Continued use of poliovirus after eradication: hyper-attenuated strains as a safe alternative for release testing of human immunoglobulins.

BACKGROUND: Wild-type poliovirus may be eradicated soon and under WHO GAPIII guidance, laboratory use will be discontinued or subject to strict containment. Per US Code of Federal Regulations, however, immunoglobulin lot release testing will still require use of replicating poliovirus. The suitability of S19 hyper-attenuated and apathogenic poliovirus strains as alternatives to the currently used wild-type virus in such a release assay was investigated. STUDY DESIGN AND METHODS: S19 poliovirus strains were propagated in a commercial setting using good virological practices and maintenance of the S19 hyper-attenuated genotype was confirmed by massively parallel sequencing. RESULTS: The attenuated phenotype of the produced S19 stocks was confirmed in a highly sensitive mouse-model. Equivalency in performance was seen in the lot release assay for the S19 and wild-type polioviruses. CONCLUSION: The deployment of such hyper-attenuated and thoroughly characterized S19 stocks in these and other essential activities might reconcile final containment measures with continued safe use of poliovirus.

Environmental Surveillance Reveals Complex Enterovirus Circulation Patterns in Human Populations.

BACKGROUND: Enteroviruses are common human pathogens occasionally associated with severe disease, notoriously paralytic poliomyelitis caused by poliovirus. Other enterovirus serotypes such as enterovirus A71 and D68 have been linked to severe neurological syndromes. New enterovirus serotypes continue to emerge, some believed to be derived from nonhuman primates. However, little is known about the circulation patterns of many enterovirus serotypes and, in particular, the detailed enterovirus composition of sewage samples. METHODS: We used a next-generation sequencing approach analyzing reverse transcriptase polymerase chain reaction products synthesized directly from sewage concentrates. RESULTS: We determined whole-capsid genome sequences of multiple enterovirus strains from all 4 A to D species present in environmental samples from the United Kingdom, Senegal, and Pakistan. CONCLUSIONS: Our results indicate complex enterovirus circulation patterns in human populations with differences in serotype composition between samples and evidence of sustained and widespread circulation of many enterovirus serotypes. Our analyses revealed known and divergent enterovirus strains, some of public health relevance and genetically linked to clinical isolates. Enteroviruses identified in sewage included vaccine-derived poliovirus and enterovirus D-68 stains, new enterovirus A71 and coxsackievirus A16 genogroups indigenous to Pakistan, and many strains from rarely reported serotypes. We show how this approach can be used for the early detection of emerging pathogens and to improve our understanding of enterovirus circulation in humans.

Report of the international conference on next generation sequencing for adventitious virus detection in biologicals.

A fundamental aspect of biological product safety is to assure absence of adventitious agents in the final product. Next-generation or high-throughput sequencing (NGS/HTS) has recently demonstrated detection of viruses that were previously missed using the recommended routine assays for adventitious agent testing of biological products. This meeting was co-organized by the International Alliance for Biological Standardization (IABS) and the U.S. Food and Drug Administration (FDA) to assess the current status and discuss the readiness of NGS for adventitious virus detection in biologics. The presentations included efforts for standardization, case studies on applications in biologics, comparison with routine virus detection assays, and current regulatory thinking. Participants identified the need for standard reference reagents, well-annotated databases, large data storage and transfer capacity, personnel with relevant expertise, particularly in bioinformatics; and harmonization of international regulations for testing biologic products and reagents used for their manufacturing. We hope this meeting summary will be of value to regulators and industry for considerations of NGS applications for adventitious virus detection in biologics.

Detection of bovine viral diarrhoea virus nucleic acid, but not infectious virus, in bovine serum used for human vaccine manufacture.

