Risks associated with the use of live-attenuated vaccine poliovirus ...

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Risks associated with the use of live-attenuated vaccine poliovirus strains and the strategies for control and eradication of paralytic poliomyelitis Expert Rev. Vaccines 11(5), 609–628 (2012)

Vaia Pliaka*, Zaharoula Kyriakopoulou and Panayotis Markoulatos University of Thessaly, School of Health Sciences, Department of Biochemistry and Biotechnology, Microbiology–Virology Laboratory, Larissa, Greece *Author for correspondence: Tel.: +30 241 056 5274 Fax: +30 241 056 5294 [email protected]

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. Keywords: immunization schedules • inactivated polio vaccine • mutations • neurovirulence • oral polio vaccine • recombination • vaccine-associated paralytic poliomyelitis • vaccine-derived polioviruses

Three types of poliovirus (PV), namely, types 1, 2 and 3, are responsible for the ­initiation of paralytic poliomyelitis. No specific treatment has been raised against the disease. Two effective vaccines against polio have been available since the 1950s: the oral PV vaccine (OPV), which is made of live-attenuated PV of three serotypes, and the inactivated PV vaccine (IPV), which is made of formalin-inactivated wild-type PVs. However, OPV was adopted throughout most of the countries mainly due to the ease of administration and lower cost. The incidence of paralytic poliomyelitis in an estimated 350,000 children in 125 endemic countries every year by the 1980s led the WHO to initiate the Global Polio Eradication Initiative (GPEI) in 1988. By 1999, this initiative had led to the annual reduction of ­paralytic poliomyelitis cases by 99%, the eradication of type2 wild PVs (WPVs) and the www.expert-reviews.com

10.1586/ERV.12.28

elimination of all PVs from three of the six WHO regions. Despite the effectiveness of OPV in reducing the transmission of paralytic poliomyelitis, its use has been associated with the emergence of vaccine-associated paralytic poliomyelitis (VAPP) cases and of vaccine-derived PVs (VDPVs) possessing neurovirulence and transmission properties similar to those of wild-type PVs. Owing to these complications, the use of OPV must cease globally and in a coordinated fashion once WPV transmission has been interrupted. This review focuses on the risks associated with the use of live-attenuated vaccine PV strains. The molecular mechanisms that contribute to the reversion of live-attenuated vaccine PV strains to neurovirulence are also discussed. Moreover, the progress of polio eradication and the strategies for control and eradication of paralytic poliomyelitis are presented.

