Plant-made polio type 3 stabilized VLPs-a candidate synthetic polio vaccine.

Poliovirus (PV) is the causative agent of poliomyelitis, a crippling human disease known since antiquity. PV occurs in two distinct antigenic forms, D and C, of which only the D form elicits a robust neutralizing response. Developing a synthetically produced stabilized virus-like particle (sVLP)-based vaccine with D antigenicity, without the drawbacks of current vaccines, will be a major step towards the final eradication of poliovirus. Such a sVLP would retain the native antigenic conformation and the repetitive structure of the original virus particle, but lack infectious genomic material. In this study, we report the production of synthetically stabilized PV VLPs in plants. Mice carrying the gene for the human PV receptor are protected from wild-type PV when immunized with the plant-made PV sVLPs. Structural analysis of the stabilized mutant at 3.6 Å resolution by cryo-electron microscopy and single-particle reconstruction reveals a structure almost indistinguishable from wild-type PV3.Despite the success of current vaccination against poliomyelitis, safe, cheap and effective vaccines remain sought for continuing eradication effort. Here the authors use plants to express stabilized virus-like particles of type 3 poliovirus that can induce a protective immune response in mice transgenic for the human poliovirus receptor.

[Immunoreactivity of chimeric proteins carrying poliovirus epitopes on the VP6 of rotavirus as a vector].

Rotavirus and poliovirus continue to present significant risks and burden of disease to children in developing countries. Developing a combined vaccine may effectively prevent both illnesses and may be advantageous in terms of maximizing compliance and vaccine coverage at the same visit. Recently, we sought to generate a vaccine vector by incorporating multiple epitopes into the rotavirus group antigenic protein, VP6. In the present study, a foreign epitope presenting a system using VP6 as a vector was created with six sites on the outer surface of the vector that could be used for insertion of foreign epitopes, and three VP6-based PV1 epitope chimeric proteins were constructed. The chimeric proteins were confirmed by immunoblot, immunofluorescence assay, and injected into guinea pigs to analyze the epitope-specific humoral response. Results showed that these chimeric proteins reacted with anti-VP6F and -PV1 antibodies, and elicited antibodies against both proteins in guinea pigs. Antibodies against the chimeric proteins carrying PV1 epitopes neutralized rotavirus Wa and PV1 infection in vitro. Our study contributes to a better understanding of the use of VP6-based vectors as multiple-epitope delivery vehicles and the epitopes displayed in this form could be considered for development of epitope-based vaccines against rotavirus and poliovirus.

Isolation and Characterization of Vaccine-Derived Polioviruses, Relevance for the Global Polio Eradication Initiative.

Stool specimens were collected from children with acute flaccid paralysis (AFP) and their contacts, and viral isolation was performed according to standard procedures. If the specimens tested positive for poliovirus, then intratypic differentiation (ITD) methods were performed on the viral isolates to determine whether the poliovirus isolates were wild or of vaccine origin, these include a poliovirus diagnostic ITD real-time PCR method and a vaccine-derived poliovirus (VDPV) screening real-time PCR method.Viral RNA was extracted from the poliovirus isolates by using the QIAamp Mini Viral RNA Extraction Kit (Qiagen) and was used for RT-PCR amplification by the standard method. The entire VP1 region of the poliovirus isolates was amplified by RT-PCR with primers that flanked the VP1-coding region. After purification of the PCR products by the QIAquick Gel Extraction Kit (Qiagen), the amplicons were bidirectionally sequenced with the ABI PRISM 3130 Genetic Analyzer (Applied Biosystems). A neurovirulence test of polioviruses isolates was carried out using PVR-Tg21 mice that expressed the human poliovirus receptor (CD155). And the temperature sensitivities of polioviruses isolates were assayed on monolayer RD cells in 24-well plates as described.

[Complete Genome Characterization of Vaccine-derived Polioviruses Isolated in Yunnan,China].

