Deep Sequencing for Evaluation of Genetic Stability of Influenza A/California/07/2009 (H1N1) Vaccine Viruses.

Virus growth during influenza vaccine manufacture can lead to mutations that alter antigenic properties of the virus, and thus may affect protective potency of the vaccine. Different reassortants of pandemic "swine" H1N1 influenza A vaccine (121XP, X-179A and X-181) viruses as well as wild type A/California/07/2009(H1N1) and A/PR/8/34 strains were propagated in embryonated eggs and used for DNA/RNA Illumina HiSeq and MiSeq sequencing. The RNA sequences of these viruses published in NCBI were used as references for alignment of the sequencing reads generated in this study. Consensus sequences of these viruses differed from the NCBI-deposited sequences at several nucleotides. 121XP stock derived by reverse genetics was more heterogeneous than X-179A and X-181 stocks prepared by conventional reassortant technology. Passaged 121XP virus contained four non-synonymous mutations in the HA gene. One of these mutations (Lys226Glu) was located in the Ca antigenic site of HA (present in 18% of the population). Two non-synonymous mutations were present in HA of viruses derived from X-179A: Pro314Gln (18%) and Asn146Asp (78%). The latter mutation located in the Sa antigenic site was also detected at a low level (11%) in the wild-type A/California/07/2009(H1N1) virus, and was present as a complete substitution in X-181 viruses derived from X-179A virus. In the passaged X-181 viruses, two mutations emerged in HA: a silent mutation A1398G (31%) in one batch and G756T (Glu252Asp, 47%) in another batch. The latter mutation was located in the conservative region of the antigenic site Ca. The protocol for RNA sequencing was found to be robust, reproducible, and suitable for monitoring genetic consistency of influenza vaccine seed stocks.

Human monoclonal antibodies that neutralize vaccine and wild-type poliovirus strains.

An essential requirement for eradication of poliomyelitis is the elimination of circulating vaccine derived polioviruses (cVDPV) and polioviruses excreted by chronically infected individuals with immunodeficiencies (iVDPV). As part of a post-eradication risk management strategy, a human monoclonal antibody (mAb) therapeutic could play a role in halting excretion in asymptomatic carriers and could be used, in combination with vaccines and antiviral drugs, to protect polio-exposed individuals. Cross-neutralizing mAbs may be particularly useful, as they would reduce the number of mAbs needed to create a comprehensive PV therapeutic. We cloned a panel of IgG mAbs from OPV-vaccinated, IPV-boosted healthy subjects. Many of the mAbs had potent neutralizing activities against PV wild-type (WT) and Sabin strains, and two of the mAbs, 12F8 and 1E4, were significantly cross-reactive against types 1 and 2 and types 1 and 3, respectively. Mapping the binding epitopes using strains resistant to neutralization (escape mutants) suggested that cross-specific PV binding epitopes may primarily reside within the canyon region, which interacts with the cellular receptor molecule CD155 and the cross-neutralizing chimpanzee/human mAb, A12. Despite their close proximity, the epitopes for the 12F8 and 1E4 mAbs on Sabin 1 were not functionally identical to the A12 epitope. When tested together, 12F8 and 1E4 neutralized a diverse panel of clinically relevant PV strains and did not exhibit interference. Virus mutants resistant to the anti-poliovirus drug V-073 were also neutralized by the mAbs. The 12F8 and 1E4 mAbs may suitable for use as anti-PV therapeutics.

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.

Deep sequencing approach for genetic stability evaluation of influenza A viruses.

Assessment of genetic stability of viruses could be used to monitor manufacturing process of both live and inactivated viral vaccines. Until recently such studies were limited by the difficulty of detecting and quantifying mutations in heterogeneous viral populations. High-throughput sequencing technologies (deep sequencing) can generate massive amounts of genetic information and could be used to reveal and quantify mutations. Comparison of different approaches for deep sequencing of the complete influenza A genome was performed to determine the best way to detect and quantify mutants in attenuated influenza reassortant strain A/Brisbane/59/2007 (H1N1) and its passages in different cell substrates. Full-length amplicons of influenza A virus segments as well as multiple overlapping amplicons covering the entire viral genome were subjected to several ways of DNA library preparation followed by deep sequencing using Solexa (Illumina) and pyrosequencing (454 Life Science) technologies. Sequencing coverage (the number of times each nucleotide was determined) of mutational profiles generated after 454-pyrosequencing of individually synthesized overlapping amplicons were relatively low and insufficiently uniform. Amplification of the entire genome of influenza virus followed by its enzymatic fragmentation, library construction, and Illumina sequencing resulted in high and uniform sequencing coverage enabling sensitive quantitation of mutations. A new bioinformatic procedure was developed to improve the post-alignment quality control for deep-sequencing data analysis.

Cross-neutralizing human anti-poliovirus antibodies bind the recognition site for cellular receptor.