© 2012 Expert Reviews Ltd

ISSN 1476-0584

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Poliomyelitis & PVs

Polioviruses belong to the Enterovirus genus of the Picornaviridae family. Enteroviruses are grouped into four species, namely, HEV-A to -D, according to the degree of their genetic relatedness [1] . Polioviruses had formed a separate group but now belong to HEV-C because of their molecular similarity [2] . The ingestion and multiplication of virus in the oropharyngeal and intestinal mucosa include the first stage of infection. After the initial multiplication of the virus in the mucosa, it moves into cervical and mesenteric lymph nodes and then to the blood, leading to a transient viremia. Most infections of humans are asymptomatic or mild including nonspecific symptoms such as sore throat, fever and malaise. However, virus replication at extraneural sites (brown fat, reticuloendothelial tissues and muscle) preserves viremia beyond the first stage, increasing the possibility of virus entry into the CNS. In rare cases (1–2% of infections), the virus invades the CNS and multiplies in motor neurons within the spinal cord, brain stem or motor cortex leading to the characteristic paralytic poliomyelitis [3] . On average, 10 days (range:  5–25 days) are needed for the appearance of paralysis [4] . In most developing countries, the major route of PV transmission is faecal–oral [5] . The virus replicates in the intestinal tract and is normally shed in the feces for 3–4 weeks, and occasionally for some weeks longer. Shedding may be discontinuous and is affected by the immune status and immune competence of the individual [6] . In areas with good hygiene and ­uncontaminated drinking water, other routes of transmission are major [5] . In these areas, PVs are mainly transmitted through upper respiratory tract secretions because the virus also replicates in the upper respiratory tract [7] . Virus can be isolated from throat swabs and washings in the early acute period of infection. Studies with nonpolio enteroviruses show that respiratory tract secretions are infectious and could lead to virus spread via direct person-to-person contact, large-particle aerosols or fomites [8,9] . The extent of the populations’ immunity gap and the presence of additional risk factors facilitating PV spread affect the duration and magnitude of PV transmission. The size of crowding, levels of hygiene, water quality and sewage handling facilities comprise factors that affect PV transmission [10] . Polioviruses are classified into three serotypes (PV1, PV2 and PV3) based on the cross-neutralization of PV isolates by polyclonal sera raised against the different PV strains [11] . Four major epitopes, known as antigenic sites 1–4 (AgS1–AgS4), have been recognized in PV capsids located on surface-exposed loops in viral structural proteins VP1, VP2 and VP3 [12] . PVs are small (diameter 30 nm), nonenveloped virions with icosahedral symmetry. Virus capsid includes 60 protomers, each consisting of one copy of capsid proteins VP1, VP2, VP3 and VP4. The viral proteins VP1, VP2 and VP3 are situated at the external side of the capsid forming the antigenic sites and the viral canyon where the cell receptor CD155 is attached during virus entry. VP4 viral protein is situated in the inner s­urface of the capsid [13] . The PV capsid covers the mRNA genome of ~7500 base pairs in length (Figure 1) . The viral genome starts with a 5′ noncoding 610

region (5′-NCR) of approximately 740 nt and ends in a 3′ noncoding region (3′-NCR) of approximately 70 nt followed by a poly (A)-sequence [14] . A cloverleaf structure, which is needed for the initiation of positive-strand RNA replication, is formed by the first 100 nt of 5′-NCR and is followed by further secondary structures forming the internal ribosomal entry site, which is essential for the cap-independent initiation of virus genome translation [14] . The 3′-NCR is crucial for the initiation of the negative-strand RNA synthesis. The protein coding region is located after the 5′-NCR and encodes a single polyprotein that is proteo-lytically cleaved into precursor proteins P1, P2 and P3, which give the final four structural proteins VP1 to VP4 (from P1) and the seven nonstructural proteins (from P2 and P3) (Figure 1) . Viral proteases (2A, 3C and 3CD) and the RNA-dependent RNA polymerase (3D) include the nonstructural proteins. In addition, the 2C viral protein is a helicase and 2B, 2BC, 3A and 3AB are essential for various functions during viral RNA replication [14] . Viral protein 3B (also known as VPg) is covalently linked to the 5′ end of the viral genome and participates in viral replication by functioning as a primer for RNA synthesis [15] . Mechanisms of genetic variation in PVs

Mutations and recombination events con-stitute the two major mechanisms that are responsible for the high rates of PVmolecular evolution.The mutation rate of PVs during genome replication is generally 10 -4 substitutions per nucleotide, which accounts for their quasispecies nature [14] . The characteristic of quasispecies provides the mutant swarms with a variety of strains with phenotypes that are possibly adaptable to a new environment [16] . The lack of a proof-reading/repair mechanism and a postreplicative error correction mechanism constitute the major biochemical causes for the limited replication fidelity in PVs [17] . Recombination frequently occurs in PV genomes and allows the elimination of adverse mutations from the viral genome leading to variants with increased fitness to an environmental change. Three different mechanisms of recombination have been proposed for PV: the ‘primer alignment- and- extension,’ the ‘breakage and ligation’ and the ‘template-switch’ mechanism [18–20] . The most widely accepted mechanism for RNA recombination in PVs is the template-switch mechanism proposed by Kirkegaard and Baltimore [19] . In this mechanism, PV RNA-dependent RNA polymerase changes template in the course of negativestrand RNA elongation. Probable causes for the slowdown and termination of the elongation are stable secondary structures [21] or nucleotide misincorporations [22] . In general, PVs exploit homologous recombination where the two parental sequences recombine in a homologous region, thus producing a new sequence with the same structure as the parental sequences [23] . A prerequisite for recombination is the coinfection of a cell with two different viral strains. Moreover, the recombination frequency depends on the similarity of the participating viral sequences [24] . Recombination occurs frequently, but not all chimeric strains survive due to the purifying selection of nonfunctional or reduced-fitness hybrids [24] . Expert Rev. Vaccines 11(5), (2012)

