Complete genomic characterization of an intertypic Sabin 3/Sabin 2 capsid recombinant

The genetic properties of strain K/2002, isolated from fecal samples of a 7-month-old child who had received his first oral poliovirus vaccine (OPV) dose at the age of 3 months, are described. Preliminary sequencing characterization of isolate K/2002 revealed an S3/S2 recombination event at the 3' end of the VP1 coding region. A recombination event resulted in the introduction of six Sabin 2 amino acid residues in a Sabin 3 genomic background. Furthermore, mutations associated with loss of the attenuated phenotype of Sabin 3 strains have been identified in the genome of isolate K/2002. The data presented here emphasize the need for careful planning of vaccination strategies, which involve stopping OPV administration in regions that are certified to be polio-free.     

Albert B. Sabin and the Development of Oral Poliovaccine

Abstract. There are many reasons for the modern interest in viral vaccines, but there is no doubt that the key role played by viral vaccines in public health is the major factor since other prophylactic or therapeutic anti-vital products simply do not exist. Viral vaccines have a long history that has been marked by successful events and by tragic accidents. Live viral vaccines are an extraordinary category of biologicals since, despite their reputed efficacy, they were developed by empirical experiments and patient epidemiological observation. From this point of view oral polio vaccine should be considered a 'miracle' since it became a major tool for public health in the 20th century, before we were able to understand the molecular basis of polio virus neurovirulence attenuation. The first evidence that polio virus can be attenuated was provided in the early 1940s by Max Theiler, but it was Hilary Koprowsky who demonstrated further in 1952, that a rodent adapted strain was safe and able to immunise a limited number of volunteers. Koprowsky studies were confirmed later during a mass field trial in Africa. However it is undeniable that the patient and systematic work of Albert B. Sabin was primordial in developing live oral attenuated poliovaccine. The excellence of Sabin's testing of poliovirus neurovirulence in the accurate studies that he developed, enabled him to select, after the cloning of viral populations by plaque assay, the best attenuated variants. It is interesting to remember that the real selective factor that allowed the isolation of attenuated variants was ignored by Sabin and was put forward by Lwoff in the Pasteur Institute, when he described the role of temperature in the selection of cold attenuated mutants. Historically, the first to perform a successful mass vaccination with Sabin oral live poliovaccine were Russian scientists. Oral live poliovaccine was in some cases the origin of paralytic accidents and Sabin strains were involved occasionally in such events. Other attenuated poliovirus strains used in clinical trials as oral vaccine, such as Cox-Lederle type 1 and Usol-D bac type 3, generated in some instances clusters of vaccinees that developed paralysis. An important achievement in the consistency of the Sabin vaccine was the transfer by Albert Sabin to the WHO of the seed material and the responsibility for surveying the quality control and licensing procedure of oral poliovaccine.     

First decade (1950-1960) of studies and trials with the polio vaccine.

The chronology of the development of polio vaccines following the first human trials of attenuated poliovirus vaccine in 1950 is described by me as a witness to the first decade of trials and tribulations following my discovery of polio vaccine in 1950. Mass vaccination trials are considered to be the most important phase of the discovery of oral polio vaccine (OPV). These took place in the Belgian Congo, Poland, Croatia, Switzerland, and finally in the former Soviet Union. By 1960, approximately 13 million individuals had been vaccinated with the Koprowski oral polio vaccine and over 11 million with the Sabin vaccine.     

Evaluation of poliovirus antibody titers in orally vaccinated semi‐captive chimpanzees in Uganda

Background To understand immunological responses in chimpanzees vaccinated with live‐attenuated vaccine (oral polio vaccine; OPV), serum neutralizing antibodies against poliovirus types 1, 2, and 3 were investigated over time. Methods The neutralizing antibody titers against poliovirus types 1, 2, and 3 were determined by microneutralization test using 100 ID₅₀ of poliovirus types 1, 2, and 3 (Sabin strains). Results Neutralizing antibodies against poliovirus types 1, 2, and 3 were detected in 85.7%, 71.4%, and 65% of the serum from 42 chimpanzees tested 9 years post‐vaccination. The neutralizing antibody titers in chimpanzees were similar to the documented levels in human studies as an indicator of vaccine efficacy. Conclusions This study reveals persistence of neutralizing antibodies in chimpanzees for at least 9 years after vaccination with OPV. This first study in chimpanzees provides useful information for the evaluation of the success of vaccination with OPV in other captive apes.     

