Cold chain and virus-free oral polio booster vaccine made in lettuce chloroplasts confers protection against all three poliovirus serotypes.

To prevent vaccine-associated paralytic poliomyelitis, WHO recommended withdrawal of Oral Polio Vaccine (Serotype-2) and a single dose of Inactivated Poliovirus Vaccine (IPV). IPV however is expensive, requires cold chain, injections and offers limited intestinal mucosal immunity, essential to prevent polio reinfection in countries with open sewer system. To date, there is no virus-free and cold chain-free polio vaccine capable of inducing robust mucosal immunity. We report here a novel low-cost, cold chain/poliovirus-free, booster vaccine using poliovirus capsid protein (VP1, conserved in all serotypes) fused with cholera non-toxic B subunit (CTB) expressed in lettuce chloroplasts. PCR using unique primer sets confirmed site-specific integration of CTB-VP1 transgene cassettes. Absence of the native chloroplast genome in Southern blots confirmed homoplasmy. Codon optimization of the VP1 coding sequence enhanced its expression 9-15-fold in chloroplasts. GM1-ganglioside receptor-binding ELISA confirmed pentamer assembly of CTB-VP1 fusion protein, fulfilling a key requirement for oral antigen delivery through gut epithelium. Transmission Electron Microscope images and hydrodynamic radius analysis confirmed VP1-VLPs of 22.3 nm size. Mice primed with IPV and boosted three times with lyophilized plant cells expressing CTB-VP1co, formulated with plant-derived oral adjuvants, enhanced VP1-specific IgG1, VP1-IgA titres and neutralization (80%-100% seropositivity of Sabin-1, 2, 3). In contrast, IPV single dose resulted in <50% VP1-IgG1 and negligible VP1-IgA titres, poor neutralization and seropositivity (<20%, <40% Sabin 1,2). Mice orally boosted with CTB-VP1co, without IPV priming, failed to produce any protective neutralizing antibody. Because global population is receiving IPV single dose, booster vaccine free of poliovirus or cold chain offers a timely low-cost solution to eradicate polio.

Molecular mechanism of poliovirus Sabin vaccine strain attenuation.

Recruitment of poliovirus (PV) RNA to the human ribosome requires the coordinated interaction of the viral internal ribosome entry site (IRES) and several host cellular initiation factors and IRES trans-acting factors (ITAFs). Attenuated PV Sabin strains contain point mutations in the PV IRES domain V (dV) that inhibit viral translation. Remarkably, attenuation is most apparent in cells of the central nervous system, but the molecular basis to explain this is poorly understood. The dV contains binding sites for eukaryotic initiation factor 4G (eIF4G) and polypyrimidine tract-binding protein (PTB). Impaired binding of these proteins to the mutant IRESs has been observed, but these effects have not been quantitated. We used a fluorescence anisotropy assay to reveal that the Sabin mutants reduce the equilibrium dissociation constants of eIF4G and PTB to the PV IRES by up to 6-fold. Using the most inhibitory Sabin 3 mutant, we used a real-time fluorescence helicase assay to show that the apparent affinity of an active eIF4G/4A/4B helicase complex for the IRES is reduced by 2.5-fold. The Sabin 3 mutant did not alter the maximum rate of eIF4A-dependent helicase activity, suggesting that this mutant primarily reduces the affinity, rather than activity, of the unwinding complex. To confirm this affinity model of attenuation, we show that eIF4G overexpression in HeLa cells overcomes the attenuation of a Sabin 3 mutant PV-luciferase replicon. Our study provides a quantitative framework for understanding the mechanism of PV Sabin attenuation and provides an explanation for the previously observed cell type-specific translational attenuation.

Manufacturing and supply of monovalent oral polio vaccines.

A new polio vaccine was developed, produced and licensed by sanofi pasteur at the request of the World Health Organization (WHO) for mass immunization campaigns in endemic countries such as Egypt. The new vaccine, monovalent oral polio vaccine 1 or mOPV1, is currently used in Egypt as a critical part of a new WHO strategy to end polio type 1 transmission by the end of the year 2005 (types 2 and 3 polioviruses have already been eliminated from Egypt). To answer this specific need, an urgent program was mounted by Sanofi pasteur to manufacture 50million doses for Egypt, in close collaboration with WHO and National Regulatory Agencies (France and Egypt). The joint efforts between manufacturer, regulators and the WHO resulted in the quickest ever vaccine development and licensure and WHO pre-qualification. The production of mOPV was based on existing tOPV but with appropriate "change control" procedures to assure the quality of the product, and to distinguish mOPV from tOPV. Key success factors included clear and careful definition of the project; close collaboration between manufacturer, regulators and WHO; and commitment and motivation of staff. As a result, development and production of mOPV1 vaccine were carried out in a drastically reduced time period, leading to the release and delivery of the first 15 million doses of mOPV1 in April 2005.