Risks associated with the use of live-attenuated vaccine poliovirus strains

Review

Poliovirus vaccines 5´-NCR Two different vaccines have been used for IRES the eradication of paralytic poliomyelitis IV since the 1950s. Formalin-IPV created by V Structural region Nonstructural region Jonas Salk et al. [25] was first made available Cloverleaf 3´-NCR VI P1 P2 P3 III in 1955. IPV comes from the inactivation of I II the three serotypes of wild-type PVs and is VP2 VP3 VP1 2A 2B 2C 3A 3C 3D used in an injectable form. In the 1970s, a VPg A Spacer A VP4 3B (VPg) new product, called enhanced potency IPV A n (eIPV), was invented by researchers at the National Institute for Public Health in The Netherlands. It was prepared from purified Figure 1. Structure of poliovirus genome and gene organization. The poliovirus genome consists of a single-stranded, positive-sense RNA molecule, that encodes a virus grown in large bioreactors and showed single polyprotein. The 5′-NCR harbors two functional domains, the cloverleaf and the significantly higher potency than the ‘Salk internal ribosome entry site, and is covalently linked to the viral protein VPg. The 3′-NCR IPV’ [26]. In the USA, the wide use of IPV is polyadenylated. The polyprotein contains (from N-terminus to C-terminus) structural led to a direct and rapid drop in deaths from (P1) and nonstructural (P2 and P3) proteins that are released from the polypeptide chain PV infections. The use of IPV in many other by proteolytic processing mediated by virally encoded proteinases. IRES: Internal ribosomal entry site; NCR: Noncoding region. countries contributed to a worldwide polio Reproduced with permission from [236] . decline. However, the incomplete inactivation of the Mahoney PV seed during vaccine production led to an incident during the first years of IPV use selection pressure leading to an attenuated phenotype [32] . The with almost 200 recipients and contacts being paralyzed [27]. This Sabin 2 strain (P2/P712, Ch, 2ab) originated from a naturally low is known as the Cutter incident and showed that new procedures neurovirulent PV2 isolate (P2/P712/56) from a healthy child [32] . need to be implemented during IPV production. Moreover, the The genetic basis of Sabin strains’ attenuated and thermosensitive incident led to a transient cessation of IPV use and reinforced claims phenotype relies on only a few substitutions in their genomes (Table in favor of a live-attenuated vaccine. Extensive clinical trials were 1) . In particular, the molecular determinants of the attenuated and performed to assess several live-attenuated strains [28,29]. In 1963, temperature-sensitive ­phenotype of Sabin 1 strain are situated in a trivalent oral PV vaccine (tOPV) created by Albert Sabin was the 5′-NCR and 3′-NCR of the genome, as well as in the regions first made available in the USA [30] . In all countries except three coding for VP1, VP3 and VP4 capsid proteins and the RNA poly(Finland, Sweden and The Netherlands), the OPV rapidly took the merase (3D) [33,34] The molecular determinants of the attenuated place of IPV for daily use due to several a­ dvantages [31]. and temperature-sensitive phenotype of Sabin 2 and 3 strains include Before introduction, the Sabin strains were carefully examined one substitution in the 5′-NCR of their genome and one or two for low neurovirulence, low rate of person-to-person transmission amino acid substitutions in capsid proteins (VP1 in both Sabin 2 and high safety in field trials [31] . In addition, the Sabin vaccine and Sabin 3, and VP3 in Sabin 3) [35,36] . strains show temperature sensitivity yielding lower viral titers at supraoptimal temperatures in comparison with wild-type PVs [31] . Safety, efficacy & immunogenicity of IPV & OPV The progenitors of attenuated Sabin 1 (P1/LSc, 2ab) and Sabin 3 Natural infection and vaccination with OPV or IPV protect (P3/Leon 12 a,b) strains were highly neurovirulent P1­/­Mahoney/41 and against poliomyelitis through the production of antibodies P3/Leon/37 strains, respectively. These viruses were passaged that inhibit viremia and infection of the CNS [37–39] . However, repeatedly in monkey cells in conditions that created extensive natural infection and vaccination with both vaccines do not