To explore the genomic characterization of 4vaccine-derived poliovirus(VDPV)strains isolated from 2acute flaccid paralysis(AFP)cases in Yunnan Province in 2010 and 2012,respectively,the complete genome sequences of the 4strains were determined. Sequence analysis revealed that the complete genome length of the type Ⅱ and type Ⅰ VDPV was 7439nt and 7441 nt, respectively. Nucleotide and amino acid sequence similarities of type II VDPV were 95.4% and 97.7%,respectively,and type I VDPV were93.9% and 97.9%,respectively as compared with those of Sabin strains. Nucleotide substitutions were found at two important attenuation sites (nt 481 and nt in type Ⅱ VDPV, and three important attenuation sites(nt480,nt2795 and nt6203)in type I VDPV. Type 2 and type 1VDPV strains had 1.0% and2.3% divergence with Sabin strains, respectively. Similarity plot analysis showed multiple recombination events in the genome of the 4strains,which showed that the recombination was common and complex. Analysis of the characteristics of VDPVs on molecular level could provide valuable information on evolutionary dynamics and lay foundation for developing scientific and feasible strategy to control VDPV.

Comparison of microarray-predicted closest genomes to sequencing for poliovirus vaccine strain similarity and influenza A phylogeny.

We evaluate sequence data from the PathChip high-density hybridization array for epidemiological interpretation of detected pathogens. For influenza A, we derive similar relative outbreak clustering in phylogenetic trees from PathChip-derived compared to classical Sanger-derived sequences. For a positive polio detection, recent infection could be excluded based on vaccine strain similarity.

[Genetic Characteristics of Type 2 Vaccine-derived Poliovirus in Shanxi Province (China) in 2014].

The World Health Organization redefined the type 2 vaccine-derived poliovirus (VDPV) in 2010. To study the genetic characteristics and evolution of type 2 VDPV under this new definition, we conducted genome sequencing and analyses of type 2 VDPVs isolated from one patient with acute flaccid paralysis in Shanxi province (China) in 2014. Nucleotide sequencing revealed that the full-length of type 2 VDPV is 7439 bases encoding 2207 amino acids with no insertion or deletion of nucleotides compared with Sabin2. One nucleotide substitution identified as a key determinant of the attenuated phenotype of the Sabin 2 strain (A-G reversion at nucleotide nt 481 in the 5-end of the untranslated region) had reverted in the Shanxi type 2 VDPV. The other known key determinant of the attenuated phenotype of the Sabin 2 strain (U-->C reversion at nt2909 in the VP1 coding region that caused a Ile143Thr substitution in VP1) had not reverted in the Shanxi VDPV. The Shanxi type 2 VDPV was S2/S1 recombinant, the crossover site of which mapped to the 3-end of the 3D region (between nt 6247 and nt 6281). A phylogentic tree based on the VP1 coding region showed that evolution of the Shanxi type 2 VDPV was independent of other type 2 VDPVs detected worldwide. We estimated that the strain circulated for approximately = 11 months in the population according to the known evolution rate. The present study confirmed that the Chinese Polio Laboratory Network could discover the VDPV promptly and that it played an important part in maintenance of a polio-free China.

Circulating vaccine-derived polioviruses in the Extreme North region of Cameroon.