Intratypic recombination among lineages of type 1 vaccine-derived poliovirus emerging during chronic infection of an immunodeficient patient

We determined the complete genomic sequences of nine type 1 immunodeficient vaccine-derived poliovirus (iVDPV) isolates obtained over a 337-day period from a poliomyelitis patient from Taiwan with common variable immunodeficiency. The iVDPV isolates differed from the Sabin type 1 oral poliovirus vaccine (OPV) strain at 1.84% to 3.15% of total open reading frame positions and had diverged into at least five distinct lineages. Phylogenetic analysis suggested that the chronic infection was initiated by the fifth and last OPV dose, given 567 days before onset of paralysis, and that divergence of major lineages began very early in the chronic infection. Key determinants of attenuation in Sabin 1 had reverted in the iVDPV isolates, and representative isolates of each lineage showed increased neurovirulence for PVR-Tg21 transgenic mice. None of the isolates had retained the temperature-sensitive phenotype of Sabin 1. All isolates were antigenic variants of Sabin 1, having multiple amino acid substitutions within or near neutralizing antigenic sites 1, 2, and 3a. Antigenic divergence of the iVDPV variants from Sabin 1 followed two major independent evolutionary pathways. The emergence of distinct coreplicating lineages suggests that iVDPVs can replicate for many months at separate sites in the gastrointestinal tract. Some isolates had mosaic genome structures indicative of recombination across and within lineages. iVDPV excretion apparently ceased after 30 to 35 months of chronic infection. The appearance of a chronic VDPV excretor in a tropical, developing country has important implications for the strategy to stop OPV immunization after eradication of wild polioviruses.     

Developments in the production and quality control of poliovirus vaccines -- historical perspectives.

Using virus grown in monkey kidney cells, Salk and his colleagues developed an inactivated poliovirus vaccine (IPV) in 1952. A large-scale field trial showed the vaccine to be safe and highly immunogenic in children, but soon after the vaccine became generally available in 1955, cases of paralytic disease were reported in recipients. Investigations showed that almost all the cases occurred in children who had received vaccine from one particular manufacturer. Extensive studies attributed the disaster to problems with inactivation. Addition of a Seitz filtration step midway during formalin inactivation and extension of the inactivation period resulted in a safe vaccine. No further paralytic cases were observed following the use of several hundred million doses of this improved vaccine. Thus, IPV was safe and caused a dramatic decline in the incidence of poliomyelitis in countries where it was used. A second generation IPV is produced in fermentors using well-characterized cell strains or continuous cell lines. The major breakthrough in the development of live poliovirus vaccine was the application of tissue culture methods for virus attenuation. By 1959 several candidate live oral poliovirus vaccines (OPV) had been developed. These were clinically tested in millions of individuals and found to be safe and effective. Since the attenuated virus strains developed by Koprowski and Cox were more neurotropic in monkeys than the Sabin strains, only the latter was licensed in the USA in 1961 and endorsed shortly after by the World Health Organization (WHO). The widespread use of Sabin's OPV in many countries hastened the development of International Requirements by WHO for OPV in 1962 to define the criteria that ensured the uniformity of batches produced by different manufacturers. These have been updated continuously in light of new information and quality control procedures. Extensive field trials have shown the risk of OPV associated polio to be less than 0.3 per million doses administered.     

Post-eradication poliovirus facility-associated community risks.

