Javier Martin

Evolution of the Sabin Strain of Type 3 Poliovirus in an Immunodeficient Patient during the Entire 637-Day Period of Virus Excretion

ABSTRACT 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.

Failure to clear persistent vaccine-derived neurovirulent poliovirus infection in an immunodeficient man

BACKGROUND Individuals who chronically excrete neurovirulent poliovirus of vaccine-origin are of considerable concern to the Global Polio Eradication programme. Chronic infection with such polioviruses is a recognised complication of hypogammaglobulinaemia. METHODS We did a series of in-vitro and in-vivo therapeutic studies, with a view to clearing persistent neurovirulent poliovirus infection in an individual with common variable immunodeficiency, using oral immunoglobulin, breast milk (as a source of secretory IgA), ribavirin, and the anti-picornaviral agent pleconaril. We undertook viral quantitation, antibody neutralisation and drug susceptibility assays, and viral gene sequencing. FINDINGS Long-term asymptomatic excretion of vaccine-derived neurovirulent poliovirus 2 was identified in this hypogammaglobulinaemic man, and was estimated to have persisted for up to 22 years. Despite demonstrable in-vitro neutralising activity of immunoglobulin and breast milk, and in-vitro antiviral activity of ribavirin, no treatment was successful at clearing the virus, although in one trial breast milk significantly reduced excretion levels temporarily. During the course of study, the virus developed reduced susceptibility to pleconaril, precluding the in-vivo use of this drug. Sequence analysis revealed the emergence of a methionine to leucine mutation adjacent to the likely binding site of pleconaril in these isolates. INTERPRETATION Chronic vaccine-associated poliovirus infection in hypogammaglobulinaemia is a difficult condition to treat. It represents a risk to the strategy to discontinue polio vaccination once global eradication has been achieved.

Isolation of an Intertypic Poliovirus Capsid Recombinant from a Child with Vaccine-Associated Paralytic Poliomyelitis

ABSTRACT The isolation of a capsid intertypic poliovirus recombinant from a child with vaccine-associated paralytic poliomyelitis is described. Virus 31043 had a Sabin-derived type 3-type 2-type 1 recombinant genome with a 5′-end crossover point within the capsid coding region. The result was a poliovirus chimera containing the entire coding sequence for antigenic site 3a derived from the Sabin type 2 strain. The recombinant virus showed altered antigenic properties but did not acquire type 2 antigenic characteristics. The significance of the presence in nature of such poliovirus chimeras and the consequences for the current efforts to detect potentially dangerous vaccine-derived poliovirus strains are discussed in the context of the global polio eradication initiative.

Co-circulation and evolution of polioviruses and species C enteroviruses in a district of Madagascar.

Between October 2001 and April 2002, five cases of acute flaccid paralysis (AFP) associated with type 2 vaccine-derived polioviruses (VDPVs) were reported in the southern province of the Republic of Madagascar. To determine viral factors that favor the emergence of these pathogenic VDPVs, we analyzed in detail their genomic and phenotypic characteristics and compared them with co-circulating enteroviruses. These VDPVs appeared to belong to two independent recombinant lineages with sequences from the type 2 strain of the oral poliovaccine (OPV) in the 5′-half of the genome and sequences derived from unidentified species C enteroviruses (HEV-C) in the 3′-half. VDPV strains showed characteristics similar to those of wild neurovirulent viruses including neurovirulence in poliovirus-receptor transgenic mice. We looked for other VDPVs and for circulating enteroviruses in 316 stools collected from healthy children living in the small area where most of the AFP cases occurred. We found vaccine PVs, two VDPVs similar to those found in AFP cases, some echoviruses, and above all, many serotypes of coxsackie A viruses belonging to HEV-C, with substantial genetic diversity. Several coxsackie viruses A17 and A13 carried nucleotide sequences closely related to the 2C and the 3Dpol coding regions of the VDPVs, respectively. There was also evidence of multiple genetic recombination events among the HEV-C resulting in numerous recombinant genotypes. This indicates that co-circulation of HEV-C and OPV strains is associated with evolution by recombination, resulting in unexpectedly extensive viral diversity in small human populations in some tropical regions. This probably contributed to the emergence of recombinant VDPVs. These findings give further insight into viral ecosystems and the evolutionary processes that shape viral biodiversity.