BACKGROUND: The World Health Organization (WHO) poliovirus eradication program includes careful surveillance of acute-flaccid paralysis (AFP) and mass and routine immunization with oral polio vaccine (OPV). In populations with low vaccine coverage, the live-attenuated Sabin strains, OPV types 1, 2 and 3, can evolve into virulent vaccine-derived polioviruses (VDPVs) and circulate in the community. Until recently, circulating VDPVs (cVDPVs) had not been reported in Cameroon despite the fact that VDPV2 outbreaks have occurred in nearby countries. OBJECTIVES: This study aimed to characterize virus isolates from four AFP patients infected with cVDPV2 in the Extreme North region of Cameroon in 2013. STUDY DESIGN: The complete VP1 region of the four VDPV strains was sequenced and the relationships with cVDPVs from neighboring countries were investigated. RESULTS: All four patients were infected by cVDPV2 strains showing 1.2-2.0% nucleotide difference compared to the reference Sabin 2 VP1 sequence. Phylogenetic analysis indicated that the VDPV strains were genetically linked to cVDPV2 lineages of the recent Chad cVDPV2 outbreak. CONCLUSIONS: The circulation of pathogenic VDPVs suggests that there are localized immunization gaps in some districts like Makary, Mada and Kolofata in Cameroon. To avoid poliomyelitis outbreaks in Cameroon, especially in the districts close to neighboring countries with ongoing cVDPV outbreaks, high polio vaccine coverage is essential.

[Investigation of a Patient with Pre-vaccine-derived Poliovirus in Shandong Province, China].

To analyze the genetic characteristics of a polio-I highly variant vaccine recombinant virus in Shandong Province (China) in 2011 and to identify isolates from healthy contacts, two stool specimens from one patient with acute flaccid paralysis (AFP) and 40 stool specimens from his contacts were collected for virus isolation. The complete genome of poliovirus and VP1 coding region of the non-polio enterovirus were sequenced. Homologous comparison and phylogenetic analyses based on VP1 sequences were undertaken among coxsackievirus (CV) B1, CV-B3 isolates, and those in GenBank. One poliovirus (P1/11186), CV-A4 and CV-A8 were isolated from the AFP patient; one CV-A2, Echovirus 3 (E-3), E-12 and E-14, ten CV-B1, and five CV-B3 strains were isolated from his contacts. These results led us to believe that there may be a human enterovirus epidemic in this area, and that surveillance must be enhanced. P1/11186 was a type-1 vaccine-related poliovirus; it combined with type-2 and type-3 polioviruses in 2A and 3A regions, respectively. There were 25 nucleotide mutations with 9 amino-acid alterations in the entire genome. There were 8 nucleotide mutations with 5 amino-acid alterations in the VP1 region compared with the corresponding Sabin strains. Homology analyses suggested that the ten CV-B1 isolates had 97.0%-100% nucleotide and 98.9%-100% amino-acid identities with each other, as well as 92.6%-100% nucleotide and 99.2%-100% amino-acid identities among the five CV-B3 isolates. Phylogenetic analyses on the complete sequences of VP1 among CV-B1 and CV-B3 isolates showed that Shandong strains, together with strains from other provinces in China, had a close relationship and belonged to the same group.

Vaccine-derived mutation in motif D of poliovirus RNA-dependent RNA polymerase lowers nucleotide incorporation fidelity.

All viral RNA-dependent RNA polymerases (RdRps) have a conserved structural element termed motif D. Studies of the RdRp from poliovirus (PV) have shown that a conformational change of motif D leads to efficient and faithful nucleotide addition by bringing Lys-359 into the active site where it serves as a general acid. The RdRp of the Sabin I vaccine strain has Thr-362 changed to Ile. Such a drastic change so close to Lys-359 might alter RdRp function and contribute in some way to the attenuated phenotype of Sabin type I. Here we present our characterization of the T362I RdRp. We find that the T362I RdRp exhibits a mutator phenotype in biochemical experiments in vitro. Using NMR, we show that this change in nucleotide incorporation fidelity correlates with a change in the structural dynamics of motif D. A recombinant PV expressing the T362I RdRp exhibits normal growth properties in cell culture but expresses a mutator phenotype in cells. For example, the T362I-containing PV is more sensitive to the mutagenic activity of ribavirin than wild-type PV. Interestingly, the T362I change was sufficient to cause a statistically significant reduction in viral virulence. Collectively, these studies suggest that residues of motif D can be targeted when changes in nucleotide incorporation fidelity are desired. Given the observation that fidelity mutants can serve as vaccine candidates, it may be possible to use engineering of motif D for this purpose.