Minimizing the risk of poliovirus transmission from the poliovirus facility to an increasingly susceptible community is crucial when global poliovirus transmission and OPV use stops. Community risks of exposure to wild poliovirus as well as Sabin strains are highest from facility personnel who are unknowingly contaminated or infected. Immunization with OPV or IPV prevents poliomyelitis, but neither vaccine fully inhibits silent infection of the gut. Facility environments maintained at low relative humidity (<50%) may reduce poliovirus survival and inhalation risk. Circulating antibodies reduce personnel infection risks from injection or virus entry through breaks in skin or mucous membranes. Community exposure risk through inhalation of contaminated air effluent is likely low in most modern facilities. Community risks through ingestion of liquid effluents are facility-specific and may range from high to low. This assessment of community risks, when combined with assessments of facility-specific hazards and the consequences of wild or Sabin poliovirus transmission, provides the foundation for effective risk management.     

Evolution of the Sabin type 1 poliovirus in humans: characterization of strains isolated from patients with vaccine-associated paralytic poliomyelitis.

Attenuated strains of the Sabin oral poliovirus vaccine replicate in the human gut and in rare cases cause vaccine-associated paralytic poliomyelitis (VAPP). Reversion of vaccine strains toward a pathogenic phenotype is probably one of the main causes of VAPP, a disease most frequently associated with type 3 and type 2 strains and more rarely with the type 1 (Sabin 1) strain. To identify the determinants and mechanisms of safety versus pathogenicity of the Sabin 1 strain, we characterized the genetic and phenotypic changes in six Sabin 1-derived viruses isolated from immunocompetent patients with VAPP. The genomes of these strains carried either few or numerous mutations from the original Sabin 1 genome. As assessed in transgenic mice carrying the human poliovirus receptor (PVR-Tg mice), all but one strain had lost the attenuated phenotype. Four strains presented only a moderate neurovirulent phenotype, probably due at least in part to reversions to the wild-type genotype, which were detected in the 5' noncoding region of the genome. The reversions found in most strains at nucleotide position 480, are known to be associated with an increase in neurovirulence. The construction and characterization of Sabin 1 mutants implicated a reversion at position 189, found in one strain, in the phenotypic change. The presence of 71 mutations in one neurovirulent strain suggests that a vaccine-derived strain can survive for a long time in humans. Surprisingly, none of the strains analyzed were as neurovirulent to PVR-Tg mice as was the wild-type parent of Sabin 1 (Mahoney) or a previously identified neurovirulent Sabin 1 mutant selected at a high temperature in cultured cells. Thus, in the human gut, the Sabin 1 strain does not necessarily evolve toward the genetic characteristics and high neuropathogenicity of its wild-type parent.     

Circulation of type 1 vaccine-derived poliovirus in the Philippines in 2001

In 2001, highly evolved type 1 circulating vaccine-derived poliovirus (cVDPV) was isolated from three acute flaccid paralysis patients and one contact from three separate communities in the Philippines. Complete genomic sequencing of these four cVDPV isolates revealed that the capsid region was derived from the Sabin 1 vaccine strain but most of the noncapsid region was derived from an unidentified enterovirus unrelated to the oral poliovirus vaccine (OPV) strains. The sequences of the cVDPV isolates were closely related to each other, and the isolates had a common recombination site. Most of the genetic and biological properties of the cVDPV isolates were indistinguishable from those of wild polioviruses. However, the most recently identified cVDPV isolate from a healthy contact retained the temperature sensitivity and partial attenuation phenotypes. The sequence relationships among the isolates and Sabin 1 suggested that cVDPV originated from an OPV dose given in 1998 to 1999 and that cVDPV circulated along a narrow chain of transmission. Type 1 cVDPV was last detected in the Philippines in September 2001, and population immunity to polio was raised by extensive OPV campaigns in late 2001 and early 2002.     

