Evolution of vaccine poliovirus strains

The paper considers different aspects of the molecular evolution of poliovirus vaccine strains: mutational and recombinant variability; mechanisms of fixation of occurring genetic changes. Matters concerning the policy of vaccination of the population against poliomyelitis are covered.     

Correlation between recombination junctions and RNA secondary structure elements in poliovirus Sabin strains

In order to test the hypothesis that RNA structural elements promote the distribution of certain types of recombination junctions in each one of the 2C and 3D poliovirus genomic regions (Sabin 3/Sabin 2 or Sabin 1 in 2C and Sabin 2/Sabin 1 or Sabin 3 in 3D), we searched in 2C and 3D regions of reference Sabin strains for high probability RNA structural elements that could promote recombination. Recombination junctions that were identified in clinical strains of this study, as well as in clinical strains of previous studies, were superimposed on RNA secondary structure models of 2C and 3D genomic regions. Furthermore, we created an in vitro model, based on double infection of cell-culture with two poliovirus strains, for the production and identification of recombinant Sabin strains in 2C and 3D regions. Our intention was to compare the results that refer to the correlation of recombination junctions and RNA secondary structures in 2C and 3D regions of clinical strains, with the respective results of the in vitro model. Most of the recombination junctions of the clinical strains were correlated with RNA secondary structure elements, which were identical between recombining Sabin strains, and also presented high predictive value. In consensus were, the respective results originated from the in vitro model. We propose that the distribution of specific types of recombination junctions in certain regions of Sabin strains is not fortuitous and is correlated with RNA secondary structure elements identical to both recombination partners. Furthermore, results of this study highlight an important role for the stem region of the RNA structure elements in promoting recombination.     

Defective interfering particles from poliovirus vaccine and vaccine reference strains

Defective interfering (DI) viral particles have been found to be associated with attenuated oral poliovirus vaccine and also with poliovirus vaccine reference strains. The DI particles replicate in low-passage human fibroblastic cells, the BSC-1 line of monkey kidney cells as well as in HeLa cells. DI particles of the attenuated polioviruses are generated after relatively few serial, high multiplicity passages of cloned, DI-free vaccine virus, whereas virulent strains of poliovirus generate a significant number of DI only after a large number of passages.     

Growth kinetic analysis of bi-recombinant poliovirus vaccine strains

Attenuated strains of Sabin poliovirus vaccine replicate in the human gut and in rare cases may cause vaccine-associated paralytic poliomyelitis (VAPP). Mutations at specific sites of the genome and recombination between Sabin strains may result in the loss of the attenuated phenotype of OPV (Oral Poliovirus Vaccine) strains and the acquisition of traits characteristic of wild polioviruses, such as increased neurovirulence and loss of temperature sensitivity. In this study, we determined the phenotypic traits such as temperature sensitivity and growth kinetics of eight OPV isolates (six bi-recombinant and two non-recombinant). The growth phenotype of each isolate as well as of Sabin vaccine strains in Hep2 cell line at two different temperatures (37 and 40°C) was evaluated using two different assays, RCT test (Reproductive Capacity at different Temperatures) and one-step growth curve analysis. Moreover, the nucleotide and amino acid positions in the genomes of the isolates that have been identified as being involved in the attenuated and thermo sensitive phenotype of Sabin vaccine strains were investigated. Mutations that result in loss of the attenuated and thermo sensitive phenotype of Sabin vaccine strains were identified in the genomes of all isolates. Both mutations and recombination events correlated well with the reverted phenotypic traits of OPV-derivatives. In the post-eradication era of wild polioviruses, the identification and the characterization (genomic and phenotypic) of vaccine-derived polioviruses become increasingly important in order to prevent cases or even outbreaks of paralytic poliomyelitis caused by neurovirulent strains.     

Correlation of RNA secondary structure and attenuation of Sabin vaccine strains of poliovirus in tissue culture.

Part of the 5' noncoding regions of all three Sabin vaccine strains of poliovirus contains determinants of attenuation that are shown here to influence the ability of these strains to grow at elevated temperatures in BGM cells. The predicted RNA secondary structure of this region (nt 464-542 in P3/Sabin) suggests that both phenotypes are due to perturbation of base-paired stems. Ts phenotypes of site-directed mutants with defined changes in this region correlated well with predicted secondary structure stabilities. Reversal of base-pair orientation had little effect whereas stem disruption led to marked increases in temperature sensitivity. Phenotypic revertants of such viruses displayed mutations on either side of the stem. Mutations destabilizing stems led to intermediate phenotypes. These results provided evidence for the biological significance of the predicted RNA secondary structure.     