Highly divergent type 2 and 3 vaccine-derived polioviruses isolated from sewage in Tallinn, Estonia.

Highly divergent vaccine-derived polioviruses (VDPVs) have been isolated from sewage in Tallinn, Estonia, since 2002. Sequence analysis of VDPVs of serotypes 2 and 3 showed that they shared common noncapsid region recombination sites, indicating origination from a single trivalent oral polio vaccine dose, estimated to have been given between 1986 and 1998. The sewage isolates closely resemble VDPVs chronically excreted by persons with common variable immunodeficiency, but no chronic excretors have yet been identified in Estonia.

Multiple independent emergences of type 2 vaccine-derived polioviruses during a large outbreak in northern Nigeria.

Since 2005, a large poliomyelitis outbreak associated with type 2 circulating vaccine-derived poliovirus (cVDPV2) has occurred in northern Nigeria, where immunization coverage with trivalent oral poliovirus vaccine (tOPV) has been low. Phylogenetic analysis of P1/capsid region sequences of isolates from each of the 403 cases reported in 2005 to 2011 resolved the outbreak into 23 independent type 2 vaccine-derived poliovirus (VDPV2) emergences, at least 7 of which established circulating lineage groups. Virus from one emergence (lineage group 2005-8; 361 isolates) was estimated to have circulated for over 6 years. The population of the major cVDPV2 lineage group expanded rapidly in early 2009, fell sharply after two tOPV rounds in mid-2009, and gradually expanded again through 2011. The two major determinants of attenuation of the Sabin 2 oral poliovirus vaccine strain (A481 in the 5'-untranslated region [5'-UTR] and VP1-Ile143) had been replaced in all VDPV2 isolates; most A481 5'-UTR replacements occurred by recombination with other enteroviruses. cVDPV2 isolates representing different lineage groups had biological properties indistinguishable from those of wild polioviruses, including efficient growth in neuron-derived HEK293 cells, the capacity to cause paralytic disease in both humans and PVR-Tg21 transgenic mice, loss of the temperature-sensitive phenotype, and the capacity for sustained person-to-person transmission. We estimate from the poliomyelitis case count and the paralytic case-to-infection ratio for type 2 wild poliovirus infections that ∼700,000 cVDPV2 infections have occurred during the outbreak. The detection of multiple concurrent cVDPV2 outbreaks in northern Nigeria highlights the risks of cVDPV emergence accompanying tOPV use at low rates of coverage in developing countries.

[Formation and identification of virus-like particles of poliovirus type I].

OBJECTIVE: To establish a method to produce virus-like particles (VLP) of poliovirus type I in Saccharomy cescerevisiae to develop potential novel recombinant vaccine against poliovirus type 1. METHODS: The genes of P1 and 3CD of poliovirus type I were optimized, synthesized and inserted into expression vector, which was further transfected into Saccharomy cescerevisiae. The extracts of yeast cells were purified by CsCl density gradient centrifugation after induction and cell lysis. RESULTS: Electrophoresis and sequencing analyses showed that the genes P1 and 3CD of poliovirus type I were successfully inserted into expression vector and encode a protein whose amino acid sequences were identical with wide-type genes of poliovirus type I. Electronic microscopy analysis showed that the VLPs of poliovirus type I could be efficiently formed in Saccharomy cescerevisiae. CONCLUSION: The VLPs of poliovirus type I could be efficiently produced by co-expression of P1 and 3CD genes in Saccharomy cescerevisiae.

Phenotypic and genomic analysis of serotype 3 Sabin poliovirus vaccine produced in MRC-5 cell substrate.