Infectious cDNA, Cell Receptors and Transgenic Mice in the Study of Sabin's Poliovirus Vaccines

Abstract. The determination of the nucleotide sequence of the poliovirus genome, the isolation of infectious poliovirus cDNAs, and the identification of the cell receptor for poliovirus and establishment of a transgenic mouse model for poliomyelitis have all contributed to our understanding of the live, attenuated Sabin poliovirus vaccines. These highly effective vaccines have been studied extensively by many laboratories to determine the molecular basis for their attenuation phenotype. For this special issue I would like to review how our research has improved the understanding of Sabin's poliovirus vaccines, and highlight Albert's influence on our work.     

Vaccine-derived polioviruses.

The Sabin oral poliovaccine (OPV) is extremely efficacious and safe, despite its inherent genetic instability. While reversion to nearly wild-type phenotype regularly occurs soon after the onset of OPV reproduction in the gastro-intestinal tract of vaccine recipients or their contacts, this is usually not a big problem, provided the vaccine is used either for mass vaccination or in populations with a relatively high level of anti-polio immunity. However, if these conditions are not met, the vaccine viruses are likely to be converted into highly transmissible agents with a nearly wild-type level of neurovirulence. Moreover, OPV viruses may persist and evolve even in adequately immunized populations. The current strategy for the "endgame" of poliovirus eradication envisions cessation of OPV usage shortly after the last isolation of a wild poliovirus. If implemented, this strategy would result in rapid growth of non-immune human populations at the time when OPV derivatives would very likely be persisting. Therefore, the planned cessation of OPV vaccination is associated with a very high, and in the author's opinion, unacceptable risk of polio outbreaks caused by OPV derivatives. The only currently available tool to curb such outbreaks is OPV, which should have been used at a global scale. Safe discontinuation of OPV vaccination will be possible only after an efficient new vaccine or an anti-poliovirus drug is available. To achieve this goal, stimulation of poliovirus research and elimination of organizational and financial obstacles preventing it are needed.     

Detection of mutants in polio vaccine viruses using pooled antipoliovirus monoclonal antibodies.

We prepared six monoclonal antibodies (mAbs) for type 1 polioviruses, and analysed their neutralizing specificities for use in safety tests in oral poliomyelitis vaccine (OPV) production. Pools of two or more individual mAbs showed high neutralizing activity against high-titre (approximately 10(7) CCID (50)/25 microl) of Sabin type 1 virus. It was demonstrated that the pooled mAbs can be utilized effectively in detection tests of adventitious viruses, which are among the safety tests in OPV production. Moreover, some pooled mAbs were shown to be capable of detecting very small amounts of type 1 virulent viruses and mutants in high-titre Sabin type 1 virus suspensions. Neutralizing antibody titres of these pooled mAbs decreased with increasing numbers of mutants containing neurovirulent activity in high-titre Sabin type 1 viruses which were repeatedly passaged in culture. It is expected that these pooled mAbs will contribute greatly to safety tests for OPV production.     

Environmental Poliovirus Surveillance during Oral Poliovirus Vaccine and Inactivated Poliovirus Vaccine Use in Córdoba Province,Argentina