Table 1. Location of attenuating nucleotides and amino acid substitutions in the genome of Sabin vaccine strains. Sabin strain

5′-NCR†

VP4

VP3

VP1

3D

3′-NCR

1

A-480-G

G-935-T Ala-65-Ser

T-2438-A Leu-225-Met

G-2795-A C-2879-T Ala-106-Thr Leu-134-Phe

T-6203-C Tyr-73-His

A-7441-G

2

G-481-A

3

C-472-T

C-2909-T Thr-143-Ile C-2034-T Ser-91-Phe

T-2493-C Ile-6-Thr

The nucleotide mutation position refers to the complete genome whereas the corresponding amino acid change is numbered according to the primary amino acid sequence of a given protein after cleavage of the polyprotein precursor. † The attenuating nucleotides and amino acids are situated after the number position. NCR: Noncoding region.

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confer protection toward reinfection. Consequently, vaccinees (mainly adult and elderly) contribute to the asymptomatic PV transmission in populations with high immunization coverage. The restriction of PV transmission is as critical as the attempts to avoid paralysis of infected individuals [40] . PV transmission is restricted by high pre-existing natural or vaccine-acquired immunity [10] . The extent of viral shedding was decreased in children who were vaccinated with IPV, who possessed ­pre-existing antibodies to the infecting serotype or who had prior intestinal infection with homologous PV [6] . Moreover, as proven by Onorato et al., the extent of PV shedding by infants to whom OPV was recently administrated is less than that of infants to whom eIPV was administrated [7] . The administration of a vaccine containing live virus and the determination of PV shedding are needed in due course to assess mucosal immunity after vaccination [41] . Infection with live virus (WPV or OPV) stimulates the production of both circulating IgG and duodenal secretory IgA antibodies [42,43] , which could increase virus load needed for reinfection and decrease the quantity and duration of virus shedding in the feces after an infection [44,45] , both of these wouldrestrict virus transmission [39] . Immunization with IPV stimulates the production of circulating antibodies in the same or higher titer in comparison with OPV. Moreover, IPV induces mucosal intestinal and nasopharyngeal IgA antibodies [7,43,46–50] . The results of five studies that evaluated the ability of IPV and OPV to prevent PV shedding from feces after OPV vaccination revealed that OPV is superior to IPV in stimulating mucosal intestinal immunity [7,51–53] . However, PV excretion was decreased after IPV vaccination as opposed to no vaccination [52] . Consequently, IPV offers negligible protection against infection of the intestinal tract [54] and PV transmission through the fecal–oral route [40] . However, IPV is comparable with OPV in preventing shedding of PV from the pharynx [7,42,55] . IPV offers protection against PV transmission through upper respiratory tract secretions as like OPV. Both vaccines show a high herd effect in high-income/hygiene populations but the herd effect induced by IPV is superior to OPV in low-to-middle income/hygiene populations. Herd effect is a direct reflection of the mucosal immunity because it reduces or aborts the transmission of the virus from the vaccinated to the unvaccinated. Therefore, the herd effect (not to be confused with secondary infections from vaccine recipients to contact persons) is the reduction of incidence of infection or disease in the unvaccinated segment of a population as a result of immunizing a proportion of that population. Although, at the individual level, OPV induces a stronger mucosal immune response than IPV, the vaccine efficacy of OPV is inferior to IPV. The excellent herd effect of IPV has been documented extensively with the remarkable and obvious effect in the USA from 1955 to 1962 [56–58] . The efficacy of IPV has been demonstrated by a massive field trial led by Salk, which involved more than 1 million participants [59] . Immunization schedules based on the sole use of IPV have been tightly controlled and have led to the eradication of poliomyelitis from Scandinavian countries and The Netherlands [60–62] . Prior to the widespread introduction of OPV, many other countries adopted exclusive and successful use of IPV [63,64] . IPV 612

