Genetic basis of attenuation of the Sabin type 3 oral poliovirus vaccine.

The poliovirus type 3 Sabin oral poliovirus vaccine strain P3/Leon/12a1b differs in nucleotide sequence from its neurovirulent progenitor P3/Leon/37 by just 10 point mutations. The contribution of each mutation to the attenuation phenotype of the vaccine strain was determined by the construction of a series of recombinant viruses from infectious cDNA clones. The neurovirulence testing of recombinant viruses indicated that the attenuation phenotype is determined by just two point mutations: a C to U in the noncoding region at position 472 and a C to U at nucleotide 2034 which results in a serine-to-phenylalanine amino acid substitution in the structural protein VP3.     

Determinants of attenuation and temperature sensitivity in the type 1 poliovirus Sabin vaccine.

To identify determinants of attenuation in the poliovirus type 1 Sabin vaccine strain, a series of recombinant viruses were constructed by using infectious cDNA clones of the virulent type 1 poliovirus P1/Mahoney and the attenuated type 1 vaccine strain P1/Sabin. Intracerebral inoculation of these viruses into transgenic mice which express the human receptor for poliovirus identified regions of the genome that conferred reduced neurovirulence. Exchange of smaller restriction fragments and site-directed mutagenesis were used to identify the nucleotide changes responsible for attenuation. P1/Sabin mutations at nucleotides 935 of VP4, 2438 of VP3, and 2795 and 2879 of VP1 were all shown to be determinants of attenuation. The recombinant viruses and site-directed mutants were also used to identify the nucleotide changes which are involved in the temperature sensitivity of P1/Sabin. Determinants of this phenotype in HeLa cells were mapped to changes at nucleotides 935 of VP4, 2438 of VP3, and 2741 of VP1. The 3Dpol gene of P1/Sabin, which contains three amino acid differences from its parent P1/Mahoney, also contributes to the temperature sensitivity of P1/Sabin; however, mutants containing individual amino acid changes grew as well as P1/Mahoney at elevated temperatures, suggesting that either some combination or all three changes are required for temperature sensitivity. In addition, the 3'-noncoding region of P1/Sabin augments the temperature-sensitive phenotype conferred by 3Dpol. Although nucleotide 2741, 3Dpol, and the 3'-noncoding region of P1/Sabin contribute to the temperature sensitivity of P1/Sabin, they do not contribute to attenuation in transgenic mice expressing the poliovirus receptor, demonstrating that determinants of attenuation and temperature sensitivity can be genetically separated.     

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.     

Reversion to neurovirulence of the live-attenuated Sabin type 3 oral poliovirus vaccine

The complete nucleotide sequence has been determined of a strain of poliovirus type 3, P3/119, isolated from the central nervous system of a victim of fatal vaccine-associated poliomyelitis. Comparison of this sequence with those obtained previously for the Sabin type 3 vaccine, P3/Leon 12a1b and its neurovirulent progenitor, P3/Leon/37, reveals that these three strains are on a direct geneaological lineage and therefore that P3/119 is a bona fide revertant of the vaccine. P3/119 differs in sequence from its attenuated vaccine parent at just seven positions. Only one of these differences, a mutation from U to C at position 472 in the presumed noncoding region of the genome, is a back mutation to the wild type sequence. Of the six other differences, three give rise to coding changes in virus structural proteins, two are silent changes in the major open reading frame of the genome and one affects the 3'-terminus just prior to the poly A tract. These differences indicate that there are three possible types of molecular change which could, singly or collectively, result in attenuation and reversion to neurovirulence of the Sabin type 3 vaccine.     

Role of mutations G-480 and C-6203 in the attenuation phenotype of Sabin type 1 poliovirus.