The cell substrate has a pivotal role in live virus vaccines production. It is necessary to evaluate the effects of the cell substrate on the properties of the propagated viruses, especially in the case of viruses which are unstable genetically such as polioviruses, by monitoring the molecular and phenotypical characteristics of harvested viruses. To investigate the presence/absence of mutation(s), the near full-length genomic sequence of different harvests of the type 3 Sabin strain of poliovirus propagated in MRC-5 cells were determined. The sequences were compared with genomic sequences of different virus seeds, vaccines, and OPV-like isolates. Nearly complete genomic sequencing results, however, revealed no detectable mutations throughout the genome RNA-plaque purified (RSO)-derived monopool of type 3 OPVs manufactured in MRC-5. Thirty-six years of experience in OPV production, trend analysis, and vaccine surveillance also suggest that: (i) different monopools of serotype 3 OPV produced in MRC-5 retained their phenotypic characteristics (temperature sensitivity and neuroattenuation), (ii) MRC-5 cells support the production of acceptable virus yields, (iii) OPV replicated in the MRC-5 cell substrate is a highly efficient and safe vaccine. These results confirm previous reports that MRC-5 is a desirable cell substrate for the production of OPV.

Highly divergent neurovirulent vaccine-derived polioviruses of all three serotypes are recurrently detected in Finnish sewage.

In Finland, surveillance of potential re-emergence of poliovirus transmission is mainly based on environmental surveillance, i.e. search for infectious poliovirus in sewage samples. Since December 2008, 21 genetically highly divergent, neurovirulent vaccine-derived polioviruses (VDPV) have been isolated from sewage in Tampere, Finland. While the source of the VDPV is unknown, characteristics of the viruses resemble those of strains isolated from immunodeficient, persistently infected persons. No cases of suspected poliomyelitis have been reported in Finland since 1985.

Characterization of a rare natural intertypic type 2/type 3 penta-recombinant vaccine-derived poliovirus isolated from a child with acute flaccid paralysis.

A type 2 vaccine-derived poliovirus (VDPV) (strain CHN1025), with a 1.1 % (10/903) difference from Sabin strain in the VP1 coding region, was isolated from a child with poliomyelitis caused by a poliovirus variant infection. The patient was from Shandong Province of China and developed acute flaccid paralysis in 1997. The child was infected with a rare and complicated penta-recombinant poliovirus with the uncommon genomic recombinant organization S2/S3/S1/S3/S1/S3. At least five successive rounds of recombination occurred in the VP1 capsid coding region and in the 2C, 3C (twice) and 3D(pol) non-capsid coding regions, respectively, during virus evolution. Strain CHN1025 had most of the characteristics of the type 2 vaccine strain; it had Sabin-specific epitopes, suggesting that the virus was antigenically indistinguishable from the Sabin 2 reference strain. Typical mutations in the 5'-untranslated region and VP1 associated with reversion to neurovirulence for Sabin 2 poliovirus were found, and the virus showed moderate neurovirulence in transgenic mice. A few nucleotide substitutions were located in the donor sequences, and two donor sequences contained no nucleotide substitutions, suggesting that these sequences were relatively new. The appearance of these mutations within approximately 192 days of at least five successive rounds of recombination events derived from a single ancestral infection illustrates the rapid emergence of new recombinants among VDPVs. This is the first report on the isolation of a type 2/type 3 poliovirus capsid recombinant with one of the five crossover sites located in the VP1 coding region.

Future of polio vaccines.

Over the past half-century, global use of highly effective vaccines against poliomyelitis brought this disease to the brink of elimination. Mounting evidence supports the argument that a high level of population immunity must be maintained after wild poliovirus circulation is stopped to preserve a polio-free status worldwide. Shifting factors in the risk-benefit-cost equation favor the creation of new poliovirus vaccines for use in the foreseeable future. Genetically stable attenuated virus strains could be developed for an improved oral poliovirus vaccine, but proving their safety and efficacy would be impractical owing to the enormous size of the clinical trials required. Novel versions of inactivated poliovirus vaccine that could be used globally should be developed. An improved inactivated poliovirus vaccine must be efficacious, inexpensive, safe to manufacture and easy to administer. Combination products containing inactivated poliovirus vaccine and other protective antigens should become part of routine childhood immunizations around the world.