Most structural information about poliovirus interaction with neutralizing antibodies was obtained in the 1980s in studies of mouse monoclonal antibodies. Recently we have isolated a number of human/chimpanzee anti-poliovirus antibodies and demonstrated that one of them, MAb A12, could neutralize polioviruses of both serotypes 1 and 2. This communication presents data on isolation of an additional cross-neutralizing antibody (F12) and identification of a previously unknown epitope on the surface of poliovirus virions. Epitope mapping was performed by sequencing of antibody-resistant mutants and by cryo-EM of complexes of virions with Fab fragments. The results have demonstrated that both cross-neutralizing antibodies bind the site located at the bottom of the canyon surrounding the fivefold axis of symmetry that was previously shown to interact with cellular poliovirus receptor CD155. However, the same antibody binds to serotypes 1 and 2 through different specific interactions. It was also shown to interact with type 3 poliovirus, albeit with about 10-fold lower affinity, insufficient for effective neutralization. Antibody interaction with the binding site of the cellular receptor may explain its broad reactivity and suggest that further screening or antibody engineering could lead to a universal antibody capable of neutralizing all three serotypes of poliovirus.

Quantitative one-step RT-PCR assay for rapid and sensitive identification and titration of polioviruses in clinical specimens.

Rapid identification and quantitation of polioviruses in clinical specimens is important for surveillance and analysis of virus shedding by vaccine recipients, which could be used to assess the level of mucosal immunity. A quantitative one step RT-PCR was developed for identification and titration of all three poliovirus serotypes. The assay could be an alternative to the traditional procedure based on cell culture isolation and subsequent determination of poliovirus serotype and virus titration. The method is based on quantitative PCR performed with reverse transcription reaction in the same tube. The multiplex assay that quantifies all three serotypes of poliovirus was found to be highly specific, sensitive, and takes only one day to complete.

MAPREC assay for quantitation of mutants in a recombinant flavivirus vaccine strain using near-infrared fluorescent dyes.

Mutant analysis by PCR and restriction enzyme cleavage (MAPREC) is a quantitative assay of revertants in batches of live viral vaccines. The assay is highly sensitive and reliable but requires radioactive isotopes, which complicates its use in quality control laboratories. To quantify mutants in the cDNA of the West Nile (WN)/Dengue 4 chimera that was proposed as a new candidate of live vaccine against West Nile disease, alternative MAPREC protocols using non-radioactive dyes were explored. To compare the utility of different fluorescent dyes for MAPREC, the G(2337)→C mutation that was revealed by microarray hybridization in WN/Dengue 4 chimera virus was used as a model. DNA fragments produced by restriction endonuclease digestion were visualized in polyacrylamide gels by visible-range fluorescent dyes including ethidium bromide (EtBr) and SYBR Green I as well as by near-infrared (NIR) dye SYTO 60 and NIR dyes 700 and 800. The MAPREC assay performed with SYTO 60 and SYBR Green I was more sensitive than with EtBr but less sensitive than with NIR dyes 700 or 800. The NIR dyes 700 and 800 exhibited a wide linear range that may enable the detection of 0.05% of mutants in viral stocks. The NIR-based MAPREC assay was validated by using World Health Organization (WHO) international references for poliovirus type 3 with known contents of mutants. Values of mutant content produced by the non-radioactive assay were similar to the values determined in a previous WHO international collaborative study. The modified MAPREC assay could be used as an alternative to the radioisotope-based standard protocol for quality control of live viral vaccines.

Microarray hybridization for assessment of the genetic stability of chimeric West Nile/dengue 4 virus.

Genetic stability is an important characteristic of live viral vaccines because an accumulation of mutants can cause reversion to a virulent phenotype as well as a loss of immunogenic properties. This study was aimed at evaluating the genetic stability of a live attenuated West Nile (WN) virus vaccine candidate that was generated by replacing the pre-membrane and envelope protein genes of dengue 4 virus with those from WN. Chimeric virus was serially propagated in Vero, SH-SY5Y human neuroblastoma and HeLa cells and screened for point mutations using hybridization with microarrays of overlapping oligonucleotide probes covering the entire genome. The analysis revealed several spontaneous mutations that led to amino acid changes, most of which were located in the envelope (E) and non-structural NS4A, NS4B, and NS5 proteins. Viruses passaged in Vero and SH-SY5Y cells shared two common mutations: G(2337) C (Met(457) Ile) in the E gene and A(6751) G (Lys(125) Arg) in the NS4A gene. Quantitative assessment of the contents of these mutants in viral stocks indicated that they accumulated independently with different kinetics during propagation in cell cultures. Mutant viruses grew better in Vero cells compared to the parental virus, suggesting that they have a higher fitness. When tested in newborn mice, the cell culture-passaged viruses did not exhibit increased neurovirulence. The approach described in this article could be useful for monitoring the molecular consistency and quality control of vaccine strains.