Of the 55 point mutations which distinguish the type 1 poliovirus vaccine strain (Sabin 1) from its neurovirulent progenitor (P1/Mahoney), two have been strongly implicated by previous studies as determinants of the attenuation phenotype. A change of an A to a G at position 480, located within the 5' noncoding region, has been suggested to be the major attenuating mutation, analogous to the mutations at positions 481 and 472 in poliovirus types 2 and 3, respectively. In addition, the change of a U to a C at position 6203, resulting in an amino acid change in the polymerase protein 3D, has also been implicated as a determinant of attenuation, albeit to a lesser extent. To assess the contributions of these mutations to attenuation and temperature sensitivity, reciprocal changes were generated at these positions in infectious cDNA clones of Sabin 1 and P1/Mahoney. Assays in tissue culture and primates indicated that the two mutations make some contribution to the temperature sensitivity of the Sabin 1 strain but that neither is a strong determinant of attenuation.     

Evolution of the Sabin strain of type 3 poliovirus in an immunodeficient patient during the entire 637-day period of virus excretion

A 20-year-old female hypogammaglobulinemic patient received monotypic Sabin 3 vaccine in 1962. The patient excreted type 3 poliovirus for a period of 637 days without developing any symptoms of poliomyelitis, after which excretion appeared to have ceased spontaneously. The evolution of Sabin 3 throughout the entire period of virus excretion was studied by characterization of seven sequential isolates from the patient. The isolates were analyzed in terms of their antigenic properties, virulence, sensitivity for growth at high temperatures, and differences in nucleotide sequence from the Sabin type 3 vaccine. The isolates followed a main lineage of evolution with a rate of nucleotide substitution that was very similar to that estimated for wild-type poliovirus during person-to-person transmission. There was a delay in the appearance of antigenic variants compared to sequential type 3 isolates from healthy vaccines, which could be one of the possible explanations for the long-term excretion of virus from the patient. The distribution of mutations in the isolates identified regions of the virus possibly involved in adaptation for growth in the human gut and virus persistence. None of the isolates showed a full reversion of the attenuated and temperature-sensitive phenotypes of Sabin 3. Information of this sort will help in the assessment of the risk of spread of virulent polioviruses from long-term excretors and in the design of therapies to stop long-term excretion. This will make an important contribution to the decision-making process on when to stop vaccination once wild poliovirus has been eradicated.     

Trypsin sensitivity of the Sabin strain of type 1 poliovirus: cleavage sites in virions and related particles.

Treatment of the Sabin strain of type 1 poliovirus with trypsin produced two stable fragments of capsid protein VP1 which remained associated with the virions. Trypsinized virus was fully infectious and was neutralized by type-specific antisera. The susceptible site in the Sabin 1 strain was between the lysine at position 99 and the asparagine at position 100. A similar tryptic cleavage occurred in the Leon and Sabin strains of type 3 poliovirus, probably at the arginine at position 100, but not in the type 1 Mahoney strain, which lacks a basic residue at either position 99 or position 100. Tryptic treatment of heat-treated virus and 14S assembly intermediates produced unique stable fragments which were different from those produced in virions. The implications of our results for future characterization of the surface structures of these particles and structural rearrangements in the poliovirus capsid are discussed.     

Determinants in the 5'' noncoding region of poliovirus Sabin 1 RNA that influence the attenuation phenotype.