Bovine viral diarrhoea virus (BVDV) is a cattle pathogen that has previously been reported to be present in bovine raw materials used in the manufacture of biological products for human use. Seven lots of trivalent measles, mumps and rubella (MMR) vaccine and 1 lot of measles vaccine from the same manufacturer, together with 17 lots of foetal bovine serum (FBS) from different vendors, 4 lots of horse serum, 2 lots of bovine trypsin and 5 lots of porcine trypsin were analysed for BVDV using recently developed techniques, including PCR assays for BVDV detection, a qRT-PCR and immunofluorescence-based virus replication assays, and deep sequencing to identify and genotype BVDV genomes. All FBS lots and one lot of bovine-derived trypsin were PCR-positive for the presence of BVDV genome; in contrast all vaccine lots and the other samples were negative. qRT-PCR based virus replication assay and immunofluorescence-based infection assay detected no infectious BVDV in the PCR-positive samples. Complete BVDV genomes were generated from FBS samples by deep sequencing, and all were BVDV type 1. These data confirmed that BVDV nucleic acid may be present in bovine-derived raw materials, but no infectious virus or genomic RNA was detected in the final vaccine products.

Isolation of Vaccine-Like Poliovirus Strains in Sewage Samples From the United Kingdom.

Background: Environmental surveillance (ES) is a sensitive method for detecting human enterovirus (HEV) circulation, and it is used worldwide to support global polio eradication. We describe a novel ES approach using next-generation sequencing (NGS) to identify HEVs in sewage samples collected in London, United Kingdom, from June 2016 to May 2017. Methods: Two different methods were used to process raw sewage specimens: a 2-phase aqueous separation system and size exclusion by filtration and centrifugation. HEVs were isolated using cell cultures and analyzed using NGS. Results: Type 1 and 3 vaccine-like poliovirus (PV) strains were detected in samples collected from September 2016 through January 2017. NGS analysis allowed us to rapidly obtain whole-genome sequences of PV and non-PV HEV strains. As many as 6 virus strains from different HEV serotypes were identified in a single cell culture flask. PV isolates contained only a small number of mutations from vaccine strains commonly seen in early isolates from vaccinees. Conclusions: Our ES setup has high sensitivity for polio and non-PV HEV detection, generating nearly whole-genome sequence information. Such ES systems provide critical information to assist the polio eradication endgame and contribute to the improvement of our understanding of HEV circulation patterns in humans.

Scientific consultation on the safety and containment of new poliovirus strains for vaccine production, clinical/regulatory testing and research. Report of a meeting held at NIBSC, Potters Bar, Hertfordshire, UK, 6/7th July 2016.

When poliomyelitis is totally eradicated from the natural world containment will be vital to prevent its re-emergence. The matter has become pressing as type 2 component of oral polio vaccine was completely withdrawn by May 2016 as wild ty[e 2 was declared eradicated. Work on polioviruses must be contained in accordance with GAPIII (the third version of the Global Action Plan of WHO). Some activities will be essential for years after eradication. Vaccine production and control, surveillance and supportive applied and academic research must all continue. Most laboratories do not currently comply with GAPIII and could not do so in the short term without disruption of essential activities including vaccine supply. The development and use of safer strains is raised in GAPIII and the meeting considered the strains available and the uses to which they could be put to facilitate compliance with the aims of GAPIII.

Genetically Thermo-Stabilised, Immunogenic Poliovirus Empty Capsids; a Strategy for Non-replicating Vaccines.

While wild type polio has been nearly eradicated there will be a need to continue immunisation programmes for some time because of the possibility of re-emergence and the existence of long term excreters of poliovirus. All vaccines in current use depend on growth of virus and most of the non-replicating (inactivated) vaccines involve wild type viruses known to cause poliomyelitis. The attenuated vaccine strains involved in the eradication programme have been used to develop new inactivated vaccines as production is thought safer. However it is known that the Sabin vaccine strains are genetically unstable and can revert to a virulent transmissible form. A possible solution to the need for virus growth would be to generate empty viral capsids by recombinant technology, but hitherto such particles are so unstable as to be unusable. We report here the genetic manipulation of the virus to generate stable empty capsids for all three serotypes. The particles are shown to be extremely stable and to generate high levels of protective antibodies in animal models.

