Radu Crainic

Natural Genetic Exchanges between Vaccine and Wild Poliovirus Strains in Humans

ABSTRACT In a previous study of poliovirus vaccine-derived strains isolated from patients with vaccine-associated paralytic poliomyelitis (VAPP) (9, 11), we reported that a high proportion (over 50%) of viruses had a recombinant genome. Most were intertypic vaccine/vaccine recombinants. However, some had restriction fragment length polymorphism (RFLP) profiles different from those of poliovirus vaccine strains. We demonstrate here that five such recombinants, of 88 VAPP strains examined, carried sequences of wild (nonvaccine) origin. To identify the parental wild donor of these sequences, we used RFLP profiles and nucleotide sequencing to look for similarity in the 3D polymerase-coding region of 61 wild, cocirculating poliovirus isolates (43 type 1, 16 type 2, and 2 type 3 isolates). In only one case was the donor identified, and it was a wild type 1 poliovirus. For the other four vaccine/wild recombinants, the wild parent could not be identified. The possibility that the wild sequences were of a non-poliovirus-enterovirus origin could not be excluded. Another vaccine/wild recombinant, isolated in Belarus from a VAPP case, indicated that the poliovirus vaccine/wild recombination is not an isolated phenomenon. We also found wild polioviruses (2 of 15) carrying vaccine-derived sequences in the 3′ moiety of their genome. All these results suggest that genetic exchanges with wild poliovirus and perhaps with nonpoliovirus enteroviruses, are also a natural means of evolution for poliovirus vaccine strains.

The natural genomic variability of poliovirus analyzed by a restriction fragment length polymorphism assay

The genomic variability of poliovirus was examined by analyzing the restriction fragment length polymorphism of a reverse-transcribed genomic fragment amplified by the polymerase chain reaction. The fragment was a 480-nucleotide sequence of the poliovirus genome coding for the N-terminal half of the capsid protein VP1, including antigenic site 1. The identification of a pair of generic primers flanking this fragment allowed its amplification in practically all the poliovirus strains tested so far (more than 150). By using the restriction enzymes HaeIII, DdeI, and HpaII, strain-specific restriction profiles could be generated for the amplified genomic fragment of each of the six reference poliovirus strains tested: one representative wild poliovirus of each of the three serotypes (P1/Mahoney, P2/Lansing, and P3/Finland/23127/84) and the three Sabin vaccine strains. When 21 poliovirus field isolates previously identified as Sabin vaccine-related were tested, they showed restriction profiles identical to those of the originating homotypic Sabin virus, demonstrating the conservation of these profiles during virus replication in humans. These profiles could thus be used as markers for Sabin-derived genotypes. To compare the distribution of poliovirus genotypes in nature before and after the introduction of poliovirus vaccines, the restriction profiles of the amplified genomic fragment of a total of 72 strains of various geographic and temporal origins were determined. Strains isolated before the introduction of polio vaccines displayed a wide diversity of genotypes. In contrast, wild (Sabin unrelated) strains isolated after vaccine introduction, during a single epidemic in a particular geographic area, showed identical or very similar restriction profiles, indicating the circulation of predominant regional genotypes. Our results indicate that the assay we developed for the analysis of the restriction fragment length polymorphism of the poliovirus genome may be used to identify and characterize poliovirus genotypes circulating in nature.

Genomic Features of Intertypic Recombinant Sabin Poliovirus Strains Excreted by Primary Vaccinees

ABSTRACT The trivalent oral poliomyelitis vaccine (OPV) contains three different poliovirus serotypes. It use therefore creates particularly favorable conditions for mixed infection of gut cells, and indeed intertypic vaccine-derived recombinants (VdRec) have been frequently found in patients with vaccine-associated paralytic poliomyelitis. Nevertheless, there have not been extensive searches for VdRec in healthy vaccinees following immunization with OPV. To determine the incidence of VdRec and their excretion kinetics in primary vaccinees, and to establish the general genomic features of the corresponding recombinant genomes, we characterized poliovirus isolates excreted by vaccinees following primary immunization with OPV. Isolates were collected from 67 children 2 to 60 days following vaccination. Recombinant strains were identified by multiple restriction fragment length polymorphism assays. The localization of junction sites in recombinant genomes was also determined. VdRec excreted by vaccinees were first detected 2 to 4 days after vaccination. The highest rate of recombinants was on day 14. The frequency of VdRec depends strongly on the serotype of the analyzed isolates (2, 53, and 79% of recombinant strains in the last-excreted type 1, 2, and 3 isolates, respectively). Particular associations of genomic segments were preferred in the recombinant genomes, and recombination junctions were found in the genomic region encoding the nonstructural proteins. Recombination junctions generally clustered in particular subgenomic regions that were dependent on the serotype of the isolate and/or on the associations of genomic segments in recombinants. Thus, VdRec are frequently excreted by vaccinees, and the poliovirus replication machinery requirements or selection factors appear to act in vivo to shape the features of the recombinant genomes.