A number of recombinants between the virulent Mahoney and attenuated Sabin strains of type 1 poliovirus were constructed by using infectious cDNA clones of the two strains. To identify a strong neurovirulence determinant(s) residing in the genome region upstream of nucleotide position 1122, these recombinant viruses were subjected to biological tests, including monkey neurovirulence tests. The results of the monkey neurovirulence tests suggested the important contribution of an adenine residue (Mahoney type) at position 480 to the expression of the neurovirulence phenotype of type 1 poliovirus. This nucleotide, however, had only a minor effect, if any, on viral temperature sensitivity. Monkey neurovirulence tests on the recombinant virus whose genome had a guanine residue (Sabin type) at position 480 and variants generated from this recombinant virus in the central nervous system of monkeys strongly suggested that only one nucleotide change, from adenine to guanine, was not sufficient for full expression of the attenuation phenotype encoded by this genome region. These results suggest that the expression of the attenuation phenotype depends on the highly ordered structure formed in the 5' noncoding sequence and that the formation of such a structure is possibly influenced by the nucleotide at position 480. Furthermore, in vitro biological tests performed on viruses recovered from the central nervous system of monkeys injected with a temperature-sensitive recombinant virus showing the small-plaque and d phenotypes revealed that most of the recovered viruses had even higher temperature sensitivities and that all of the recovered viruses that had acquired the large-plaque phenotype had lost the d phenotype to some extent. These results indicate that there may be an unknown selection pressure(s) in the central nervous system and that common determinants might be involved in the expression of the small-plaque and d phenotypes.     

Mapping of mutations contributing to the temperature sensitivity of the Sabin 1 vaccine strain of poliovirus.

The temperature-sensitive and attenuated phenotypes of the Sabin type 1 vaccine strain of poliovirus result from numerous point mutations which occurred in the virulent Mahoney virus parent. One of these mutations is located in a 3D polymerase (3Dpol) codon (U-6203-->C, Tyr-73-->His) and is involved in attenuation in common mice (M. Tardy-Panit, B. Blondel, A. Martin, F. Tekaia, F. Horaud, and F. Delpeyroux, J. Virol. 67:4630-4638, 1993). This mutation also appears to contribute to temperature sensitivity, in association with at least 1 other of the 10 mutations of the 3'-terminal part of the genome including the 3Dpol coding and 3' noncoding regions. To map the other mutation(s), we constructed poliovirus mutants by mutagenesis and recombination of Mahoney and Sabin 1 cDNAs. Characterization of these poliovirus mutants showed that a second mutation in a 3Dpol codon (C-7071-->U, Thr-362-->Ile) contributes to temperature sensitivity. A mutation in the 3' noncoding region of the genome (A-7441-->G), alone or linked to another mutation (U-7410-->C), also appeared to be involved in this phenotype. The temperature-sensitive effect associated with the 3'-terminal part of the Sabin 1 genome results from the cumulative and/or synergistic effects of at least three genetic determinants, i.e., the His-73 and Ile-362 codons of 3Dpol and nucleotide G-7441. Sequence analysis of strains isolated from patients with vaccine-associated paralytic poliomyelitis showed that these genetic determinants are selected against in vivo, although the Ile-362 codon appeared to be more stable than either the His-73 codon or G-7441. These genetic determinants may contribute to the safety of Sabin 1 in vaccines.     

Genetic analysis of the attenuation phenotype of poliovirus type 1.

Seven different recombinant viruses from the virulent Mahoney and the attenuated Sabin parental strains of type 1 poliovirus were constructed in vitro by using infectious cDNA clones. Monkey neurovirulence tests (lesion score, spread value, and incidence of paralysis) using these recombinant viruses revealed that the loci influencing attenuation were spread over several areas of the viral genome, including the 5' noncoding region. In vitro phenotypic marker tests corresponding to temperature sensitivity of growth (rct marker), plaque size, and dependency of growth on bicarbonate concentration (d marker) were performed to identify the genomic loci of these determinants and to investigate their correlation with attenuation. Determinants of temperature sensitivity mapped to many areas of the viral genome and expressed strong but not perfect correlation with attenuation. Recombinant viruses with Sabin-derived capsid proteins showed a small-plaque phenotype, and their growth was strongly dependent on bicarbonate concentration, suggesting that these determinants map to the genomic region encoding the viral capsid proteins. Plaque size and the d marker, however, were found to be poor indicators of attenuation. Moreover, virion surface characteristics such as immunogenicity and antigenicity had little or no correlation with neurovirulence. Nevertheless, viruses carrying Sabin-derived capsid proteins had an apparent tendency to exhibit less neurovirulence in tests on monkeys compared with recombinants carrying Mahoney-derived capsid proteins. Our results suggest that the extent of viral multiplication in the central nervous system of the test animals might be one of the most important factors determining neurovirulence. Moreover, we conclude that the expression of the attenuated phenotype of the Sabin 1 strain of poliovirus is the result of several different biological characteristics. Finally, none of the in vitro phenotypic markers alone can serve as a good indicator of neurovirulence or attenuation.     