Retrospective molecular and phenotypic analysis of poliovirus vaccine strains isolated in Greece

The live oral poliovirus vaccine (OPV) strains are genetically unstable, causing, in rare cases, vaccine-associated paralytic poliomyelitis. Reversions of the known attenuating mutations in OPV strains and intertypic recombination have been identified as the underlying causes of the increased neurovirulence of poliovirus isolates. In this study, three OPV isolates (one non-recombinant and two recombinants) were tested in order to correlate phenotypic traits such as temperature sensitivity (Rct test) and growth kinetics (one-step growth curve test) with mutations and recombination events of the viral genome. Moreover, the immunity level of the western Greek population aged 1–40 years was evaluated against OPV isolates and Sabin vaccine strains, with a microneutralization assay. Members of the 1–40-year age group (both pooled and individual sera) showed no significant differences in neutralization test (NT) titres against OPV isolates in comparison with the Sabin vaccine strains. However, all three OPV isolates showed reverted phenotypic traits in Rct or one-step growth curve assays. The results of our study revealed a significant decrease in immunity level from the 1–10-year age group to the 21–30-year age group (pooled sera) for both poliovirus types 1 and 3. For both poliovirus types, the highest NT titres were observed in the 1–10-year age group, and the lowest NT titre was observed in the 21–30-year age group, towards poliovirus type 3. Our study underlines the need for immunological studies in all age groups, in order to allow reconsideration of the current vaccination policies and to avoid epidemics caused by the circulation of highly evolved OPV derivatives.     

Three cases of paralytic poliomyelitis associated with type 3 vaccine poliovirus strains in Bulgaria

Oral poliovirus vaccine (OPV) can cause, in extremely rare cases vaccine‐associated paralytic poliomyelitis in recipients, or contacts of vaccinees. Three cases of vaccine‐associated paralytic poliomyelitis (two contacts and one recipient) occurred in the Bourgas region of Bulgaria in the spring of 2006. The first two cases, notified as acute flaccid paralysis, were 55 days old unvaccinated twin brothers, having been in contact with vaccinees. The third case concerned a 4‐month‐old infant who had received the first OPV dose 37 days prior to the onset of illness. Complete clinical, epidemiological, virological, serological and molecular investigations of the children with paralysis and their contacts were undertaken. In all the three cases type 3 polioviruses were isolated from fecal samples and characterized as Sabin‐like poliovirus strains. Type 3 polioviruses isolated from the twin brothers demonstrated by sequence analysis U‐to‐C back mutation at nt 472 of the 5′ UTR, known to correlate with neurovirulence, and mutation in the VP1 region. Type 3 poliovirus isolated from the third child demonstrated in the 3D sequenced region a recombination with Sabin type 1 poliovirus. In the latter region, three silent mutations and one, resulting in amino acid substitution, were also observed. The clinical, epidemiological and virological data and the neurological sequelae observed 60 days following the onset of paralysis, confirmed the diagnosis of vaccine‐associated paralytic poliomyelitis in all the three patients. J. Med. Virol. 81:1661–1667, 2009. © 2009 Wiley‐Liss, Inc.     

Assessment of efficacy of a live oral poliovirus vaccine for virulent Sabin-like poliovirus 1 strains in Japan

Virulent Sabin-like poliovirus (VSLP) was isolated from river and sewage waters between October 1993 and September 1995 in Toyama Prefecture, Japan (Yoshida et al., Lancet 356, 1461-1463, 2000). In this study, to assess the possibility of an epidemic of poliomyelitis caused by a VSLP in Japan under the current vaccination policy of administration of live attenuated oral poliovirus vaccine (OPV), we determined titers of serum neutralizing antibodies to poliovirus 1 (PV-1) strains Sabin (vaccine strain), Mahoney (wild-type strain) and G4-12 (VSLP) in various groups of residents of Toyama Prefecture, Japan. The seropositivity and geometric mean neutralizing antibody titers against these strains in the individuals who obtained two doses of OPV were 99.1%, 94.5% and 95.5%, respectively, and 564, 186 and 194, respectively. Although the antibody titers to G4-12 were lower compared with those to Sabin, these results indicate that the OPV vaccination policy in Japan has been effective in preventing poliomyelitis caused by VSLPs. These results also suggest that (i) an epidemic of poliomyelitis caused by a VSLP has not occurred in Japan due to herd immunity, and (ii) the possibility of reemergence of VSLPs will be prevented if sufficient herd immunity is acquired immediately after completion of the OPV vaccination in accordance with the poliomyelitis eradication program.     

Characterization of poliovirus vaccine strains isolated in the country with high level of immunization coverage

The frequency of vaccine poliovirus isolation from children aged under 3 years was studied in Belarus, a country with a high level of immunization against poliomyelitis. Antigenic and genetic characteristics of the isolated strains were studied. Vaccine poliovirus detection rate was high (11.8%). Polioviruses were isolated from children immunized recently (27.2%), immunized more than 2 months before (7.5%), and from non-immunized children (9.8%). An appreciable number (36.1%) of the isolated strains were antigenically and/or genetically modified derivatives of Sabin virus. Epidemiological data and genetic characteristics of the isolated polioviruses indicate that some of them can be sufficiently transmissive for maintaining their "silent" circulation even in a population with a high level of immunization.     