[Analysis on identification and genetic character of type I vaccine-derived poliovirus in Shanxi province in 2007].

OBJECTIVE: To describe the source of vaccine-derived poliovirus (VDPV) and the effect on local polio-free status, the VP1 coding region was sequenced and analyzed for type I VDPV in Shanxi province in 2007. METHOD: The virus isolation was performed to double stool specimens from one case acute flaccid paralysis (AFP) patient. VP1 coding region of the isolated stain was sequenced and analyzed. The phylogenetic tree was constructed based on VP1 region sequence between Shanxi strains and other type I VDPVs. RESULT: 2 type I + II +III strains were isolated from double stool specimens from the AFP patient in Shanxi Province in 2007. VP1 sequencing of the two stains revealed > 1.0% divergence from the VP1 region of P I /Sabin vaccine strain. According to WHO criteria, the two stains were identified as type I vaccine-derived poliovirus (VDPV). Phylogenetic analysis based on VP1 coding sequence showed that the evolution distance of Shanxi type I VDPV was far away from other VDPVs detected in China. Moreover, no evidence supported the AFP patient as immunodeficiency patient. So Shanxi type I VDPVs were classified into ambiguous VDPV(aVDPV). CONCLUSION: Considering the genetic character for Shanxi type I VDPV and the local OPV coverage, we highly suspected that an immunodeficiency patient in local area who long-term excreted VDPVs existed and resulted in the patient infection of VDPV in Shanxi in 2007. In the post era of polio eradication, the detection and management for the possible existing patient of long-term excretion VDPV should be strengthened.

Estimating the extent of vaccine-derived poliovirus infection.

BACKGROUND: Eight outbreaks of paralytic polio attributable to circulating vaccine-derived poliovirus (cVDPV) have highlighted the risks associated with oral poliovirus vaccine (OPV) use in areas of low vaccination coverage and poor hygiene. As the Polio Eradication Initiative enters its final stages, it is important to consider the extent to which these viruses spread under different conditions, so that appropriate strategies can be devised to prevent or respond to future cVDPV outbreaks. METHODS AND FINDINGS: This paper examines epidemiological (temporal, geographic, age, vaccine history, social group, ascertainment), and virological (type, genetic diversity, virulence) parameters in order to infer the numbers of individuals likely to have been infected in each of these cVDPV outbreaks, and in association with single acute flaccid paralysis (AFP) cases attributable to VDPVs. Although only 114 virologically-confirmed paralytic cases were identified in the eight cVDPV outbreaks, it is likely that a minimum of hundreds of thousands, and more likely several million individuals were infected during these events, and that many thousands more have been infected by VDPV lineages within outbreaks which have escaped detection. CONCLUSIONS: Our estimates of the extent of cVDPV circulation suggest widespread transmission in some countries, as might be expected from endemic wild poliovirus transmission in these same settings. These methods for inferring extent of infection will be useful in the context of identifying future surveillance needs, planning for OPV cessation and preparing outbreak response plans.

Virus attenuation by genome-scale changes in codon pair bias.

As a result of the redundancy of the genetic code, adjacent pairs of amino acids can be encoded by as many as 36 different pairs of synonymous codons. A species-specific "codon pair bias" provides that some synonymous codon pairs are used more or less frequently than statistically predicted. We synthesized de novo large DNA molecules using hundreds of over-or underrepresented synonymous codon pairs to encode the poliovirus capsid protein. Underrepresented codon pairs caused decreased rates of protein translation, and polioviruses containing such amino acid-independent changes were attenuated in mice. Polioviruses thus customized were used to immunize mice and provided protective immunity after challenge. This "death by a thousand cuts" strategy could be generally applicable to attenuating many kinds of viruses.