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R Crainic

Publications and source records attributed to R Crainic.

At least 55 records · Page 3Linked to original sources
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

Engineering a poliovirus type 2 antigenic site on a type 1 capsid results in a chimaeric virus which is neurovirulent for mice.

Poliovirus type 2 (PV-2) Lansing strain produces a fatal paralytic disease in mice after intracerebral injection, whereas poliovirus type 1 (PV-1) Mahoney strain causes disease only in primates. Atomic models derived from the three-dimensional crystal structure of the PV-1 Mahoney strain have been used to locate three antigenic sites on the surface of the virion. We report here the construction of type 1-type 2 chimaeric polioviruses in which antigenic site 1 from the PV-1 Mahoney strain was substituted by that of the PV-2 Lansing strain by nucleotide cassette exchange in a cloned PV-1 cDNA molecule. These chimaeras proved to have mosaic capsids with composite type 1 and type 2 antigenicity, and induced a neutralizing response against both PV-1 and PV-2 when injected into rabbits. Moreover, a six-amino-acid change in PV-1 antigenic site 1 was shown to be responsible for a remarkable host-range mutation in so far as one of the two type 1-type 2 chimaera was highly neurovirulent for mice.

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↗
PubMed1988

Addition of a foreign oligopeptide to the major capsid protein of poliovirus.

Insertion mutants of type 3 poliovirus (Sabin strain) were constructed that encode additional amino acid sequences at the level of residue 100 of the capsid polypeptide VP1 within the neutralization site 1, corresponding to a loop on the capsid surface. The addition of a tri- or hexapeptide did not hamper virus viability. The antigenic pattern of insertion mutants was only modified locally: all mutants lost reactivity of neutralization site 1 with the corresponding monoclonal antibodies, while the reactivity of sites 2 and 3 was unaffected by the insertion. We have shown for one of the mutants--vFG68--that the antigenic specificity of the neutralization site 1 was replaced by a new one. Although vFG68 differs from its parental Sabin strain only by the addition of three amino acids within VP1, neutralizing antibodies specific for vFG68 were induced by the native virion as well as by the heat-denatured mutated virions. Our results demonstrate that an oligopeptide of three or six amino acids can lengthen VP1 at the level of antigenic site 1 without affecting virus multiplication and that this foreign peptide is exposed on the virion surface.

Amino Acid Sequence↗
PubMed1988

Presentation and immunogenicity of the hepatitis B surface antigen and a poliovirus neutralization antigen on mixed empty envelope particles.

Insertion of a synthetic DNA fragment encoding a poliovirus neutralization epitope into the S gene encoding the major envelope protein of hepatitis B virus has yielded hybrid (HBsPolioAg) particles closely resembling authentic 22-nm antigen (HBsAg) particles by expression of the modified gene in mammalian cells. In mice, these hybrid particles induce neutralizing antibodies against poliovirus but only weak immune response to HBsAg (F. Delpeyroux, N. Chenciner, A. Lim, Y. Malpièce, B. Blondel, R. Crainic, S. Van der Werf, and R. E. Streeck, Science 233:472-474, 1986). By cotransfection with different plasmids carrying either modified or unmodified S genes, we have now obtained mixed particles presenting both HBsAg and HBsPolioAg. When such particles were inoculated into rabbits, antibodies to both poliovirus and to HBsAg were induced. Moreover, the titers of neutralizing antibodies to poliovirus induced by HBsPolioAg were much higher than those previously obtained in mice. The design of multivalent particles carrying various peptide sequences or presenting several heterologous epitopes may therefore be possible.

Animals↗
PubMed1987

Antigenic and biochemical characterization of poliovirus type 2 isolated from two cases of paralytic disease.

Three isolates of poliovirus type 2 were obtained from rectal and pharyngeal swabs of two patients with paralytic poliomyelitis from Southern Italy (1981/1983). Neither one of the patients had received oral poliovirus vaccine, although a systematic vaccination campaign is conducted in the area. Neutralization tests with strain-specific antisera and analysis of the structural proteins by SDS-PAGE indicated that the three isolates were typical Sabin-like poliovirus type 2. Moreover, neutralization assays by means of a panel of well-characterized monoclonal antibodies indicated that the three isolates carried an antigenic mosaic where both wild-type and Sabin-specific epitopes were represented. The T1 fingerprints of the RNA of the three isolates diverged slightly from the standard Sabin reference map. A comparison of the oligonucleotide fingerprints of the viruses isolated from the pharynx and the feces of one patient indicated that mutations appeared as early as during replication in the intestinal tract.

Antibodies, Monoclonal↗
PubMed1986

A poliovirus neutralization epitope expressed on hybrid hepatitis B surface antigen particles.

The hepatitis B virus (HBV) envelope protein carrying the surface antigen (HBsAg) is assembled with cellular lipids in mammalian cells into empty viral envelopes. In a study to evaluate the capacity of such particles to present foreign peptide sequences in a biologically active form, in-phase insertions were created in the S gene encoding the major envelope protein. One of the sequences inserted was a synthetic DNA fragment encoding a poliovirus neutralization epitope. Mammalian cells expressing the modified gene secreted hybrid particles closely resembling authentic 22-nanometer HBsAg particles. These particles reacted with a poliovirus-specific monoclonal antibody and induced neutralizing antibodies against poliovirus. The results indicate that empty viral envelopes of HBV may provide a means for the presentation of peptide sequences and for their export from mammalian cells.

Animals↗
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

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↗
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↗
PubMed1983

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.

Animals↗
PubMed1983

A poliovirus type 1 neutralization epitope is located within amino acid residues 93 to 104 of viral capsid polypeptide VP1.

Using nuclease Bal31, deletions were generated within the poliovirus type 1 cDNA sequences, coding for capsid polypeptide VP1, within plasmid pCW119. The fusion proteins expressed in Escherichia coli by the deleted plasmids reacted with rabbit immune sera directed against poliovirus capsid polypeptide VP1 (alpha VP1 antibodies). They also reacted with a poliovirus type 1 neutralizing monoclonal antibody C3, but reactivity was lost when the deletion extended up to VP1 amino acids 90-104. Computer analysis of the protein revealed a high local density of hydrophilic amino acid residues in the region of VP1 amino acids 93-103. A peptide representing the sequence of this region was chemically synthesized. Once coupled to keyhole limpet hemocyanin, this peptide was specifically immunoprecipitated by C3 antibodies. The peptide also inhibited the neutralization of poliovirus type 1 by C3 antibodies. We thus conclude that the neutralization epitope recognized by C3 is located within the region of amino acids 93-104 of capsid polypeptide VP1.

Amino Acid Sequence↗
PubMed1983

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.

Amino Acid Sequence↗
PubMed1983

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.

Antibodies, Monoclonal↗