNA present in crude and purified killed poliovaccine. The results showed that 1.7 x 10(5) pg/ml of DNA is present in crude vaccine while in purified vaccine this product can not be detected (the limit of the detection method was 1.1 x 10(2) pg/ml). The theoretical problems of the risk of the formation of poliovirus pseudotypes and the potential contamination of the vaccine with a type C retrovirus are examined.

A critical view of cell substrates with regard to cell transformation and cancer.

The basic heuristic method in experimental medicine of proceeding from animals to human beings to facilitate the understanding of the etiology of a disease, is not completely relevant to viral oncogenesis in particular. The situation does not diminish scientific interest in tumor viruses because the study of these oncogens opens the way to identification of cancer genes and the understanding of their functions, expression and regulation in transformed cells. The results of these investigations are essential in comprehending animal tumor biology and from this, within limits, extrapolating to human neoplasia. However, the results of epidemiological, cellular and molecular approaches to human cancer all suggest a multiple and complex origin of this disease. In light of our current knowledge of cell transformation and cancer, the nature of permanent heteroploid cell lines is discussed in relation to their use as substrates for production of biologicals.

Immunofluorescence technique for detection of human cytomegalovirus (HCMV) antibodies against HCMV-induced late antigens with elimination of immunoglobulin G-receptor staining.

An immunofluorescence technique is described which permits the elimination of nonspecific cytoplasmic staining from late antigen preparations by using in situ a buffer containing Nonidet P-40.

Quantitation of cell DNA in the evaluation of heteroploid cells as substrate for the preparation of killed viral vaccines.

To evaluate the risk of using heteroploid cell lines as substrates for viral vaccine production, the presence of cell DNA in poliovirus suspensions was examined. The time course of [3H]-thymidine-labeled HeLa cell DNA release during lytic infection with type 1 poliovirus was investigated. More DNA was found in filtered supernatants of poliovirus-inoculated cultures than in control cell supernatants. DNA concentration increased with time, paralleling virus release, but did not exceed 1.5% of the total DNA content of the culture. Only about 10% of this cell DNA was resistant to DNase treatment. By both ion-exchange chromatography and rate-zonal centrifugation it was possible to remove practically all cell DNA contaminating filtered poliovirus suspensions. Results obtained in this study permitted quantitative evaluation of cell substrate DNA present in poliovirus suspensions during successive steps of killed poliovirus vaccine preparation. Based on the sensitivity of our method, the amount of residual DNA was estimated at less than 0.02 pg per dose of purified vaccine.

Presence and quantification of cell substrate DNA in inactivated poliovirus vaccine.

In order to follow the fate of cell substrate DNA in inactivated poliovirus vaccine (IPV), experiments simulating different steps in IPV preparation were performed. For this purpose, 3H-thymidine-labeled HeLa cell DNA released during lytic infection with Mahoney type 1 poliovirus strain was used as tracer. Low speed centrifugation (1500 xg for 15 min.) of crude virus suspension removed on the average 98.5% of the total labeled cell substrate DNA. Repeated freezing and thawing of the crude virus suspension before centrifugation did not increase the residual 1.5% radioactivity found in the supernatant. This level of contamination was not significantly influenced by filtration of the supernatant through a 0.22 micrometer pore size membrane filter. DEAE-Sepharose CL-6B chromatography of poliovirus suspensions containing large amounts of 3H-thymidine labeled HeLa cell extracts completely removed the contaminating substrate DNA. These data showed that IPV free of cell substrate DNA can easily be obtained by a usual purification procedure.

Cloning of the herpes simplex virus type 1 thymidine kinase gene in E. coli K 12: a selective marker for gene transfer into animal cells.

The Herpes Simplex Virus type 1 (HSV 1) thymidine kinase (TK) gene, carried by a 3.6 kb Bam H1 DNA fragment, was isolated and ligated to Bam H1 cleaved bacterial plasmid pBR 322. The TK gene, cloned in E. coli, is functional when it is transferred back into Eukaryotic cells, and can thus be used as a selective marker for gene transfer.