Long-Term Excretion of Vaccine-Derived Poliovirus by a Healthy Child

ABSTRACT A child was found to be excreting type 1 vaccine-derived poliovirus (VDPV) with a 1.1% sequence drift from Sabin type 1 vaccine strain in the VP1 coding region 6 months after he was immunized with oral live polio vaccine. Seventeen type 1 poliovirus isolates were recovered from stools taken from this child during the following 4 months. Contrary to expectation, the child was not deficient in humoral immunity and showed high levels of serum neutralization against poliovirus. Selected virus isolates were characterized in terms of their antigenic properties, virulence in transgenic mice, sensitivity for growth at high temperatures, and differences in nucleotide sequence from the Sabin type 1 strain. The VDPV isolates showed mutations at key nucleotide positions that correlated with the observed reversion to biological properties typical of wild polioviruses. A number of capsid mutations mapped at known antigenic sites leading to changes in the viral antigenic structure. Estimates of sequence evolution based on the accumulation of nucleotide changes in the VP1 coding region detected a “defective” molecular clock running at an apparent faster speed of 2.05% nucleotide changes per year versus 1% shown in previous studies. Remarkably, when compared to several type 1 VDPV strains of different origins, isolates from this child showed a much higher proportion of nonsynonymous versus synonymous nucleotide changes in the capsid coding region. This anomaly could explain the high VP1 sequence drift found and the ability of these virus strains to replicate in the gut for a longer period than expected.

Molecular epidemiology of silent introduction and sustained transmission of wild poliovirus type 1, Israel, 2013.

Poliovirus vaccine coverage in Israel is over 90%. The last nine birth cohorts have been vaccinated exclusively with inactivated polio vaccine (IPV). However, between February and July 2013 type 1 wild poliovirus (WPV1) was detected persistently in 10 and intermittently in 8 of 47 environmental surveillance sites in southern and central Israel and in 30 stool samples collected during July from healthy individuals in southern Israel. We report results of sequence and phylogenetic analyses of genes encoding capsid proteins to determine the source and transmission mode of the virus. WPV1 capsid protein 1 nucleotide sequences were most closely related to South Asia (SOAS) cluster R3A polioviruses circulating in Pakistan in 2012 and isolated from Egyptian sewage in December 2012. There was no noticeable geographical clustering within WPV1-positive sites. Uniform codon usage among isolates from Pakistan, Egypt and Israel showed no signs of optimisation or deoptimisation. Bayesian phylogenetic time clock analysis of the entire capsid coding region (2,643 nt) with a 1.1% evolutionary rate indicated that Israeli and Egyptian WPV1-SOAS lineages diverged in September 2012, while Israeli isolates split into two sub-branches after January 2013. This suggests one or more introduction events into Israel with subsequent silent circulation despite high population immunity.

Characterization and standardization of Sabin based inactivated polio vaccine: proposal for a new antigen unit for inactivated polio vaccines.

GMP-batches of Sabin-IPV were characterized for their antigenic and immunogenic properties. Antigenic fingerprints of Sabin-IPV reveal that the D-antigen unit is not a fixed amount of antigen but depends on antibody and assay type. Instead of the D-antigen unit we propose standardization of IPV based on a combination of protein amount for dose and D-antigenicity for quality of the vaccine. Although Sabin-IPV type 2 is less immunogenic than regular wild type IPV type 2, the immunogenicity (virus neutralizing titers) per microgram antigen for Sabin-IPV type 2 is in the same order as for wild type serotypes 1 and 3. The latter observations are in line with data from human trials. This suggests that a higher dose of Sabin-IPV type 2 to compensate for the lower rat immunogenicity may not be necessary.

Twenty-Eight Years of Poliovirus Replication in an Immunodeficient Individual: Impact on the Global Polio Eradication Initiative.

