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F Horaud

Publications and source records attributed to F Horaud.

At least 37 records · Page 2Linked to original sources
PubMed1991

European project concerning the guidelines on medicinal products derived from human blood and plasma.

The Biotechnology/Pharmacy Working Party (BPWP) decided in October 1989 to put on its working programme the elaboration of a guideline on medicinal products derived from human blood and plasma. This has been determined by the adoption of Directive 89/381/EEC extending the scope of the EEC pharmaceutical legislation to medicinal products derived from human plasma. Because Directive 89/381/EEC will come into force on 1 Jan. 1992, the existing products derived from plasma should be reviewed in order to harmonize them with the EEC requirements before 1 Jan. 1993. In this paper, I give an account of the main points and questions addressed by the BPWP draft in order to stimulate comments from producers and regulatory agencies.

Biological Products↗
PubMed1990

Poliovirus permissivity and specific receptor expression on human endothelial cells.

To test the role of the endothelial cells (EC) in the poliomyelitis pathogenesis, their sensitivity to poliovirus infection was determined at different times after isolation from the human umbilical vein. While 80% of EC were permissive for poliovirus after 4 days of in vitro primary cultures, only 6% of freshly isolated EC were susceptible to poliovirus infection. A progressive development of this susceptibility was observed during the first 3 days of culture. In an attempt to explain the mechanism of the appearance of cell permissivity for poliovirus, the expression of the poliovirus receptor on EC was studied by cytofluorometric analysis using an anti-receptor monoclonal antibody. The number of poliovirus receptor molecules in the EC population was found to increase with time. This paralleled the increase of the poliovirus-binding capacity of EC cultures. In contrast, the efficiency of viral internalization did not appear to be dependent on the age of culture. These results indicate that the development of EC permissivity for poliovirus in vitro is mainly dependent on the expression of poliovirus receptor on the cell surface.

Cells, Cultured↗
PubMed1990

Mapping of mutations associated with neurovirulence in monkeys infected with Sabin 1 poliovirus revertants selected at high temperature.

Poliovirus type 1 neurovirulence is difficult to analyze because of the 56 mutations which differentiate the neurovirulent Mahoney strain from the attenuated Sabin strain. We have isolated four neurovirulent mutants which differ from the temperature-sensitive parental Sabin 1 strain by only a few mutations, using selection for temperature resistance: mutant S(1)37C1 was isolated at 37.5 degrees C, S(1)38C5 was isolated at 38.5 degrees C, and S(1)39C6 and S(1)39C10 were isolated at 39.5 degrees C. All four mutants had a positive reproductive capacity at supraoptimal temperature (Rct+ phenotype). Mutant S(1)37C1 induced paralysis in two of four cynomolgus monkeys, and the three other mutants induced paralysis in four of four monkeys. The lesion score increased from the S(1)37C1 mutant to the S(1)39 mutants. To map the mutations associated with thermoresistance and neurovirulence, we sequenced all regions in which the Sabin 1 genome differs from the Mahoney genome. The S(1)37C1 mutant had one mutation in the 5' noncoding region and another in the 3' noncoding region. Mutant S(1)38C5 had these mutations plus another mutation in the 3D polymerase gene. The S(1)39 mutants had three additional mutations in the capsid protein region. The mutations were located at positions at which the Sabin 1 and Mahoney genomes differ, except for the mutation in the 5' noncoding region. The noncoding-region mutations apparently confer a low degree of neurovirulence. The 3D polymerase mutation, which distinguishes S(1)38C5 and S(1)39 mutants from S(1)37C1, is probably responsible for the high neurovirulence of S(1)38C5 and S(1)39 mutants. The capsid region mutations may contribute to the neurovirulence of the S(1)39 mutants, which was the highest among the mutants.

Animals↗
PubMed1989

Molecular pathogenesis of type 2 poliovirus in mice.

