The Use of Next-Generation Sequencing for the Quality Control of Live-Attenuated Polio Vaccines.

BACKGROUND: Next-generation sequencing (NGS) analysis was compared to the current MAPREC (mutational analysis by polymerase chain reaction and restriction enzyme cleavage) assay for quality control of live-attenuated oral polio vaccine (OPV). METHODS: MAPREC measures reversion of the main OPV attenuating mutations such as uracil (U) to cytosine (C) at nucleotide 472 in the 5' noncoding region of type 3 OPV. Eleven type 3 OPV samples were analyzed by 8 laboratories using their in-house NGS method. RESULTS: Intraassay, intralaboratory, and interlaboratory variability of NGS 472-C estimates across samples and laboratories were very low, leading to excellent agreement between laboratories. A high degree of correlation between %472-C results by MAPREC and NGS was observed in all laboratories (Pearson correlation coefficient r = 0.996). NGS estimates of sequences at nucleotide 2493 with known polymorphism among type 3 OPV lots also produced low assay variability and excellent between-laboratory agreement. CONCLUSIONS: The high consistency of NGS data demonstrates that NGS analysis can be used as high-resolution test alternative to MAPREC, producing whole-genome profiles to evaluate OPV production consistency, possibly eliminating the need for tests in animals. This would be very beneficial for the quality assessment of next-generation polio vaccines and, eventually, for other live-attenuated viral vaccines.

Safety, immunogenicity and lot-to-lot consistency of a new Bivalent Oral Polio Vaccine (bOPV) in healthy Infants: Results of a Phase III, observer blind, randomized, controlled clinical study.

BACKGROUND: Poliomyelitis infection continues to be endemic in few countries despite rigorous efforts for eradication. A new Bivalent Oral Polio Vaccine (BBio bOPV) was tested in a Phase III Clinical study. METHODS: An observer blind, randomized, controlled clinical study was conducted comparing BBio bOPV with a licensed bOPV (SII bOPV). Initially in Part 1, 40 children 5-6 years of age were given a single dose of either vaccine in 1:1 ratio. In Part 2, 1080 infants of 6-8 weeks of age were received in 1:1:1:1 ratio one of the 3 lots of BBio bOPV or SII bOPV at 6, 10 and 14 weeks of age. Blood samples were collected to assess neutralizing antibody responses against Polio Type 1 and 3 viruses. Safety of the vaccines were recorded. RESULTS: All children were seroprotected against both Type 1 and Type 3 polioviruses post-vaccination. More than 96% of the infants demonstrated seroconversion as well as seroprotection against both types of polioviruses. The geometric mean titres (GMT) for Type 1 and Type 3 antibodies were comparable between the groups. The 3 lots of BBio bOPV generated similar GMTs of Type 1 and Type 3 antibodies. In total 387 participants reported at least one adverse event and 18 serious adverse events. None of these events were vaccine related. CONCLUSIONS: The new bOPV vaccine demonstrated immunogenicity that was non-inferior to a licensed bOPV vaccine. Consistency in immune response by 3 consecutively manufactured lots was also demonstrated. The vaccine did not cause any adverse event. Clinicaltrials.gov.identifier: NCT02766816.

Monitoring and Validation of the Global Replacement of tOPV with bOPV, April-May 2016.

The phased withdrawal of oral polio vaccine (OPV) associated with the Polio Eradication and Endgame Strategic Plan 2013-2018 began with the synchronized global replacement of trivalent OPV (tOPV) with bivalent OPV (bOPV) during April - May 2016, a transition referred to as the "switch." The World Health Organization's (WHO) Strategic Advisory Group of Experts (SAGE) on Immunization recommended conducting this synchronized switch in all 155 OPV-using countries and territories (which collectively administered several hundred million doses of tOPV each year via several hundred thousand facilities) to reduce risks of re-emergence of vaccine-derived polioviruses. Safe execution of this switch required implementation of an associated independent monitoring strategy, the primary objective of which was verification that tOPV was no longer available for administration post-switch. This strategy had to be both practical and rigorous such that tOPV withdrawal could be reasonably employed and confirmed in all countries and territories within a discreet timeframe. Following these principles, WHO recommended that designated monitors in each of the 155 countries and territories visit all vaccine stores as well as a 10% sample of highest-risk health facilities within two weeks of the national switch date, removing any tOPV vials found. National governments were required to provide the WHO with formal validation of execution and monitoring of the switch. In practice, all countries reported cessation of tOPV by 12 May 2016 and 95% of countries and territories submitted detailed monitoring data to WHO. According to these data, 272 out of 276 (99%) national stores, 3,741 out of 3.968 (94%) regional stores, 16,144 out of 22,372 (72%) district level stores, and 143,050 out of 595,401 (24%) of health facilities were monitored. These data, along with field reports suggest that monitoring and validation of the switch was efficient and effective, and that the strategies used during the process could be adapted to future stages of OPV withdrawal.