Molecular evolution of oral poliovirus vaccine strains during multiplication in humans and possible implications for global eradication of poliovirus

The oral poliovirus vaccine (OPV) has been effectively used in the control of poliomyelitis and in the eradication of wild polioviruses. Although there are many advantages in using attenuated OPV strains in the campaign to eradicate poliomyelitis, several studies have demonstrated that there are some disadvantages such as (a) excretion by vaccines of OPV-derived polioviruses with genomic modifications known to increase the neurovirulence, (b) appearance of vaccine-associated paralytic poliomyelitis (VAPP) and other adverse effects in vaccinees, (c) occurrence of persistent infections caused by OPV-derived strains in immunodeficient patients with VAPP, (d) transmission of OPV-derived polioviruses to susceptible individuals which develop VAPP, and (e) detection of OPV-derived polioviruses in the environment, which could be a source of infection for humans in the future. Different studies indicate that it is important to consider the possibility of persistent infections and excretion of OPV-derived polioviruses for long periods by humans, and also the survival in the environment of OPV-derived polioviruses excreted by humans, which could be transmitted and circulate in a non-immune population after stopping poliovirus vaccination. The findings reported here may have important implications for global poliomyelitis eradication initiative and indicate that surveillance of OPV-derived strains will also be important in the final step of eradication of poliomyelitis from the planet.     

Evolution of attenuating mutations in dengue-2 strain S16803 PDK50 vaccine and comparison of growth kinetics with parent virus

A live-attenuated dengue-2 virus strain S16803 vaccine candidate that is immunogenic and safe in humans was derived by 50 passages in primary dog kidney (PDK) cells. To identify mutations associated with attenuation of the dengue-2 PDK50 vaccine strain, we determined the nucleotide changes that arose during PDK passage of the dengue-2 virus. Thirteen mutations distinguished the PDK50 virus from low-passage parent resulting in amino acid substitutions in the premembrane (E89G), envelope (E202K, N203D), nonstructural proteins NS1 (A43T), NS2A (L181F), NS2B (I26V), and NS4B (I/T108T, L112F). In addition, the PDK50 virus contained a C to T change of nucleotide 57 in the 5′ non-coding region and four silent mutations of nucleotides 591, 987, 6471, and 8907. An infectious PDK50 cDNA clone virus was produced and characterized for growth kinetics in monkey (LLC-MK₂, Vero) and mosquito (C6/36) cells. Identification of mutations in the vaccine strain and availability of an infectious clone will permit systematic analysis of the importance of individual or collective mutations on attenuation of dengue virus.     

A seroprevalence study of poliovirus antibody against a collection of recombinant and non-recombinant poliovirus vaccine strains in the population of southern Greece

In this study, the serological status of the southern Greek population in the 1–10-year, 11–20-year, 21–30-year and 31–40-year age groups with regard to Sabin vaccine strains and a collection of 15 recombinant and four non-recombinant poliovirus vaccine strains was determined. For all three poliovirus types, the highest neutralization test (NT) titres were observed in the 1–10-year age group, indicating a good response to vaccination. In general, the serological status of the population of southern Greece with regard to poliovirus is better for types 1 and 2 than for type 3. The presence of the lowest NT titre in the 21–30-year age group against poliovirus type 3 suggests the need for a booster dose of monovalent Sabin3 vaccine to ensure personal and herd immunity.     

Clinical development of a novel inactivated poliomyelitis vaccine based on attenuated Sabin poliovirus strains

Following achievement of polio eradication, the routine use of all live-attenuated oral poliovirus vaccines should be discontinued. However, the costs per vaccine dose for the alternative inactivated poliovirus vaccine (IPV) are significantly higher and the current production capacity is not sufficient for worldwide distribution of the vaccine. In order to achieve cost-prize reduction and improve affordability, IPV production processes and dose-sparing strategies should be developed to facilitate local manufacture at a relatively lower cost. The use of attenuated Sabin instead of wild-type polio strains will provide additional safety during vaccine production and permits production in low-cost settings. Sabin-IPV is under development by several manufacturers. This article gives an overview of results from clinical trials with Sabin-IPV and discusses the requirements and challenges in the clinical development of this novel IPV.     