Construction of viable deletion and insertion mutants of the Sabin strain of type 1 poliovirus: function of the 5'' noncoding sequence in viral replication.

A number of deletion and insertion sequences were introduced into the 5' noncoding sequence (742 nucleotides long) of the genome of the Sabin strain of type 1 poliovirus by using an infectious cDNA clone of the virus strain. The genomes of all three poliovirus serotypes contained highly homologous sequences (nucleotide positions 509 to 639) as well as highly variable sequences (positions 640 to 742) in the 5' noncoding region. The viability of mutant viruses was tested by transfecting mutant cDNA clones into African green monkey kidney cells and then estimating the plaque sizes displayed on the cells. The results suggested that the highly variable sequence next to the VP4 coding region did not play an important role, at least in the in vitro culture system used, that the loci of highly conserved nucleotide sequences were not always expected to be the genome regions essential for viral replication, that the sequence between positions 564 and 599 carried genetic information to maintain the efficiency of certain steps in viral replication, and that the sequence between positions 551 to 563 might play an essential role in viral replication. Four-base deletion or insertion mutations were introduced into relatively variable sequences in the genome region upstream of position 509. The results suggest that variable sequences do not always indicate that the corresponding genome regions are less important. Apparent revertants (large-plaque variants) were easily generated from one of the viable mutants with the small-plaque phenotype. The determination of nucleotide sequences of the revertant genomes revealed the second mutation site. The results suggested that the different loci at around positions 200 and 500 might specifically interact with each other. This interaction may result in the formation of a functional structure that influences the efficiency of certain steps in the viral replication.     

Correlation of virus polypeptide structure with attenuation of poliovirus type 1.

Several avirulent samples of poliovirus type 1 derived in the process attenuating the neurovirulent Mahoney strain show an altered virus capsid polypeptide, VP-1, on polyacrylamide gel electrophoresis of sodium dodecyl sulfate-disrupted virions.     

In vitro phenotypic markers of a poliovirus recombinant constructed from infectious cDNA clones of the neurovirulent Mahoney strain and the attenuated Sabin 1 strain.

Infectious cDNA corresponding to the entire genome of the attenuated Sabin strain of type 1 poliovirus has been inserted into EcoRI site of bacterial plasmid pBR325. Two consecutive PstI fragments (nucleotide positions 1814 to 3421) of the infectious cDNA of the Sabin 1 strain were replaced by the corresponding DNA fragments prepared from an infectious DNA clone of the genome of the virulent Mahoney strain of poliovirus type 1. The exchanged segment encodes capsid protein VP1 and part of capsid protein VP3, a region in which a large number of amino acid differences between the attenuated Sabin and the parental, neurovirulent Mahoney strain cluster. The recombinant virus was obtained by DNA transfection of HeLa S3 cells, and several in vitro phenotypes of the virus were compared with those of the parental viruses. The recombinant virus was recognized by a neutralizing monoclonal antibody specific to the Mahoney strain. Growth of the Sabin strain of poliovirus has been shown to be quite dependent upon the bicarbonate concentration (d marker). The growth of the recombinant virus, however, was not highly dependent upon the concentration of bicarbonate in cell culture media, and thus resembled that of the Mahoney strain. On the other hand, the temperature-sensitive multiplication (rct marker) and the small-plaque morphology of the recombinant virus corresponded to the phenotype of the Sabin 1 strain. The in vitro recombination of infectious cDNA clones of genomic RNA and subsequent analysis of the growth properties of the recombinant virus have allowed us to correlate specific mutations in the genome of an RNA virus with certain biological characteristics of that virus.     

