WO2014020356A1 - Method for quality controlling vaccine - Google Patents

Abstract

Methods of isolating and sequencing vaccine or pathogen nucleic acid directly from samples which contain host nucleic acid in vast excess,to monitor for evolution of deleterious variants are described.

Description

METHOD FOR QUALITY CONTROLLING VACCINE

Field of the Invention

The present invention relates to methods of screening for evolution of an infectious agent, for example a pathogen, or of a vaccine. Background of the Invention

Pathogens existing within a host organism are constantly evolving in order to survive within their host, and in the case of infectious disease-causing pathogens, including viruses, that evolution may not only be driven by positive selection pressure through interaction with the host, but also be driven by pharmaceutical treatment. Through natural selection, genetic variants of a pathogen can not only survive in a host and survive medical treatment, but in some cases thrive and develop complete resistance. Genetic variation defines pathogenic population structures and can be used to identify paths of transmission.

Thus, identification and characterisation of minority variants as they evolve due to selection pressure is of significant interest in order to fully understand these resistance and transmission pathways.

Similarly, it has long been known that viral vaccines are also subject to evolutionary pressures and that these pressures can affect the composition of live attenuated vaccines. For example, the live attenuated varicella-zoster virus vaccine (vOka) has been administered in numerous countries over the past 15 years. The vaccine comprises a mixed population of vOKA variants and causes both varicella- and zoster-like rashes to occur in 5% of healthy children (rising to 50% in leukemic children).

Viral genome copies per millilitre of sample from an individual can number in the billions yet the relative proportion is minute in comparison to the host genome. Direct sequencing of mixed human and viral nucleic acids yields only very small numbers (<0.1 %) of sequence reads that can be mapped to the viral genome. The two primary methods for isolating viral nucleic acid from a mixture of viral and host nucleic acid rely on the production of microgram quantities of viral nucleic acid, either by PCR or by in vitro virus culture. However, both methods are known to alter virus population structures either through the introduction of mutations, deletions or rearrangements, or through replication advantages of virus subsets during culture. Thus, these methods are not suitable for monitoring - at the single nucleotide level - vaccine or pathogen evolution in a host due to the tendency to introduce errors or positively selected mutations during the amplification step required to produce enough pathogenic or vaccine genomic material for sequencing. These prior techniques are hindered by a lack of resolution (read depth) and a comprehensive analysis of diversity within and between vaccine batches is still lacking. Similarly, new techniques are required to monitor pathogenesis in a host or between hosts in order to determine transmission chains and more effectively treat individuals infected with a pathogen. Summary of the Invention

According to a first aspect of the invention there is provided a method of quality controlling a batch of vaccine, the method comprising: a) providing at least one set of vaccine-specific polynucleotides each comprising an immobilization tag; b) contacting under hybridising conditions the at least one set of vaccine- specific polynucleotides with a sample obtained from a patient who has been inoculated with the batch of vaccine; c) exposing the mixture from b) to a solid surface provided with a binding partner specific to the immobilization tag to isolate vaccine genomic material hybridised to the vaccine-specific polynucleotides; d) sequencing at least part of the vaccine genomic material from step c); e) determining the identity of at least one polymorphic position in the vaccine genomic material from step c); wherein a mutation in the at least one polymorphic position in the vaccine genomic material relative to at least one reference sequence for that batch of vaccine is indicative that a variant of the vaccine has been selected for.

According to an alternative first aspect of the invention there is provided a method of quality controlling a batch of vaccine, the method comprising: a) providing at least one set of vaccine-specific polynucleotides each comprising an immobilization tag; b) contacting under hybridising conditions the at least one set of vaccine- specific polynucleotides with a sample; c) exposing the mixture from b) to a solid surface provided with a binding partner specific to the immobilization tag to isolate vaccine genomic material hybridised to the vaccine-specific polynucleotides; d) sequencing at least part of the vaccine genomic material from step c); e) determining the identity of at least one polymorphic position in the vaccine genomic material from step c); wherein a mutation in the at least one polymorphic position in the vaccine genomic material relative to at least one reference sequence for that batch of vaccine is indicative that a variant of the vaccine has been selected for.

In this alternative method of the first aspect, the sample may be a sample of the batch of vaccine. Alternatively, the sample may be a sample obtained from a patient inoculated with the batch of vaccine.

The methods of the first aspect allow the monitoring of batches of vaccine to ensure that a batch of vaccine, which has undergone one or more disadvantageous mutations that may result in an adverse reaction in an individual, is no longer administered. Accordingly, the methods of the first aspect may further comprise destroying a batch of vaccine when the methods indicate that a variant of that vaccine have been selected for. Alternatively, when a variant of the vaccine has been selected for, the methods of the first aspect may comprise administering an alternative batch of vaccine to an individual in need thereof, wherein it is known that no variant has been selected for in the alternative batch of vaccine. In the method of the first aspect wherein the sample is obtained from a patient inoculated with the batch of vaccine, the second batch of vaccine may be administered to a second individual in need thereof.