This study compares the presence of environmental poliovirus in two Argentinean populations using oral poliovirus vaccine (OPV) or inactivated poliovirus vaccine (IPV). From January 2003 to December 2005, Córdoba City used IPV in routine infant immunizations, with the exception of intermittent OPV use in August 2005. Between May 2005 and April 2006, we collected weekly wastewater samples in Córdoba City and the province''s three major towns, which continued OPV use at all times. Wastewater samples were processed and analyzed for the presence of poliovirus according to WHO guidelines. During the months of IPV use in Córdoba City, the overall proportion of poliovirus-positive samples was 19%. During an intermittent switch from IPV to OPV, this proportion increased to 100% within 2 months. During the 3 months when IPV was reintroduced to replace OPV, a substantial proportion of samples (25%) remained positive for poliovirus. In the OPV-using sites, on average, 54% of samples were poliovirus positive. Seventy-seven percent of poliovirus isolates showed at least one mutation in the VP1-encoding sequence; the maximum genetic divergence from the Sabin strain was 0.7%. Several isolates showed mutations on attenuation markers in the VP1-encoding sequence. The frequency or type of virus mutation did not differ between periods of IPV and OPV use or by virus serotypes. This study indicates that the sustained transmission of OPV viruses was limited during IPV use in a middle-income country with a temperate climate. The continued importation of poliovirus and genetic instability of vaccine strains even in the absence of sustained circulation suggest that high poliovirus vaccine coverage has to be maintained for all countries until the risk of reintroduction of either wild or vaccine-derived poliovirus is close to zero worldwide.In the context of the near achievement of poliomyelitis eradication and anticipated cessation of oral poliovirus (PV) vaccine (OPV), the World Health Organization (WHO) has recommended the use of inactivated PV vaccine (IPV) in countries that have IPV production facilities or other countries where immunization programs fulfill certain financial and logistic criteria (37). IPV has been shown to be safe and immunogenic in children in both developed and developing countries.(34) IPV diminishes the excretion of PV by children challenged with the Sabin strain of PV only moderately. The questions of whether and to which extent Sabin PV that is reintroduced into a population immunized with IPV could establish circulation, mutate to vaccine-derived PV (VDPV), and consequently cause poliomyelitis remain important. No such emergence of VDPV in developed countries using IPV has been reported. However, suboptimal hygienic conditions and insufficient vaccine coverage in middle- or low-income countries could favor the establishment of PV circulation after reintroduction, as indicated by recent VDPV outbreaks in populations with low OPV coverage (27, 38).Argentina currently uses OPV in the childhood immunization program according to recommendations from the Pan-American Health Organization. The last case of poliomyelitis due to wild-type PV was reported in Argentina in 1984 and in Córdoba Province in 1971 (24). In Córdoba City, the capital of Córdoba Province, standalone IPV (Imovax Polio; Sanofi Pasteur) replaced OPV (Polioral; Novartis Vaccines) in the routine childhood immunization program (2, 4, and 6 months of age plus a booster at 18 months age) from 1 January 2003 to 31 December 2005, while the surrounding provinces continued to use OPV. Due to an IPV shortage between 10 August and 7 September 2005, OPV was used in the capital during this period. We conducted environmental PV surveillance in Córdoba Province from May 2005 to April 2006 to describe environmental PV circulation and molecular characteristics of PV depending on the vaccine used. In the present evaluation, we also describe the dynamic of PV circulation around the change of IPV-OPV-IPV-OPV in the capital. This observation can contribute evidence regarding the dynamics of PV circulation and its implication for global immunization policy after polio eradication.     

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.     

Antigenic evolution of vaccine-derived polioviruses: changes in individual epitopes and relative stability of the overall immunological properties

The Sabin oral poliovirus vaccine (OPV) readily undergoes changes in antigenic sites upon replication in humans. Here, a set of antigenically altered descendants of the three OPV serotypes (76 isolates) was characterized to determine the driving forces behind these changes and their biological implications. The amino acid residues of OPV derivatives that lie within or close to the known antigenic sites exhibited a marked tendency to be replaced by residues characteristic of homotypic wild polioviruses, and these changes may occur very early in OPV evolution. The specific amino acid alterations nicely correlated with serotype-specific changes in the reactivity of certain individual antigenic sites, as revealed by the recently devised monoclonal antibody-based enzyme-linked immunosorbent assay. In comparison to the original vaccine, small changes, if any, in the neutralizing capacity of human or rabbit sera were observed in highly diverged vaccine polioviruses of three serotypes, in spite of strong alterations of certain epitopes. We propose that the common antigenic alterations in evolving OPV strains largely reflect attempts to eliminate fitness-decreasing mutations acquired either during the original selection of the vaccine or already present in the parental strains. Variability of individual epitopes does not appear to be primarily caused by, or lead to, a significant immune evasion, enhancing only slightly, if at all, the capacity of OPV derivatives to overcome immunity in human populations. This study reveals some important patterns of poliovirus evolution and has obvious implications for the rational design of live viral vaccines.     