A collaborative study to establish the 3rd WHO International Standard for hepatitis B virus for nucleic acid amplification techniques.

Nucleic acid amplification techniques (NAT) are routinely used for clinical diagnostics and monitoring hepatitis B virus (HBV) infections, and are implemented on a voluntary basis for blood screening. A collaborative study was performed to evaluate a replacement WHO International Standard for HBV for the standardization of NAT. Two lyophilised HBV candidates were evaluated by 16 laboratories worldwide, alongside the existing HBV International Standard. The overall mean potency estimates for the candidate samples 1 and 2, relative to sample 3 (2nd HBV International Standard), from quantitative assays, were 5.93 and 5.98 log10 International Units (IU)/mL respectively. The variability in individual laboratory mean estimates for samples 1-3 for quantitative assays was ∼0.3 log10 IU/mL. The inter-laboratory variability for qualitative assays was higher. Accelerated thermal degradation studies indicate that both lyophilised candidates are stable and suitable for long-term use. Overall, the results suggested that both candidates were suitable as replacement International Standards. Sample 1 (NIBSC code 10/264) was established as the 3rd WHO International Standard for HBV for NAT with an assigned potency of 850,000 IU/mL (∼5.93 log10 IU/mL), when reconstituted in 0.5 mL of nuclease-free water. It is intended for the calibration (in IU) of secondary reference materials used in HBV NAT.

A collaborative study to establish the 1st WHO International Standard for Epstein-Barr virus for nucleic acid amplification techniques.

Variability in viral load measurements using nucleic acid amplification techniques (NAT) has a significant impact on the management of Epstein-Barr virus (EBV)-associated diseases, and has highlighted a need for standardisation of these measurements. The aim of this collaborative study was to evaluate the suitability of a range of candidate reference materials to harmonise EBV viral load measurements in a wide range of NAT assays. Candidate materials included lyophilised and liquid whole virus preparations of the EBV B95-8 strain, and preparations of Namalwa and Raji cells. Variability between the individual laboratory mean estimates for each candidate was 2.5 log10 copies/mL. The agreement between laboratories was improved when the potency of each candidate was expressed relative to the lyophilised B95-8 preparation. The results of the study indicate the suitability of this candidate as the 1st WHO International Standard for EBV for NAT. It was established in October 2011 by the WHO's Expert Committee on Biological Standardisation with an assigned potency of 5 × 10(6) International Units (IU) (NIBSC code 09/260). It is intended to be used for the calibration of secondary reference materials, used in EBV NAT assays, in IU, thereby improving the comparability of patient viral load measurements.

A collaborative study to establish the 1st WHO International Standard for human cytomegalovirus for nucleic acid amplification technology.

Variability in the performance of nucleic acid amplification technology (NAT)-based assays presents a significant problem in the diagnosis and management of human cytomegalovirus (HCMV) infections. Here we describe a collaborative study to evaluate the suitability of candidate reference materials to harmonize HCMV viral load measurements in a wide range of NAT assays. Candidate materials comprised lyophilized Merlin virus, liquid Merlin virus, liquid AD169 virus, and purified HCMV Merlin DNA cloned into a bacterial artificial chromosome. Variability in the laboratory mean HCMV concentrations determined for virus samples across the different assays was 2 log10. Variability for the purified DNA sample was higher (>3 log10). The agreement between laboratories was markedly improved when the potencies of the liquid virus samples were expressed relative to the lyophilized virus candidate. In contrast, the agreement between laboratories for the purified DNA sample was not improved. Results indicated the suitability of the lyophilized Merlin virus preparation as the 1st WHO International Standard for HCMV for NAT. It was established in October 2010, with an assigned potency of 5 × 10(6) International Units (IU) (NIBSC code 09/162). It is intended to be used to calibrate secondary references, used in HCMV NAT assays, in IU.