is available either as a standalone vaccine or in combination vaccines, which also show high immunogenicity. Since 1977, IPV either alone or in a combination vaccine that also contains diphtheria, pertussis, and tetanus vaccine (DPT), has been tested in more than 100 clinical trials carried out in 24 developing and 16 developed countries with the immune response observed in no less than 10,000 persons [65,66] . In the past decade, 40 countries have exclusively used IPV in routine national immunization programs [64,65] revealing the excellent safety and efficacy of the old IPV and ­particularly the new eIPV. Different vaccination programs, number of doses given, time periods between two following doses and different ages of the vaccinees have been evaluated in clinical trials of IPV. Generally, three doses induce a better immune response than two. Moreover, starting primary vaccination at an older age, for example, 2 months, is more immunogenic than starting at 6 weeks of age, when the existence of maternal antibodies restricts immune response. In addition, the use of a sequential IPV–OPV schedule protects against all three PV serotypes similar to a full IPV schedule [67] . Longer time periods between two following doses induce an enhanced immune response; for example, a 6-month interval is better than a 2-month interval. It has been stated that a primary vaccination schedule including three doses of IPV followed by a booster dose between 13 and 18 months of age leads to a good persistence of antibodies until the age of 7 years [63] . However, there are no studies to show that long-lasting immunity is feasible unless a booster dose of IPV is administered after the first 2 years of life [68] . In infants, 85% showed protective levels of immunity against all three PV serotypes after IPV vaccination in 18 studies carried out in various settings with a percentage of seroconversion close to 100% [63] . A study was carried out in Cuba by the WHO to examine the immunological properties of IPV in tropical developing countries. In particular, three doses of IPV (at 6, 10 and 14 weeks of age) or two doses of IPV (at 8 and 16 weeks of age) were administered to 178 children [52] . A month after the vaccination schedule, the immunity levels in serum and mucosa were evaluated. A percentage of seroconversion of no less than 90% was achieved after the administration of three doses of IPV against PV serotypes 1 and 3, but not against serotype 2. Although both IPV and OPV are effective in preventing infection from wild-type PVs, the GPEI selected OPV over the IPV for both routine and supplementary immunization activities [69] . The oral administration of OPV facilitated the employment of many volunteers to apply annual National Immunization Days, contrary to IPV, which would demand injection by skilled health staff. The significantly lower cost of OPV (12 months), following WPV importation before 2010 (Angola, Chad, and the Democratic Republic of the Congo), and 13 countries (Congo, Kazakhstan, Liberia, Mali, Nepal, Niger, Russian Federation, Senegal, Mauritania, Tajikistan, Turkmenistan, Uganda and Sierra Leone) in which WPV cases were confirmed because of new importations during 2010 (Figure 2) . The polio eradication partnership has planned 3 years of intensive activities during 2010–2012, targeting foci of persistent WPV transmission in each country [303] .

