Development of inactivated poliovirus vaccine derived from Sabin strains.

In the course of Sabin-inactivated poliovirus vaccine (S-IPV) development, we have established high-yield virus production techniques based on Vero cell micro-carrier cultures. Development of specific ELISA tests to quantify the antigen content of S-IPV has been achieved. To adjust the immunogenicity of S-IPV so as to be comparable with the conventional-IPV, a new formulation was determined using a potency test using rats. The reformulated S-IPV was shown to be efficacious for the immunization of monkeys.     

Use of elution marker for the intratypic characterization of poliovirus strains.

Elution marker was used for intratypic characterization of poliovirus strains with Al(OH)3 gel as adsorbent. The virion suspensions to be tested were partially purified by chromatography and labelled with 32P. In the labelled preparations of wild virus strains practically all radioactivity was found in virus-specific bond, whereas in those of the vaccine strains and isolates of vaccine origin a considerable, but variable, proportion of the activity was bound to residual cell components. For this reason, the EC50 value (i.e. the phosphate molarity corresponding to the 50% adsorption equilibrium of virions), for the vaccine and vaccine-like strains showed a wide scattering. The activity bound to cell components was removable from the gel with 0.005 M phosphate buffer, whereas the elution maxima for all the poliovirus strains examined so far were over 0.02 M. The elution marker was calculated for 25 type-2 and 30 type-3 strains, all isolated during or soon after vaccination periods from cases suspect of poliomyelitis, both with and without taking into account the cell-bound activity. In the latter case, the EC50 values agreed with that for the corresponding reference vaccine strain much better than in the former; furthermore, the type-2 reference vaccine strain and the type-2 isolates, indistinguishable from the wild reference strain by the original method, could easily be differentiated on the basis of the corrected EC50 value. The possibility that the lack of an appreciable proportion of cell-bound radioactivity may reflect the pathogenicity of poliovirus strains is discussed.     

Restricted growth of attenuated poliovirus strains in cultured cells of a human neuroblastoma.

Cultured cells of a human neuroblastoma, SK-N-MC, were found to be highly resistant to Sabin attenuated poliovirus types 1 and 2 strains; no appreciable cytopathic effect was observed, and the total harvest was generally in the order of 1 PFU per cell or less. On the other hand, related neurovirulent strains of these antigenic types produced a relatively good (2 orders of magnitude higher) yield in a markedly protracted infectious cycle. The limited growth of the attenuated virus in the neuroblastoma cells appeared to be confined to a minor cell subpopulation. Experiments with intratypic (type 1) poliovirus recombinants suggested that the major genetic determinants limiting reproduction of the attenuated polioviruses in the neuroblastoma cells are located in the 5' half of the viral RNA, although the 3' half also appears to contribute somewhat to this phenotype. The possibility that neuroblastoma cells may represent an in vitro model for studying poliovirus neurovirulence is briefly discussed.     

Differential inhibitory effects of actinomycin D among strains of poliovirus

Schaffer, Frederick L. (University of California, Berkeley), and Marjorie Gordon. Differential inhibitory effects of actinomycin D among strains of poliovirus. J. Bacteriol. 91:2309-2316. 1966.-Actinomycin D exerted a differential effect on the ability of strains of poliovirus to replicate in HeLa cells. LSc-2ab was studied as an example of a strain markedly inhibited by actinomycin; MEF(1) served as a control strain with minimal inhibition. The effect was noted at an actinomycin concentration of 0.1 mug/ml, but 2.5 mug/ml was used for most studies. Variability in the effect was attributed, in part, to physiological factors. Actinomycin was effective when present during the first 2 hr of LSc infection, but had little effect if present at later times. It did not block adsorption or initiation of ecilpse. It did block synthesis of ribonucleic acid in LSc-infected cells. Several possible modes of action are discussed, the most attractive being that actinomycin blocks synthesis of some cell component, the concentration of which is more critical for replication of some poliovirus strains than others.     

Isolation of recombinant vaccine strains of poliovirus from patients with poliomyelitis

Oligonucleotide maps of some poliovirus type 2 strains isolated from polio cases, while being clearly related to that of the Sabin vaccine type 2 strain, exhibited, nevertheless, marked differences from the reference (vaccine) map. Several large oligonucleotides derived from 4 such strains were subjected to enzymatic sequencing. The results strongly suggest that all of them were intertypic recombinants between the Sabin strains. The 5'-parts of the genomes of these strains were derived from the type 2 vaccine whereas the 3'-parts were of type 1 (in 2 strains) or type 3 (in other 2 strains) origin.     

Natural genetic exchanges between vaccine and wild poliovirus strains in humans

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.     

Isolation of apparently wild strains of poliovirus type 1 from sewage in the Ottawa area.

In the first 4 months of 1974, 140 gauze pad samples of sewage collected in the Ottawa area were analysed by the BS-C-1 cell system for the presence of viruses pathogenic for humans. Viruses were isolated from 111 (79%) of the samples. Of the 72 (65%) isolates identified by serology and electron microscopic examination, 56 (78%) were reoviruses and 16 (22%), enteroviruses. The enterovirus isolates included one coxsackievirus B4, one vaccine strain of poliovirus type 3, nine vaccine strains of poliovirus type 1 and five strains of poliovirus type 1 that proved by serodifferentiation and temperature marker tests to be different from vaccine strains. The fact that these strains were present in the community sewage in readily detectable concentrations at a time when immunity against polioviruses is declining in such communities is a cause for concern.     