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.     

A mutation present in the amino terminus of Sabin 3 poliovirus VP1 protein is attenuating.

The attenuated phenotype of Sabin 3 poliovirus compared with its neurovirulent progenitor strain has been largely accounted for by mutations in the genome at positions 472 and 2034 (G. D. Westrop, K. A. Wareham, D. M. A. Evans, G. Dunn, P. D. Minor, D. I. Magrath, F. Taffs, S. Marsden, M. A. Skinner, G. C. Schild, and J. W. Almond, J. Virol. 63:1338-1344, 1989). By sequencing vaccine virus RNA, we recently identified another Sabin 3-specific mutation at position 2493 (U----C), which predicts an Ile----Thr change at the sixth residue of VP1 (C. Weeks-Levy, J. M. Tatem, S. J. DiMichele, W. Waterfield, A. F. Georgiu, and S. J. Mento, Virology 185:934-937, 1991). Viruses generated by using cDNAs which represent the vaccine sequence (LED3) and a derivative (VR318) possessing a single base change to the wild-type nucleotide (U) at 2493 were used to determine the impact of the 2493 mutation on virus phenotype. The VP1 proteins of LED3 and VR318 viruses were distinguishable by denaturing electrophoretic analysis. LED3 produced smaller plaques in Vero cells than VR318 virus did. Neurovirulence testing of these cDNA-derived viruses in monkeys demonstrated that the 2493 mutation in LED3 virus is attenuating.     

Comparison of the airborne survival of calf rotavirus and poliovirus type 1 (Sabin) aerosolized as a mixture

A mixture of a cell culture-adapted strain (C-486) of calf rotavirus and poliovirus type 1 (Sabin) was prepared in tryptose phosphate broth containing 0.1% uranine (physical tracer) and antifoam at a final concentration of 0.001%. By using a six-jet Collison nebulizer, the mixture was aerosolized into a 300-liter stainless-steel rotating (4 rpm) drum. The temperature of the air inside the drum was kept at 20 +/- 1 degrees C, and the virus aerosols were held at the following three levels of relative humidity (RH): low (30 +/- 5%), medium (50 +/- 5%), and high (80 +/- 5%). An all-glass impinger, containing 10.0 ml of tryptose phosphate broth with antifoam, was used to collect samples of air from the drum. Both viruses were propagated and quantitated in MA-104 cells. The calf rotavirus was found to survive well at mid-range RH, where 60% of the infectious virus could be detected even after 24 h of virus aerosolization. At the low RH, the half-life of the infectious rotavirus was ca. 14 h. On the other hand, no infectious poliovirus could be recovered from the drum air at the low and medium RH. At the high RH, more than 50% of the infectious rotavirus became undetectable within 90 min of aerosolization. In contrast to this, the half-life of the poliovirus at the high RH was about 10 h. These data, based on the aerosolization of virus mixtures, therefore suggest that there is a pronounced difference in the way RH influences the airborne survival of these two types of viruses held under identical experimental conditions.     

The Sabin live poliovirus vaccination trials in the USSR, 1959.

Widespread use of the Sabin live attenuated poliovirus vaccine has had tremendous impact on the disease worldwide, virtually eliminating it from a number of countries, including the United States. Early proof of its safety and effectiveness was presented in 1959 by Russian investigators, who had staged massive trials in the USSR, involving millions of children. Their positive results were at first viewed in the United States and elsewhere with some skepticism, but the World Health Organization favored proceeding with large-scale trials, and responded to the claims made by Russian scientists by sending a representative to the USSR to review in detail the design and execution of the vaccine programs and the reliability of their results. The report that followed was a positive endorsement of the findings and contributed to the acceptance of the Sabin vaccine in the United States, where it has been the polio vaccine of choice since the mid-1960s.