[Study on the failure time of the vaccine vial monitor used for oral poliovirus vaccine at 25 ℃ and 37 ℃].

Objective: To evaluate the failure time of vaccine vial monitor (VVM) used for oral poliovirus vaccine (OPV) at 25 ℃ and 37 ℃. Methods: 160 copies of VVM were produced by a company, the model was QM5D37A, samples were taken from different batches by using the method of random number table . 100 bottles of vaccine were produced by a domestic company, and samples were taken from different batches by using the method of random number table. 160 copies of labels were placed in the incubator at 25 ℃ and 37 ℃, which were used to measure the mutative color of the active region. When the values of color were equal to 40, the color of active region was the same with the reference color, and the VVM was failed. 100 bottles of vaccine were placed in the incubator at 25 ℃ and 37 ℃, which were used to measure the vaccine titer. When total vaccine titer was less than 6.12 CCID50 or vaccine titer of typeⅠ was less than 6.0 CCID50 or vaccine titer of type Ⅲ was less than 5.5 CCID50, the vaccine was failed. We drew the graph of mutative color to calculate the failure time range of VVM According to the graph , we can determine that whether the failure time of VVM was later than the time of vaccines by the data of OPV . Results: The earliest failure time of OPV was 21 days at 25 ℃, and the number of samples was one; The earliest Failure time of VVM was 12.5 days at 25 ℃, and it was less than the earliest failure time of OPV. The earliest failure time of OPV was 4.0 days at 37 ℃, and the number of samples was one; The earliest Failure time of VVM was 3.1 days at 37 ℃, and it was equal to the earliest failure time of OPV. Conclusion: We could know that the failure time of VVM was always earlier than the failure time of vaccines at the same temperatures . The latest failure time of VVM was equal to the earliest failure time of vaccines at 37 ℃. All of the failure times of samples were earlier than that of vaccines at 25 ℃.

Allele-specific PCR for quantitative analysis of mutants in live viral vaccines.

Monitoring consistency of genetic composition of oral polio vaccine (OPV) is a part of its quality control. It is performed by mutant analysis by PCR and restriction enzyme cleavage (MAPREC) used to quantify neurovirulent revertants in the viral genome. Here an alternative method based on quantitative PCR is proposed. Allele-specific quantitative polymerase chain reaction (asqPCR) uses a "tethered" oligonucleotide primer consisting of two specific parts connected by a polyinosine stretch. Homogeneous DNA from plasmids containing wild Leon/37 and attenuated Sabin 3 sequences with 100% 472(C) and 100% 472(T) could only be amplified using homologous primers. Real-time implementation of the allele-specific PCR resulted in sensitive detection of 472(C) revertants with the limit of quantitation of less than 0.05%. Monovalent vaccine batches and international viral references for MAPREC test were used to validate the method. asqPCR performed with the WHO references and monovalent batches of vaccine showed that the new method could measure accurately and reproducibly the content of revertants producing values comparable to MAPREC results. This suggests that asqPCR could be used as an alternative to MAPREC for lot release of OPV. The method could also be used for the quantitation of other mutants in populations of microorganisms.

Calibration of Ph. Eur. BRP batch 4 for oral poliomyelitis vaccine (OPV).

Poliomyelitis (polio) is a highly infectious disease that affects mostly young children and which may lead to paralysis and death. Prevalence of polio has considerably decreased. However, the effort of global eradication through immunisation needs to be continued to prevent infection risks in non-vaccinated populations by wild, as well as vaccine-derived, polioviruses. In addition, the stockpile of oral poliomyelitis vaccine (OPV) must be maintaine for emergency cases even after eradication. To this end relevant reference standards must be available for the quality control (QC) of polio vaccines. Stocks of the European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) batch 3 for the assay of OPV are dwindling and therefore a collaborative study was initiated by the European Directorate for the Quality of Medicines and HealthCare (EDQM) with the goal to establish a replacement batch as a working standard. Ten laboratories took part in the collaborative study to calibrate the candidate BRP (cBRP), a commercial trivalent OPV containing live attenuated poliovirus types 1, 2 and 3 (Sabin strain), against the WHO 2(nd) International Reference Reagent (IRR) for OPV and, for the sake of continuity, to compare it to the BRP batch 3. Based on the results of the collaborative study, the cBRP appears suitable as a reference standard and the potencies assigned are 7.28, 6.34, 7.01, and 7.52 log10CCID50/mL (CCID50: 50 % Cell Culture Infective Dose) for poliovirus type 1, 2 and 3 and for the total virus content respectively. The cBRP was adopted by the Ph. Eur. Commission at its 146(th) session in June 2013 as Ph. Eur. BRP batch 4 for OPV.