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.

Natural genetic recombination between co-circulating heterotypic enteroviruses

Natural recombination in poliovirus is a frequent phenomenon. In practice, whenever different genotypes have the opportunity to infect the same individual, a high proportion of viruses with recombinant genomes are excreted. To determine whether enteroviruses other than poliovirus can naturally produce viable virions with recombinant genomes, we studied the molecular features of two distant regions of the viral genomes - the VP1 coding region and the 3D polymerase coding region - of the echovirus serotypes associated with a large outbreak of aseptic meningitis. Nucleotide sequences of nine epidemic strains [belonging to echovirus serotypes 4 (E4), 7 (E7) and 30 (E30)] in the two genomic regions (300 nt of VP1 and 520 nt of 3D polymerase) were compared to prototype and field strains, and phylogenetic trees were generated from alignments. In the VP1 region, each of the three epidemic serotypes clustered with the homotypic prototype strain, whereas in the 3D polymerase region, E7 and E30 grouped as a single cluster, distant from the two corresponding prototype strains. This suggests that one of these two E7 and E30 strains has evolved through recombination with the other or that both have acquired the 3D polymerase coding region from a common ancestor. Our results suggest that such genetic recombinations between different echovirus serotypes are possible when multiple epidemic strains are circulating simultaneously.

Structure of the complex between the Fab fragment of a neutralizing antibody for type 1 poliovirus and its viral epitope.

The crystal structure of the complex between the Fab fragment of C3, a neutralizing antibody for poliovirus, and a peptide corresponding to the viral epitope has been determined at 3.0 Å resolution. Although this antibody was originally raised to heat inactivated (noninfectious) virus particles, it strongly neutralizes the Mahoney strain of type 1 poliovirus. Eleven peptide residues are well-defined in the electron-density map and form two type I β-turns in series. At the carboxyl end, the peptide is bound snugly in the antibody-combining site and adopts a conformation that differs significantly from the structure of the corresponding residues in the virus. Structural comparisons between the peptide in the complex and the viral epitope suggests that on binding to infectious virions, this antibody may induce structural changes important for neutralization.

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.

Influence of host related factors on the antibody response to trivalent oral polio vaccine in Tunisian infants

The low efficiency of trivalent oral polio vaccine (TOPV) in inducing protective antibody titres to polio3 is a problem of great importance in many regions of the world. A prospective study was conducted in 121 Tunisian infants aged 3 months during routine immunization with TOPV under carefully controlled conditions. Seroconversion rates to polio1, polio2 and polio3, one month after the third dose, were 94.7, 100 and 89.5%, respectively. The kinetics of the antibody response showed delayed and more difficult responses to polio3 compared to polio2 and polio1. The following host related factors, previously suggested to interfere with the immune response, were assessed: maternal antibodies; breast-feeding; concurrent enteric infections; and other illnesses. The main factor associated with the lack of seroconversion was concurrent infection with non-polio enteroviruses (NPE) which was found in 50% of non-responders to polio1 and/or to polio3 during the vaccination protocol whereas no NPE was isolated in vaccine responders. The other studied factors seemed not to interfere in the infants according to the locally adopted vaccination schedule and to the specific socio-economic conditions.

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.

Characterization of persistent poliovirus mutants selected in human neuroblastoma cells.

Six Sabin-derived persistent poliovirus mutants were selected in human neuroblastoma IMR-32 cells. The mutants had a titer 30 to 10(5) times lower in nonneural HEp-2c cells than in IMR-32 cells. When the growth cycles of persistent viruses in the two cell lines were compared, the most striking feature was a delay of 2 to 4 hr in virus release from HEp-2c cells. In Hep-2c cells, type 1 mutants could spontaneously establish a persistent infection in the absence of any exogenous viral inhibitor. Mutations at a rate of 1 every 210 nucleotides had accumulated in the genome of the type 1 mutants selected in neuroblastoma cells, modifying cell specificity and conferring the ability to persist in some nonneural cells. These results indicate that mutants of poliovirus with highly modified biological properties can be selected in vitro in cells of neural origin.