Lansing strain of poliovirus type 2 (PV-2) produces a fatal paralytic disease in mice after intracerebral inoculation. To identify virus-containing nerve cells in poliovirus-infected mice, we developed a technique of in situ hybridization for viral RNA with a poliovirus-specific riboprobe. Large numbers of genomes were found in motoneurons and their processes, as well as in ghosts of neurons with or without inflammatory cells. This indicates that the death of motoneurons is due to a direct effect of viral replication. The presence of viral RNA in neuronal processes suggests an axonal dissemination of poliovirus within the central nervous system (CNS). Detection of viral genomes in small cells located in anterior and in posterior horns shows that motoneurons are not the only cells susceptible to poliovirus in the CNS. To see if PV-2/Lansing still exits in nature, PV-2 isolated from the CNS and from the gut of two human cases of paralytic poliomyelitis were characterized by determining epitope maps and phenotypic markers and by sequencing regions of the genome coding for antigenic sites and the 5' non-coding region. Comparison of virus isolated from the intestine and from the CNS of the same subject revealed a high degree of homology, suggesting that virus isolated from the gut is representative of virus in the neuronal lesions. The isolate from a case had more homology to PV-2/Lansing, whereas the other isolate showed more homology to PV-2/Sabin. These results show that, despite intensive vaccination with oral polio vaccine for 25 years, wild PV-2/Lansing-like viruses, pathogenic for mice, still circulate in nature ad induce human paralytic cases.

Animals↗
PubMed1989

Molecular pathogenesis of neural lesions induced by poliovirus type 1.

Using in situ hybridization techniques for viral RNA and employing a specific riboprobe, we have detected virus in neural cells of monkeys infected with poliovirus type 1 (PV-1) by the intraspinal route. In monkeys paralysed after inoculation of a neurovirulent revertant of PV-1/Sabin strain, viral RNA was detected in motor neurons and their processes, and in polymorphonuclear and small neural cells. Quantitative in situ hybridization provided evidence of viral replication in individual cells suggesting that the death of motor neurons was due to the direct effect of poliovirus replication in these cells. The histological study of neural lesions of monkeys paralysed after infection with the attenuated Sabin strain of PV-1 revealed two major differences compared to monkeys infected with a virulent strain: (i) the number of destroyed motor neurons was reduced and limited to the site of inoculation and (ii) the inflammatory reaction was localized but more intense. An account is given of the difference in histopathology induced by virulent and attenuated strains of PV-1 in the central nervous system.

Animals↗
PubMed1988

Construction and characterization of hybrid hepatitis B antigen particles carrying a poliovirus immunogen.

The hepatitis B surface antigen (HBsAg) has the unique property of assembling with cellular lipids into spherical or elongated particles of 22 nm diameter which are secreted by mammalian cells expressing HBsAg. We have studied the structural requirements for particle formation and secretion by creating in-phase insertions into different regions of the S gene of the hepatitis B virus, coding for HBsAg. Modified genes were integrated into an appropriate vector and expressed in mouse L cells. Various single and double inserts in the two major hydrophilic domains of HBsAg were compatible with particle synthesis and secretion. The level of secretion was influenced by the length of the insert, its primary structure, and the site of insertion into the HBsAg molecule. One of the inserted sequences was a synthetic DNA fragment encoding a continuous type 1 poliovirus neutralization epitope (the C3 epitope). Mammalian cells expressing the modified hepatitis B virus S gene secreted hybrid particles carrying the poliovirus antigen. The hybrid polio-HBsAg particles reacted with a monoclonal antibody specific for the C3 epitope and induced poliovirus neutralizing antibodies at low, but significant, titers in mice and at high titers in rabbits. However, the immune response to HBsAg was weaker to hybrid particles than to unmodified HBsAg particles. By cotransfection with two different plasmids carrying either modified or unmodified genes, we obtained phenotypically mixed particles containing both polio-HBsAg and HBsAg molecules. Inoculated into rabbits, the mixed particles induced high antibody titers against both poliovirus and HBsAg.

Amino Acid Sequence↗
PubMed1986

Antigenic and molecular properties of type 3 poliovirus responsible for an outbreak of poliomyelitis in a vaccinated population.