There are currently huge efforts by the World Health Organization and partners to complete global polio eradication. With the significant decline in poliomyelitis cases due to wild poliovirus in recent years, rare cases related to the use of live-attenuated oral polio vaccine assume greater importance. Poliovirus strains in the oral vaccine are known to quickly revert to neurovirulent phenotype following replication in humans after immunisation. These strains can transmit from person to person leading to poliomyelitis outbreaks and can replicate for long periods of time in immunodeficient individuals leading to paralysis or chronic infection, with currently no effective treatment to stop excretion from these patients. Here, we describe an individual who has been excreting type 2 vaccine-derived poliovirus for twenty eight years as estimated by the molecular clock established with VP1 capsid gene nucleotide sequences of serial isolates. This represents by far the longest period of excretion described from such a patient who is the only identified individual known to be excreting highly evolved vaccine-derived poliovirus at present. Using a range of in vivo and in vitro assays we show that the viruses are very virulent, antigenically drifted and excreted at high titre suggesting that such chronic excreters pose an obvious risk to the eradication programme. Our results in virus neutralization assays with human sera and immunisation-challenge experiments using transgenic mice expressing the human poliovirus receptor indicate that while maintaining high immunisation coverage will likely confer protection against paralytic disease caused by these viruses, significant changes in immunisation strategies might be required to effectively stop their occurrence and potential widespread transmission. Eventually, new stable live-attenuated polio vaccines with no risk of reversion might be required to respond to any poliovirus isolation in the post-eradication era.

Investigation of porcine circovirus contamination in human vaccines.

DNA from porcine circovirus type 1 (PCV1) and 2 (PCV2) has recently been detected in two vaccines against rotaviral gastroenteritis from manufacturers A and B. We investigated if PCV1 sequences are present in other viral vaccines. We screened seeds, bulks and final vaccine preparations from ten manufacturers using qRT-PCR. We detected 3.8 × 10³ to 1.9 × 10⁷ PCV1 DNA copies/milliliter in live poliovirus seeds for inactivated polio vaccine (IPV) from manufacturer A, however, following inactivation and purification, the finished IPV was PCV1-negative. PCV1 DNA was not detectable in live polio preparations from other vaccine producers. There was no detectable PCV1 DNA in the measles, mumps, rubella and influenza vaccines analysed including material supplied by manufacturer A. We confirmed that the PCV1 genome in the rotavirus vaccine from manufacturer A is near full-length. It contains two mutations in the PCV cap gene, which may result from viral adaptation to Vero cells. Bulks of this vaccine contained 9.8 × 10¹⁰ to 1.8 × 10¹¹ PCV1 DNA copies/millilitre and between 4.1 × 10⁷ and 5.5 × 10⁸ DNA copies were in the final doses. We found traces of PCV1 and PCV2 DNA in the rotavirus vaccine from manufacturer B. This highlights the issue of vaccine contamination and may impact on vaccine quality control.

Isolation and Characterization of Circulating Type 1 Vaccine-Derived Poliovirus from Sewage and Stream Waters in Hispaniola

Twenty-one cases of acute flaccid paralysis (AFP) were reported on the island of Hispaniola in 2000. Laboratory analysis confirmed the presence of circulating vaccine-derived poliovirus (cVDPV) type 1 in stool samples obtained from patients. As a complement to the active search for cases of AFP, environmental sampling was conducted during November and December 2000, to test for cVDPV in sewage, streams, canals, and public latrines. Fifty-five environmental samples were obtained and analyzed for the presence of polioviruses by use of cell culture followed by neutralization and reverse-transcription polymerase chain reaction. Of the 23 positive samples, 10 tested positive for poliovirus type 1, 7 tested positive for poliovirus type 2, 5 tested positive for poliovirus type 3, and 1 tested positive for both poliovirus type 2 and type 3. By sequence analysis of the complete viral capsid gene 1 (VP1), a 2.1%-3.7% genetic sequence difference between 7 type 1 strains and Sabin type 1 vaccine strain was found. Phylogenetic analysis showed that these viruses are highly related to cVDPV isolated from clinical cases and form distinct subclusters related to geographic region. Our findings demonstrate a useful role for environmental surveillance of neurovirulent polioviruses in the overall polio eradication program.