Gestación y realización de la primera campaña nacional de vacunación antipoliomielítica oral en España / Gestation and conduct of the first national campaign of oral polio vaccination in Spain

El presente trabajo recoge la intervención del Dr Luis Valenciano Clavel en la jornada que se celebró el pasado 2 de julio de 2013 bajo el título Celebración del 50 aniversario de la instauración de las campañas de vacunación antipoliomielitis en España. (Homenaje al Dr D Florencio Pérez Gallardo), en el Salón de Actos Ernest Lluch del Ministerio de Sanidad, Servicios Sociales e Igualdad. El Dr Luis Valenciano Clavel narra su experiencia y participación directa, junto a Florencio Pérez Gallardo, en las campañas de vacunación contra el virus de la poliomielitis, tras retornar de su estancia en centros sanitarios de Alemania y asumir la dirección del Laboratorio de Diagnóstico de Poliomielitis de la Escuela Nacional de Sanidad, que tras el éxito de la campaña de vacunación antipoliomielítica, dio origen al actual Centro Nacional de Virología, pivote del actual Instituto de Salud Carlos III (AU)

This paper presents the intervention of Dr Luis Valenciano Clavel in the act that was held on July 2, 2013 under the title Celebrating the 50th anniversary of the establishment of poliovirus vaccination campaigns in Spain. (Tribute to Dr D Florencio Perez Gallardo), in Ernest Lluch Hall of the Ministry of Health, Social Services and Equality. Dr Luis Valenciano Clavel describes his experience and direct participation, along with Florencio Pérez Gallardo, during the first oral polio vaccination campaign in Spain, after returning from his stay in health centers of Germany and assuming the leadership of the Polio Diagnostic Laboratory of theNational School of Public Health. The success of the polio vaccination campaign, it gave rise to the current National Center of Virology, pivot of the current Institute of Health Carlos III (AU)

Caution needed in using oral polio vaccine beyond the cold chain: vaccine vial monitors may be unreliable at high temperatures.

BACKGROUND & OBJECTIVES: Stabilized live attenuated oral polio vaccine (OPV) is used to immunize children up to the age of five years to prevent poliomyelitis. It is strongly advised that the cold-chain should be maintained until the vaccine is administered. It is assumed, that vaccine vial monitors (VVMs) are reliable at all temperatures. VVMs are tested at 37°C and it is assumed that the labels reach discard point before vaccine potency drops to >0.6 log10. This study was undertaken to see if VVMs were reliable when exposed to high temperatures as can occur in field conditions in India. METHODS: Vaccine vials with VVMs were incubated (10 vials for each temperature) in an incubator at different temperatures at 37, 41, 45 and 49.5°C. Time-lapse photographs of the VVMs on vials were taken hourly to look for their discard-point. RESULTS: At 37 and 41°C the VVMs worked well. At 45°C, vaccine potency is known to drop to the discard level within 14 h whereas the VVM discard point was reached at 16 h. At 49.5°C the VVMs reached discard point at 9 h when these should have reached it at 3 h. CONCLUSION: Absolute reliance cannot be placed on VVM in situation where environmental temperatures are high. Caution is needed when using 'outside the cold chain' (OCC) protocols.

Clustered lot quality assurance sampling: a tool to monitor immunization coverage rapidly during a national yellow fever and polio vaccination campaign in Cameroon, May 2009.

We used the clustered lot quality assurance sampling (clustered-LQAS) technique to identify districts with low immunization coverage and guide mop-up actions during the last 4 days of a combined oral polio vaccine (OPV) and yellow fever (YF) vaccination campaign conducted in Cameroon in May 2009. We monitored 17 pre-selected districts at risk for low coverage. We designed LQAS plans to reject districts with YF vaccination coverage <90% and with OPV coverage <95%. In each lot the sample size was 50 (five clusters of 10) with decision values of 3 for assessing OPV and 7 for YF coverage. We 'rejected' 10 districts for low YF coverage and 14 for low OPV coverage. Hence we recommended a 2-day extension of the campaign. Clustered-LQAS proved to be useful in guiding the campaign vaccination strategy before the completion of the operations.

WHO Working Group discussion on revision of the WHO recommendations for the production and control of poliomyelitis vaccines (oral): TRS Nos. 904 and 910. Report of Meeting held on 20-22 July 2010, Geneva, Switzerland.