Virus isolated from an outbreak of poliomyelitis in Finland has been examined serologically and at the molecular level. The causative agent was an antigenically unusual strain of type 3 poliovirus, which was unrelated to the strains used to manufacture either live or killed poliovaccines. It is likely that the antigenic properties of the virus played a part in establishing a limited outbreak of poliomyelitis in a vaccinated population.

Amino Acid Sequence↗
PubMed1986

Mutations conferring resistance to neutralization with monoclonal antibodies in type 1 poliovirus can be located outside or inside the antibody-binding site.

Antigenic variants resistant to eight neutralizing monoclonal antibodies were selected from wild (Mahoney) and attenuated (Sabin) type 1 infectious poliovirions. Cross-immunoprecipitation revealed interrelationships between epitopes which were not detected by cross-neutralization. Operational analysis of antigenic variants showed that seven of eight neutralization epitopes studied were interrelated. Only one neutralization epitope, named Kc, varied independently from all the others. This latter, recognized by C3 neutralizing monoclonal antibody, was present not only on infectious virions but also on heat-denatured (C-antigenic) particles and on isolated capsid protein VP1. Loss of the neutralization function of an epitope did not necessary result from the loss of its antibody-binding capacity. Such potential, but not functional, neutralization epitopes exist naturally on Mahoney and Sabin 1 viruses. Their antibody-binding property could be disrupted by isolating antigenic variants in the presence of the nonneutralizing monoclonal antibody and anti-mouse immunoglobulin antibodies. Single-point mutations responsible for the acquisition of resistance to neutralization in the antigenic variants were located by sequence analyses of their genomes. Mutants selected in the presence of C3 neutralizing monoclonal antibody always had the mutation located inside the antibody-binding site (residues 93 through 103 of VP1) at the amino acid position 100 of VP1. On the contrary, antigenic variants selected in the presence of neutralizing monoclonal antibodies reacting only with D-antigenic particles had mutations situated in VP3, outside the antibody-binding site (residues 93 through 103 of VP1). The complete conversion of the Mahoney to the Sabin 1 epitope map resulted from a threonine-to-lysine substitution at position 60 of VP3.

Amino Acid Sequence↗
PubMed1985

Comparative expression of the hepatitis B surface antigen gene in biochemically transformed human, simian and murine cells.

In this paper, we show that the pattern of expression of the human hepatitis B surface antigen (HBs Ag) gene, transfected along with a dominant selectable marker into mammalian cells, is complex. In human hepatoma (HepG2) cells, late transient expression occurs and permanent expression takes place at high frequencies in the selected clones. In HeLa and human xeroderma pigmentosum (GM4312A)-derived cells, the late transient expression is barely seen or absent and permanent expression is only seen in a few selected clones. In monkey kidney Vero cells, late transient expression has been described and we show in this report that only 5% of the selected clones are capable of expressing HBs Ag in a permanent manner. In most of the Vero clones, the absence of HBs Ag expression is mainly due to HBs Ag gene rearrangements. We have selected and amplified more than 500 transfected Vero clones and have characterized in detail one clone (GAR1412) which is a permanent high-level HBs Ag expressor.

Animals↗
PubMed1985

Mapping of type 1 poliovirus neutralization epitopes.

Several independently variable neutralization epitopes were identified on type 1 poliovirus by testing various field isolates with a panel of homotypic monoclonal antibodies. According to their distribution among viruses tested, neutralization epitopes were classified as strain-specific or variable (V) and cross-reacting or constant (K). Operational, relationship between different neutralization epitopes was analysed by selecting mutants resistant to neutralization with monoclonal antibodies from wild Mahoney and attenuated Sabin type 1 poliovirus. The results of this study and their implication in the characterization of poliovirus antigenic structure by neutralization epitope formulae is discussed.

Antibodies, Monoclonal↗
PubMed1985

Antigenic modification of attenuated Sabin type 1 poliovirus by in vitro passages at supraoptimal temperatures.