Spread of vaccine-derived poliovirus from a paralytic case in an immunodeficient child: an insight into the natural evolution of oral polio vaccine

Sabin strains used in the manufacture of oral polio vaccine (OPV) replicate in the human organism and can give rise to vaccine-derived polioviruses. The increased neurovirulence of vaccine derivatives has been known since the beginning of OPV use, but their ability to establish circulation in communities has been recognized only recently during the latest stages of the polio eradication campaign. This important observation called for studies of their emergence and evolution as well as extensive surveillance to determine the scope of this phenomenon. Here, we present the results of a study of vaccine-derived isolates from an immunocompromised poliomyelitis patient, the contacts, and the local sewage. All isolates were identified as closely related and slightly evolved vaccine derivatives with a recombinant type 2/type 1 genome. The strains also shared several amino acid substitutions including a mutation in the VP1 protein that was previously shown to be associated with the loss of attenuation. Another mutation in the VP3 protein resulted in altered immunological properties of the isolates, possibly facilitating virus spread in immunized populations. The patterns and rates of the accumulation of synonymous mutations in isolates collected from the patient over the extended period of excretion suggest either a substantially nonuniform rate of mutagenesis throughout the genome, or, more likely, the strains may have been intratypic recombinants between coevolving derivatives with different degrees of divergence from the vaccine parent. This study provides insight into the early stages of the establishment of circulation by runaway vaccine strains.     

Recombinant Sabin environmental isolates in Greece and Cyprus

Aims: Twenty‐one polioviruses (PVs) Sabin strains were isolated from sewage treatment plants from Metamorphosis, Athens, Greece during the time period from May to October 1996, and from two other sites located at Nicosia and Limassol in Cyprus between April and December 2003 were retrospectively investigated for the detection of recombinant PVs. Methods and Results: Three PVs isolates were found as tripartite recombinants, S3/S2/S1 in the 2C genomic viral region. The first recombination site S3/S2 was located close to the 5′ end of 2C while the second recombination site S2/S1 was located towards the 3′ end of 2C .Such recombination is a rare event producing a tripartite hybrid 2C protein. Three more PVs isolates were characterized as bipartite S2/S1 recombinants and one as S2/S3 bipartite recombinant. Conclusions: Detection of recombinant circulating vaccine‐derived PVs (cVDPVs) is crucial, since increased transmissibility over that of the parental Sabin strains has been proposed to be the result of recombination events. Significance and Impact of the Study: Importation of recombinant cVDPVs evolved derivatives pose a serious threat to public health and environmental surveillance should be implemented during and after PVs eradication.     

Poliovirus surveillance: isolation of polioviruses in Japan, 1980-1991. A report of the National Epidemiological Surveillance of Infectious Agents in Japan.

This report presents an overall distribution of poliovirus isolations in Japan, where poliomyelitis has been under control over two decades as a result of legal administration of two doses of the trivalent live oral poliovirus vaccine of the Sabin strains (OPV) to children under 48 months of age. During the past 12 years from 1980 through 1991, a total of 1,126 poliovirus isolations from humans and 268 isolations from sewage/river water were reported by respectively 49 and nine of the participating laboratories. Type 2 was most frequently isolated from children after administration of one dose of OPV, followed by type 1 and type 3. On the contrary, after the second dose of OPV, the rate of isolation of type 3 exceeded those of type 2 and type 1. Seasonal and age distribution of poliovirus isolations from both humans and sewage/river water paralleled the OPV vaccination schedule in Japan. One percent of the isolations were, however, from infants younger than the vaccination-scheduled ages and 5% were from children older than those ages, including one each from 15 and 16 years olds. The data indicate that the poliovirus has silently been disseminated from vaccinated children to others and the community, thus suggesting repeated transmission of the viruses. The fact that some elder children had poliovirus colonization in their alimentary tracts indicates a potential risk of infection of such a population when exposed to a wild virus and of becoming a source of transmission to others.