Comparative pre-clinical and clinical experience with oral polio vaccine produced on MRC-5 cells or on primary monkey kidney cells.

The need to avoid using primates has prompted the replacement of primary monkey kidney cells (PMKC) as a substrate for oral polio vaccine (OPV) production. Here, we report on OPV produced on MRC-5 cells using an industrial process capable of producing over 1 billion doses. All serotypes produced on MRC-5 cells proved satisfactory in the monkey and transgenic mice neurovirulence tests. All the type 3 MRC-5 lots tested by Mutant Analysis by PCR and Restriction Enzyme Cleavage (MAPREC) had a 472-C content below the acceptable limit and similar to that of PMKC derived lots. The safety/reactogenicity and immunogenicity profiles following vaccination in infants and children were similar for OPV MRC-5 and OPV PMKC vaccine lots. Excretion rates and prevalence of revertants for the three serotypes following vaccination were also similar for both vaccines. These data support the use of MRC-5 cells as an alternative to PMKC for OPV production.

Comparative study of poliovirus excretion after vaccination of infants with two oral polio vaccines prepared on Vero cells or on primary monkey kidney cells.

A comparative study was designed to assess the bioequivalence of 2 oral poliovaccines (OPV) produced on 2 different cell systems: primary monkey kidney (PMK) cells and the Vero cell line. The Vero cell line has been used to overcome the problem of obtaining a regular supply of high quality monkeys that are devoid of latent viruses. For this study, 9 children were vaccinated with PMK-OPV and 12 children with Vero-OPV. The comparison covered poliovirus excretion, reversion of polioviruses in the 5'-noncoding region, and immunogenicity. Major molecular markers in the 5'-noncoding region related to neurovirulence already had been identified at position 480 for type 1, position 481 for type 2, and position 472 for type 3 poliovirus. Two nucleic-acid based methods were designed for studying these positions: a RT-PCR followed by sequencing, which required preliminary culture and cloning; and a type-specific nested PCR followed by sequencing, which enabled direct detection and genotyping of polioviruses. Twenty-eight stool specimens were analyzed by this second method with no PCR inhibition problem. The use of Vero cell line did not modify the global pattern of poliovirus excretion, reversion frequency, or seroconversion. These results provide additional support for the use of the well-characterized Vero cell line in OPV manufacturing.

Problems encountered in the delivery and storage of OPV in an African country.

Southern Africa may be developing into another poliomyelitis-free zone. Apparent control has been achieved in spite of suboptimal coverage, absence of supplementary strategies, vulnerability to importation and major outbreaks of other waterborne pathogens such as typhoid. A number of serological studies have demonstrated significant susceptibility, especially in rural populations, and studies of three southern African epidemics have shown, in two of them, that epidemics can occur in spite of relatively high levels of serological immunity and vaccine coverage if the burden of virus is sufficiently great. Two studies comparing vaccine coverage to serological immunity have shown that only 78% of fully immunized children in one study and 81% in the other study developed antibodies to all three types of poliomyelitis. In two investigations where vaccine samples were recalled for potency testing from peripheral clinics, almost a half and almost a third, respectively, of vaccine vials had titres below WHO recommended cut-off limits. Major logistical and organizational difficulties in storage and transport of vaccine under cold chain conditions were present in South Africa. These are being rectified and energetic attempts are being made, aided by a strong governmental commitment to improve vaccine coverage and to institute mass immunization campaigns. Nevertheless the need for the development of a more stable OPV for developing and tropical countries still remains a major objective in the global eradication of poliomyelitis.

Thermal inactivation of oral polio vaccine: contribution of RNA and protein inactivation.

Heating the Sabin strains of poliovirus at 42 to 45 degrees C caused inactivation, loss of native antigen, and release of the viral RNA (vRNA). The loss of virion infectivity exceeded the loss of vRNA infectivity (as measured by transfection) by roughly 2 log10. Pirodavir inhibited the loss of native antigen and RNA release and reduced the loss of virion infectivity to the same level as the loss of vRNA infectivity. Thermoinactivation thus involves an RNA and a protein component, and pirodavir protected only against the latter.