Oral poliomyelitis vaccine (OPV) is a critical part of the polio eradication programme. A high number of doses are administered each year with an impact on billions of citizens worldwide. It is therefore essential that written standards concerning OPV are up to date and widely available. The World Health Organization (WHO) publishes technical guidance on the quality, safety and efficacy of vaccines intended to assist national regulatory authorities (NRAs), national control laboratories (NCLs) and manufacturers. As part of its programme, on 20-22 July 2010 WHO convened a working group meeting to initiate the revision of the WHO recommendations on the production and control of OPV as presently outlined in the Technical Reports Series (TRS) issues Nos. 904 and 910 [1,2]. The attendees included experts from academia, NRAs/NCLs and industry involved in the study, manufacture, and authorization and testing/release of OPV from countries around the world including representatives from China, the European Union, Indonesia, Japan, Mexico, and the USA. The objective was to review the state of knowledge concerning production and control of OPV, with a focus on neurovirulence testing, to determine how the existing guidelines should be updated and what recommendations should be made for the future. The outcomes of this meeting will be taken into consideration in future revision of the WHO TRS.

Massively parallel sequencing for monitoring genetic consistency and quality control of live viral vaccines.

Intrinsic genetic instability of RNA viruses may lead to the accumulation of revertants during manufacture of live viral vaccines, requiring rigorous quality control to ensure vaccine safety. Each lot of oral poliovirus vaccine (OPV) is tested for neurovirulence in animals and also for the presence of neurovirulent revertants. Mutant analysis by PCR and restriction enzyme cleavage (MAPREC) is used to measure the frequency of neurovirulent mutations at the 5' untranslated region (UTR) of the viral genome that correlate with the level of neurovirulence determined by the monkey neurovirulence test. However, MAPREC can only monitor mutations at a few genomic loci and miss mutations at other sites that could adversely affect vaccine quality. Here we propose to use massively parallel sequencing (MPS) for sensitive detection and quantification of all mutations in the entire genome of attenuated viruses. Analysis of vaccine samples and reference preparations demonstrated a perfect agreement with MAPREC results. Quantitative MPS analysis of validated reference preparations tested by MAPREC produced identical results, suggesting that the method could take advantage of the existing reference materials and be used as a replacement for the MAPREC procedure in lot release of OPV. Patterns of mutations present at a low level in vaccine preparations were characteristic of seed viruses used for their manufacture and could be used for identification of individual batches. This approach may represent the ultimate tool for monitoring genetic consistency of live viral vaccines.

Advances in vaccine stability monitoring technology.

Electronic time-temperature indicator (eTTI) monitors can be programmed to exactly follow the stability characteristics of vaccines with a high degree of realism. The monitors have a visual output, enabling vaccine status to be assessed at a glance, and can also output more detailed statistical data. When packaged with vaccine vials in groups of about 10 vials per box, the eTTI can remain with a vaccine throughout most of the vaccine's lifetime. The monitors can detect essentially all cold-chain breaks, and can detect issues, such as inadvertent freezing, that are presently not detected by other vaccine stability monitors such as Vaccine Vial Monitors (VVM).

The key role of vaccine vial monitors in routine and mass immunisation.

The vaccine vial monitor is an excellent monitoring device to assure that only non-heat damaged vaccine is administered to women and children. They help to extend access to immunisation, they increase the efficiency and reduce wastage in the cold-chain and, when visible on all vaccine vials of all types, they will help to streamline distribution systems for immunisation services of the 21st century in all developing countries.

Assessment of vaccine wastage during a pulse polio immunization programme in India.

A study to assess the wastage factor of oral polio vaccine (OPV) in the Pulse Polio Immunization (PPI) programme of the Government of India was undertaken by the Indian Council of Medical Research (ICMR) at approximately 31,000 immunization booths all over the country. The study was conducted through the network of 31 Human Reproduction Research Centres (HRRCs) and other ICMR institutes. Wastage at the point of administration of OPV was estimated to be 14.5% with a wastage factor of 1.17 which is well below the assumed wastage of 33% and the corresponding wastage factor of 1.5 in the PPI programme. The wastage and wastage factor as estimated in the present study were also less than the wastage of 25% and the wastage factor of 1.33 recommended by the World Health Organization. Minimum wastage (6.3%) at Kanchipuram and maximum wastage (22.1%) at Kanpur were observed. Further, the wastage of unopened vials and vials during use was also observed following colour changes on the vaccine vial monitor (VVM), indicating poor cold-chain maintenance at the immunization site. In total, 13 booths reported wastage of nine or more unopened vials, whereas 19 booths reported wastage of nine or more vials during use because of colour changes on VVM. Other reasons for wastage of vaccine were also observed from a sample of booths. The technology of introducing VVM on OPV vials for monitoring the cold-chain proved useful in situations in which mass vaccination programmes such as PPI are carried out.