Mutants were selected from Sabin type 1 attenuated poliovirus (LSc2ab strain), capable of growing at a high temperature (39.5 degrees C). They proved to be neurovirulent for monkeys. No correlation was found between neurovirulence and antigenic structure in Sabin type 1 virus as demonstrated by the analysis of the neutralization epitope formulae of thermo-resistant, neurovirulent mutants derived from LSs2ab strain. The lack of correlation between the antigenic pattern of the virus and the virulence was also confirmed by a mutant resistant to neutralization with monoclonal antibodies derived from the wild Mahoney parent of the Sabin type 1 virus. This mutant continued to be neurovirulent in spite of the complete conversion of its neutralization epitope formula to the Sabin virus pattern.

Animals↗
PubMed1985

Stable HBV surface antigen expression by Vero cell clones after transfection.

After transfection of Vero cells with the hepatitis B surface (HBs) antigen gene and the dominant selective marker amino-glycoside 3' phosphotransferase (APH3'), a late transient HBs expression is observed during the first weeks after transfection. We isolated 300 G418-resistant cell clones transformed by plasmids carrying the two genes. Only 6% of them were found to express the HBs antigen stably (versus 65% in murine cells). The HBs antigen expression is maximal after cells have reached confluency, and this level of antigen expression remains stable for months when cells are fed twice weekly without trypsinization. One clone - GAR 1412 - has been studied in greater detail. The cells (10(6] are not tumorigenic for nude mice, and the excreted HBs antigen, which is under the form 22 nm spherical particles, is immunogenic in Balb/C mice.

Animals↗
PubMed1985

A rabies vaccine produced in a non-tumorigenic rabbit cell line.

From the trypsinized skin of a rabbit embryo, a fibroblastic cell line has been obtained (FEL cells). The primary seed lot has been established at 11th passage. The tests for tumorigenicity on nu/nu mice were negative (10(4) cell/animal) at passage level 11th and 50th. Cell growth and morphology were not altered when 100 passages were performed. FEL cells were tested for their susceptibility to rabies virus. The production of virus on FEL cells was investigated after infection by the P.V. rabies virus strain adapted to several cell types (bovine fetal kidney, Vero and BHK cells). For the evaluation of an experimental rabies vaccine produced with this substrate, FEL cells were compared to Vero cells. The synthesis of glycoprotein (EIA) and the immunogenicity (NIH test) were similar for the vaccines obtained with both cell systems. FEL cells could therefore be an alternative to Vero cells for the production of a cell culture inactivated rabies vaccine.

Animals↗
PubMed1984

Neutralization epitope patterns of poliovirus strains isolated from paralytic cases.

Monoclonal antibodies were used in previous work to define neutralization epitopes (NE) of poliovirus during its natural variation upon interhuman passages. Two kind of NE were found: constant (K) epitopes, present on practically all poliovirus strains of the same serotype, and variable (V) epitopes, which were strain-specific either for wild-laboratory strains (Mahoney, MEF1, and Saukett) (VW epitopes), or for attenuated Sabin strains (VS epitopes), or for attenuated Sabin strains (VS epitopes). In the present paper we analyzed the NE formulae of poliovirus strains of each of the three serotypes isolated from paralytic cases that have occurred after mass vaccination with oral poliovaccine (Sabin). Type 1 isolates from both the central nervous system and stools showed the existence of the four VW epitopes characteristic for Mahoney virus. However, in some cases, variation of the K epitope formula was noticed. As concerns the paralytic cases where type 2 poliovirus was isolated, the situation was more complex due to the existence of three different VS epitopes. Various patterns of VS epitopes were encountered, making difficult the definition of the strains origin. Most of the type 3 strains isolated from paralytic cases contained one or both VS epitopes, some of them having an identical NE formula to the Sabin type 3 vaccine virus. These data showed that the use of strain-specific neutralizing monoclonal antibodies for the characterization of strain origin is more complex than generally agreed. The multitude of NE coexisting on the same virus particle and their variation during viral replication in the human intestine call for cautious interpretation of the results obtained in poliovirus intratypic differentiation by neutralizing monoclonal antibodies.

Antibodies, Monoclonal↗