An Introduction to Poliovirus: Pathogenesis, Vaccination, and the Endgame for Global Eradication.

Poliomyelitis is caused by poliovirus, which is a positive strand non-enveloped virus that occurs in three distinct serotypes (1, 2, and 3). Infection is mainly by the fecal-oral route and can be confined to the gut by antibodies induced either by vaccine, previous infection or maternally acquired. Vaccines include the live attenuated strains developed by Sabin and the inactivated vaccines developed by Salk; the live attenuated vaccine (Oral Polio Vaccine or OPV) has been the main tool in the Global Program of Polio eradication of the World Health Organisation. Wild type 2 virus has not caused a case since 1999 and type 3 since 2012 and eradication seems near. However most infections are entirely silent so that sophisticated environmental surveillance may be needed to ensure that the virus has been eradicated, and the live vaccine can sometimes revert to virulent circulating forms under conditions that are not wholly understood. Cessation of vaccination is therefore an increasingly important issue and inactivated polio vaccine (IPV) is playing a larger part in the end game.

Development of a candidate reference material for adventitious virus detection in vaccine and biologicals manufacturing by deep sequencing.

BACKGROUND: Unbiased deep sequencing offers the potential for improved adventitious virus screening in vaccines and biotherapeutics. Successful implementation of such assays will require appropriate control materials to confirm assay performance and sensitivity. METHODS: A common reference material containing 25 target viruses was produced and 16 laboratories were invited to process it using their preferred adventitious virus detection assay. RESULTS: Fifteen laboratories returned results, obtained using a wide range of wet-lab and informatics methods. Six of 25 target viruses were detected by all laboratories, with the remaining viruses detected by 4-14 laboratories. Six non-target viruses were detected by three or more laboratories. CONCLUSION: The study demonstrated that a wide range of methods are currently used for adventitious virus detection screening in biological products by deep sequencing and that they can yield significantly different results. This underscores the need for common reference materials to ensure satisfactory assay performance and enable comparisons between laboratories.

Development of Lentivirus-Based Reference Materials for Ebola Virus Nucleic Acid Amplification Technology-Based Assays.

The 2013-present Ebola virus outbreak in Western Africa has prompted the production of many diagnostic assays, mostly based on nucleic acid amplification technologies (NAT). The calibration and performance assessment of established assays and those under evaluation requires reference materials that can be used in parallel with the clinical sample to standardise or control for every step of the procedure, from extraction to the final qualitative/quantitative result. We have developed safe and stable Ebola virus RNA reference materials by encapsidating anti sense viral RNA into HIV-1-like particles. The lentiviral particles are replication-deficient and non-infectious due to the lack of HIV-1 genes and Envelope protein. Ebola virus genes were subcloned for encapsidation into two lentiviral preparations, one containing NP-VP35-GP and the other VP40 and L RNA. Each reference material was formulated as a high-titre standard for use as a calibrator for secondary or internal standards, and a 10,000-fold lower titre preparation to serve as an in-run control. The preparations have been freeze-dried to maximise stability. These HIV-Ebola virus RNA reference materials were suitable for use with in-house and commercial quantitative RT-PCR assays and with digital RT-PCR. The HIV-Ebola virus RNA reference materials are stable at up to 37°C for two weeks, allowing the shipment of the material worldwide at ambient temperature. These results support further evaluation of the HIV-Ebola virus RNA reference materials as part of an International collaborative study for the establishment of the 1st International Standard for Ebola virus RNA.

Production of high titer attenuated poliovirus strains on the serum-free PER.C6(®) cell culture platform for the generation of safe and affordable next generation IPV.