Characterization of CHAT and Cox type 1 live-attenuated poliovirus vaccine strains

CHAT and Cox type 1 live-attenuated poliovirus strains were developed in the 1950s to be used as vaccines for humans. This paper describes their characterization with respect to virulence, sensitivity for growth at high temperatures, and complete nucleotide and amino acid sequences. The results are compared to those for their common parental wild virus, the Mahoney strain, and to those for two other poliovirus strains derived from Mahoney, the Sabin 1 vaccine strain and the mouse-adapted LS-a virus. Analysis of four isolates from cases of vaccine-associated paralytic poliomyelitis related to the CHAT vaccine revealed genetic and phenotypic properties of the CHAT strain following replication in the human gut. CHAT-VAPP strain 134 contained a genome highly evolved from that of CHAT (1.1% nucleotide differences), suggesting long-term circulation of a vaccine-derived strain in the human population. The molecular mechanisms of attenuation and evolution of poliovirus in humans are discussed in the context of the global polio eradication initiative.     

Genomic features of intertypic recombinant sabin poliovirus strains excreted by primary vaccinees

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.     

High diversity of poliovirus strains isolated from the central nervous system from patients with vaccine-associated paralytic poliomyelitis.

To establish the etiology of vaccine-associated paralytic poliomyelitis (VAPP), isolates from the central nervous system (CNS) from eight patients with VAPP were compared with stool isolates from the same patients. The vaccine (Sabin) origin was checked for all of the available isolates. Unique and similar strains were recovered from paired stool and CNS samples for five of the eight VAPP cases and the three wild-type cases included in the study. In the remaining three VAPP cases, the stool samples and, in one case, the CNS samples contained mixtures of strains. In two of these cases an equivalent of the CNS isolate was found among the strains separated by plaque purification from stool mixtures, and in one case different strains were isolated from CNS and stool. This shows that the stool isolate in VAPP might not be always representative of the etiologic agent of the neurological disease. A wide variety of poliovirus vaccine genomic structures appeared to be implicated in the etiology of VAPP. Of nine CNS vaccine-derived strains, four were nonrecombinant and five were recombinant (vaccine/vaccine or even vaccine/nonvaccine). The neuropathogenic potential of the isolates was evaluated in transgenic mice sensitive to poliovirus. All of the CNS-isolated strains lost the attenuated phenotype of the Sabin strains. However, for half of them, the neurovirulence was lower than expected, suggesting that the degree of neurovirulence for transgenic mice is not necessarily correlated with the neuropathogenicity in humans.     

A recombinant virus between the Sabin 1 and Sabin 3 vaccine strains of poliovirus as a possible candidate for a new type 3 poliovirus live vaccine strain.

Biological tests including the monkey neurovirulence test performed on recombinants between the virulent Mahoney and attenuated Sabin 1 strains of type 1 poliovirus indicated that the genome region encoding mainly the viral capsid proteins had little correlation with the neurovirulence or attenuation phenotype of the virus. The results suggested that new vaccine strains of type 2 and type 3 polioviruses may be constructed in vitro by replacing the sequence encoding the antigenic determinants in viral capsid proteins of the Sabin 1 genome by the corresponding sequences of the type 2 and type 3 genome, respectively. Accordingly, we constructed recombinants between the Sabin 1 and Sabin 3 strains of poliovirus in which genome sequences of the Sabin 1 strain encoding most or all capsid proteins were replaced by the corresponding genome sequences of the Sabin 3 strain. One of the recombinant viruses thus constructed was fully viable and showed antigenicity and immunogenicity identical to those of type 3 poliovirus. The monkey neurovirulence tests and in vitro phenotypic marker tests (temperature sensitivity of growth, sodium bicarbonate concentration dependency of growth under agar overlay, and size of plaque) were performed on the recombinant virus. The stability of the virus in regard to the temperature sensitivity phenotype was also tested. The results suggested that the recombinant virus is a possible candidate for a new type 3 poliovirus vaccine strain.     

Postabsorption neutralization of poliovirus.

Nineteen neutralizing murine monoclonal antibodies against poliovirus type 1, including representatives reacting with each of the antigenic sites on the virion, were tested for their abilities to neutralize the virus either before or after attachment to susceptible cells. All antibodies neutralized unattached virus; six had reasonable titers of postabsorption neutralization (PAN). Experiments with antibodies lacking PAN activity showed that Fc-specific rabbit anti-mouse antibodies could confer PAN activity. PAN was shown to involve the prevention of the cell-mediated conversion of virus to 135S and 80S particles. Evidence that one of the PAN-positive antibodies probably bound bivalently to preabsorbed virions, whereas a PAN-negative antibody bound monovalently, is presented. Two PAN-positive antibodies were added to an excess of virus in suspension, and only one antibody caused the virus to aggregate.     

Two initiation sites for translation of poliovirus RNA in vitro: comparison of LSc and Mahoney strains.

Previous studies in our laboratory provided evidence that the initiation of translation by the Mahoney strain of poliovirus type 1 RNA in vitro occurs at two unique sites. This study shows that the LSc strain of poliovirus type 1, a multistep, temperature-sensitive mutant of the Mahoney strain, also utilizes two sites for the initiation of translation in vitro. Incorporation of formyl-[35S]methionine into the amino terminus of newly synthesized polypeptides revealed the production of two labeled tryptic peptides which are identical in size and electrophoretic mobility with those produced by Mahoney virus. The polypeptides containing amino-terminal label showed similar patterns on sodium dodecyl sulfate-acrylamide gels, although one of the LSc polypeptides had a slightly faster mobility. The relative proportion of initiation at each site varied with the magnesium concentration for both viruses, but the LSc strain favored initiation at one site more so than did the Mahoney strain.