In addition, the amino acid substitutions in antigenic sites may be involved in optimal virus cell recognition because of the structural overlapping of antigenic sites and the capsid regions attach the PV receptor [98] . It is likely that the combination of a number of factors such as evasion of immune pressure, ability to bind the cell receptor and improvement of fitness to grow in the gut contribute to the selection of mutations during the evolution of Sabin vaccine strains in humans. These factors might be different in individuals with immunodeficiency, resulting in viruses with different mutation profiles [103–105] . The second regular event in the early stage of Sabin strains’ replication in the gut is intratypic or intertypic recombination. The detection of intratypic recombination in only slightly evolved genomes is uncommon, but instead, Risks associated with the use of live-attenuated intertypic recombinants have been shown to be excreted very soon vaccine PV strains after vaccination [35,106–110] . Thus, viral strains ­showing increased Genetic instability of Sabin vaccine strains is the major risk for growth ability and neurovirulence prevail and are shed in the safe use of OPV. Short excretion periods limit the ­person-to-person environment via vaccinees. The Sabin vaccine strains are differspread of OPV strains. The immunologically normal OPV recipi- ent from their parental strains in terms of biological properties in ents usually excrete viruses for 3–4 weeks in the feces and for addition to neurovirulence. These properties are useful as in vitro some days in the pharyngeal secretions [6] . Hence, the viral phenotypic markers to control the quality of new vaccines before strains excreted in the feces of vaccinated individuals after the neurovirulence assays performed in monkeys. Temperature sensireplication of OPV strains (Sabin 1, 2 and, 3) in their gut will tivity and plaque size have been used as in vitro phenotypic markbe referred to as OPV derivatives. During their replication, the ers for PV neurovirulence in previous studies [110–113] . The onestrains undergo rapid genetic changes. The first mutations are step growth curve experiment has also been used as a marker of adaptive and include reversions of neurovirulence-attenuating PV replication efficiency, which is correlated with neurovirulence sites. Sabin strains isolated from healthy vaccinees, their contacts [33,114–117] . In previous studies, the correlation of phenotypic traits or environmental samples have frequently incorporated these such as thermosensitivity and growth kinetics of OPV derivatives fitness-increasing reversions [98–100] . Moreover, OPV derivatives with genomic modifications such as reversion of the attenuatfrequently accumulate mutations in antigenic sites during the ing mutations and recombinations showed that both could affect early stages of their replication, which result in changes in their their phenotypic traits and give rise to ­neurovirulent viral strains antigenic structure and properties as judged by their reactivity [33–36,110,113,115–120] . The mutant analysis by PCR and restriction with monoclonal antibodies [101,102] . It has been assumed that it enzyme cleavage (MARPEC) test, which includes the identificarepresents a virus strategy to avoid the host’s immune response. tion of mutations at the main determinants of ­attenuation, has also been correlated with PV neurovirulence [121] . Consequently, genomic sequencing, mainly of the 5′-NCR and VP1 genomic regions, where the main determinants of attenuation are situated, constitutes an indirect in vitro phenotypic marker for the identification of possibly neurovirulent OPV derivatives [122] . The infrequent VAPP cases in OPV recipients or their close contacts have been associated with the accumulation of reverted attenuating determinants and/or recombination events during the Sabin vaccine strains’ Wild-type polio virus type 1 replication in the human gut [75] . WHO has Wild-type polio virus type 3 termed VAPP as poliomyelitis which appears in a vaccinee during the time period of 7–30 Endemic countries days after a dose or in a person who is in Importation countries c ­ ontact with a vaccinee during the time No data period of 7–60 days after the dose was given. Data as of 8 March 2011 VAPP appears infrequently, calculated as one Figure 2. Transmission of wild-type polioviruses worldwide in 2010. case per million births by WHO. Moreover, Reprinted with permission from [84] . it has also been referred as approximately one 614

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case per 750,000 vaccinees in the USA, one per 400,000 in Norway, England and Wales and one per 143,000 in India [123] . The risk of the first dose is significantly higher than that of the subsequent doses [124] , and children with B-cell i­ mmunodeficiency have a particularly elevated risk for VAPP (3000-fold higher risk [125]). VAPP can occur in the OPV recipient or it can be community-acquired due to the limited spread of Sabin strains to close contacts of vaccinees even in a well-vaccinated population [126] . In general, VAPP patients excrete viral strains showing only limited genetic variation from the original Sabin strains (99% VP1 sequence identity) to the original OPV strains [157,158] . Immunologically normal OPV recipients typically excrete viruses for approximately 3–4 weeks. Short excretion periods and high population immunity normally limit the person-to-person spread of these OPV-like viruses [158] . Infrequent isolates showing ≤99% VP1 sequence identity to the original OPV strains are described as ‘vaccine-derived PVs’, present phenotypic properties similar to those of WPV and can circulate, particularly in underimmunized communities, causing outbreaks or sporadic cases of paralytic poliomyelitis [75,159] . Although this standard appears arbitrary, as isolates with