BACKGROUND: As poliovirus eradication draws closer, alternative Inactivated Poliovirus Vaccines (IPV) are needed to overcome the risks associated with continued use of the Oral Poliovirus Vaccine and of neurovirulent strains used during manufacture of conventional (c) IPV. We have previously demonstrated the susceptibility of the PER.C6(®) cell line to cIPV strains; here we investigated the suspension cell culture platform for growth of attenuated poliovirus strains. METHODS: We examined attenuated Sabin strain productivity on the PER.C6(®) cell platform compared to the conventional Vero cell platform. The suitability of the suspension cell platform for propagation of rationally-attenuated poliovirus strains (stabilized Sabin type 3 S19 derivatives and genetically attenuated and stabilized MonoCre(X) strains), was also assessed. Yields were quantified by infectious titer determination and D-antigen ELISA using either serotype-specific polyclonal rabbit sera for Sabin strains or monoclonal cIPV-strain-specific antibodies for cIPV, S19 and MonoCre(X) strains. RESULTS: PER.C6(®) cells supported the replication of Sabin strains to yields of infectious titers that were in the range of cIPV strains at 32.5°C. Sabin strains achieved 30-fold higher yields (p<0.0001) on the PER.C6(®) cell platform as compared to the Vero cell platform in infectious titer and D-antigen content. Furthermore, Sabin strain productivity on the PER.C6(®) cell platform was maintained at 10l scale. Yields of infectious titers of S19 and MonoCre(X) strains were 0.5-1 log10 lower than seen for cIPV strains, whereas D-antigen yield and productivities in doses/ml using rationally-attenuated strains were in line with yields reported for cIPV strains. CONCLUSIONS: Sabin and rationally-attenuated polioviruses can be grown to high infectious titers and D-antigen yields. Sabin strain infection shows increased productivity on the PER.C6(®) cell platform as compared to the conventional Vero cell platform. Novel cell platforms with the potential for higher yields could contribute to increased affordability of a next generation of IPV vaccines needed for achieving and maintaining poliovirus eradication.

Assaying the Potency of Influenza Vaccines.

The potency of vaccines must be determined to ensure that the appropriate dose is given. The manufacture and assessment of influenza vaccines are complicated by the continuously changing nature of the pathogen, which makes efficacy estimates difficult but also confounds attempts to produce a well-validated, consistent potency assay. Single radial diffusion has been used for decades and provides a relatively simple way to measure the amount of biologically active materials present in the vaccine. It requires reagents, which are updated on a regular, frequently yearly, basis and alternative methods continue to be sought.

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.

High resolution identity testing of inactivated poliovirus vaccines.

BACKGROUND: Definitive identification of poliovirus strains in vaccines is essential for quality control, particularly where multiple wild-type and Sabin strains are produced in the same facility. Sequence-based identification provides the ultimate in identity testing and would offer several advantages over serological methods. METHODS: We employed random RT-PCR and high throughput sequencing to recover full-length genome sequences from monovalent and trivalent poliovirus vaccine products at various stages of the manufacturing process. RESULTS: All expected strains were detected in previously characterised products and the method permitted identification of strains comprising as little as 0.1% of sequence reads. Highly similar Mahoney and Sabin 1 strains were readily discriminated on the basis of specific variant positions. Analysis of a product known to contain incorrect strains demonstrated that the method correctly identified the contaminants. CONCLUSION: Random RT-PCR and shotgun sequencing provided high resolution identification of vaccine components. In addition to the recovery of full-length genome sequences, the method could also be easily adapted to the characterisation of minor variant frequencies and distinction of closely related products on the basis of distinguishing consensus and low frequency polymorphisms.

Live attenuated vaccines: Historical successes and current challenges.

Live attenuated vaccines against human viral diseases have been amongst the most successful cost effective interventions in medical history. Smallpox was declared eradicated in 1980; poliomyelitis is nearing global eradication and measles has been controlled in most parts of the world. Vaccines function well for acute diseases such as these but chronic infections such as HIV are more challenging for reasons of both likely safety and probable efficacy. The derivation of the vaccines used has in general not been purely rational except in the sense that it has involved careful clinical trials of candidates and subsequent careful follow up in clinical use; the identification of the candidates is reviewed.