RETRACTED ARTICLE: Evidence for recombination between vaccine and wild-type mumps virus strains

Recombination of mumps virus (MuV) has rarely been reported. In this study, phylogenetic and recombination analyses were performed on 30 complete MuV genomes, including 17 vaccine and 13 wild-type strains. One potentially significant recombination event was found to have occurred between the lineage represented by the vaccine strain L3/Russia/Vector (AY508995) as the minor parent and wild MuV strain Drag94 (AY669145) as the major parent, and this led to a recombinant, 9218/Zg98 (EU370206), a wild-type MuV strain isolated from a 3-year-old boy with parotitis. In summary, we found a recombinant of MuV derived from vaccine and wild-type MuV strains.     

DTaP-sIPV联合疫苗中SabinIPV 免疫原性研究

目的 探讨吸附无细胞百白破-Sabin株脊髓灰质炎联合疫苗（DTaP-sIPV）的制备工艺,并比较不同配比的DTaP-sIPV中Sabin株脊髓灰质炎灭活疫苗（SabinIPV）三针基础免疫大鼠的中和抗体效价,为确定联合疫苗的最佳制备工艺及抗原剂量配比提供参考.方法 用不同配比的SabinIPV,制备两批DTaP-sIPV,进行各项指标的检定及稳定性试验,并联合单独的Sabin IPV和GSK制备的DTaP-wIPV对56只Wistar大鼠进行3针免疫,每针间隔1个月,每次免疫后30 d采血并分离血清,采用微量中和试验测定血清中抗脊髓灰质炎病毒3个型别的中和抗体效价.结果 两批DTaPsIPV的各项检定指标均符合〈中国药典〉三部（2005版）要求,且稳定性良好.大鼠经3针基础免疫后,其Ⅰ、Ⅱ、Ⅲ型脊髓灰质炎病毒中和抗体的几何平均滴度均显著上升,3针免疫后抗体阳转率已经达到100%.结论 经此制备的DTaP-sIPV安全、稳定、有效,且DTaP-sIPV中的SabinIPV在大鼠中有良好的免疫效果,经3针免疫可产生高水平的中和抗体.     

Risks associated with the use of live-attenuated vaccine poliovirus strains and the strategies for control and eradication of paralytic poliomyelitis

The Global Polio Eradication Initiative was launched in 1988 with the aim to eliminate paralytic poliomyelitis. Two effective vaccines are available: inactivated polio vaccine (IPV) and oral polio vaccine (OPV). Since 1964, OPV has been used instead of IPV in most countries due to several economic and biological advantages. However, in rare cases, the live-attenuated Sabin strains of OPV revert to neurovirulence and cause vaccine-associated paralytic poliomyelitis in vaccinees or lead to emergence of vaccine-derived poliovirus strains. Attenuating mutations and recombination events have been associated with the reversion of vaccine strains to neurovirulence. The substitution of OPV with an improved new-generation IPV and the availability of new specific drugs against polioviruses are considered as future strategies for outbreak control and the eradication of paralytic poliomyelitis worldwide.     

Intertypic recombination in poliovirus: genetic and biochemical studies

Poliovirus strains of type 1 and type 3 carrying genetically mapped ts mutations and differring in growth response to guanidine have been used to infect HeLa cells. With four heterotypic pairs of the mutants, recombinants with the crossover points between the loci coding for the antigenic properties, on the one hand, and for the sensitivity to guanidine, on the other, have been obtained. The recombinants have been identified on the basis of their phenotypic properties and, in particular, of the pattern of inheritance of unselected markers. One recombinant has been characterized by fingerprinting virus-specific polypeptides. It has been found that the capsid proteins (VP2, VP3, and VP1) of this recombinant originate from the type 3 parent, whereas the nonstructural polypeptides (X, 2, and 4) are inherited from the type 1 parent. Implications of the poliovirus intertypic recombination are discussed.     

Childhood immunizations: position on the enhanced inactivated poliovirus vaccine and live attenuated oral poliovirus vaccine dilemma.

Recent review of the polio vaccines (live attenuated oral poliovirus vaccine [OPV] and enhanced inactivated poliovirus vaccine [eIPV]) for children has generated much debate between infectious disease experts and public health officials. Poliomyelitis was a common medical condition in the 1940s and 1950s, and the success of OPV in eradicating poliomyelitis from the United States and even the Western hemisphere cannot be disputed. However, the adverse condition of vaccine-associated paralytic poliomyelitis (VAPP) has been reported in eight to nine cases per year as a result of exclusively using OPV in the United States. The dilemma has been how to continue the elimination of wild-type poliovirus paralytic poliomyelitis in the United States and worldwide while minimizing the occurrence of VAPP. Clinical trials have supported that eIPV and OPV provide similar protection for humoral immunity. However, OPV provides superior gastrointestinal immunity, which is a public health benefit for vulnerable populations. Recommendations among experts have concluded that the sequential eIPV/OPV is the preferred schedule, with eIPV only or OPV only as alternative equally acceptable schedules. Therefore, factors such as cost, compliance, and access to health care must be considered by parents and providers when selecting a polio vaccine regimen, especially among underserved populations.