By selection of a variant, it is meant that an event has occurred, either prior or post- inoculation, that has resulted in that variant becoming established in the patient. The variant may be a pathogenic variant. The selection of the variant may result in an adverse-vaccine reaction. The selection of the variant may be an adverse-vaccine reaction event.

The vaccine may be a viral vaccine.

The sample may be obtained from a vaccine rash.

The sample may be obtained up to two weeks after inoculation. The sample may be obtained at least two weeks after inoculation, for example at least four weeks after inoculation.

The variant may comprise a minority isoform of the vaccine present in the batch of vaccine.

The variant may comprise a denovo mutation in the vaccine genomic material relative to the at least one reference sequence.

The method may further comprise subjecting the sample to a pre-treatment step before contacting it under hybridising conditions with the set of vaccine-specific polynucleotides. The pre-treatment step may comprise fragmenting the sample.

The vaccine may be a RNA viral vaccine or a DNA viral vaccine. The vaccine may be an attenuated form of the virus.

The nucleotide identities of a plurality of polymorphic positions in the vaccine genomic material may be determined in step e). The mutation at the at least one polymorphic position may be a non-synonymous mutation or a synonymous mutation.

The vaccine-specific polynucleotides may comprise ribopolynucleotides or deoxyribopolynucleotides. The at least one set of vaccine-specific polynucleotides may comprise a plurality of overlapping polynucleotides spanning a vaccine genomic region of interest. The at least one set of vaccine-specific polynucleotides may comprise a plurality of overlapping polynucleotides complementary to a vaccine genomic region of interest.

A plurality of sets of vaccine-specific polynucleotides may be provided. The plurality of sets of vaccine-specific polynucleotides may be specific for the same vaccine.

Each set of the plurality of sets of vaccine-specific polynucleotides may be specific to a particular isoform of the vaccine.

Each set of the plurality of sets of vaccine-specific polynucleotides may be specific for a different vaccine.

The immobilization tag may comprise biotin and the binding partner may comprise streptavidin. The solid surface may comprise magnetic beads.

The vaccine may be the varicella Oka vaccine. The at least one polymorphic position of the varicella Oka vaccine may be selected from Table 1. According to a second aspect of the invention there is provided a method of determining evolution of a pathogen, the method comprising: a) providing at least one set of pathogen-specific polynucleotides each comprising an immobilization tag; b) contacting under hybridising conditions the at least one set of pathogen- specific polynucleotides with a first sample obtained at a first timepoint from an individual infected with the pathogen; c) exposing the mixture from b) to a solid surface provided with a binding partner specific to the immobilization tag to isolate pathogenic genomic material hybridised to the pathogen-specific polynucleotides; d) sequencing at least part of the pathogenic genomic material; e) determining the nucleotide identity of at least one polymorphic position in the pathogenic genomic material; and f) repeating steps a) to e) with at least a second set of pathogen-specific polynucleotides each comprising an immobilization tag and a second sample obtained at a second timepoint from an individual infected with the pathogen; wherein a mutation in the at least one polymorphic position in the pathogenic genomic material is indicative that the pathogen has evolved.

The method of the second aspect selectively enriches the pathogenic genomic material relative to the host genomic material without any error-prone amplification of the pathogenic genomic material. This subsequently allows deep sequencing of the pathogenic genomic material, with improved read depth such that single nucleotide mutations can be reliably observed.

Monitoring of mutations at any given SNP site enables identification of mutations evolving as a result of host immune pressure or drug resistance, or associated with vaccine and drug adverse events. The method of the second aspect also enables identification of SNPs which may be associated with the tissue tropism of a pathogen or virus, thus providing information regarding the pathogenesis of the virus or pathogen and the preferred tissue for infection, and the subsequent tailoring of medical treatment.

The method of the second aspect also enables the identification of SNPs associated with virulence, that is the SNP or multiple SNPs which confer increased pathogenicity, such that medical treatment can again be tailored.

Accordingly, the methods of the second aspect may further comprise a step of administering to the individual a medicament to which the pathogen has not developed resistance. Tailoring of medical treatment, i.e. administration of a particular medicament based on a laboratory analysis of a sample from an individual and mutation resistance of a pathogen to a pharmaceutical drug based on resistance is routinely carried out by medical practitioners. The first and second samples may be obtained from the same individual.

The first and second samples may be obtained from different individuals.

The sample may comprise host genomic material and pathogenic genomic material.

The second timepoint may be at least two weeks after the first timepoint. The second timepoint may be at least four weeks after the first timepoint. The method may further comprise subjecting the first and/or second sample to a pre- treatment step before contacting it under hybridising conditions with the set of pathogenic-specific polynucleotides.

The pre-treatment step may comprise fragmenting the sample.

The pre-treatment step may comprise whole genome amplification as a first pre- treatment step.

The sample may not be subjected to amplification by PCR as a first pre-treatment step.

The sample may not be subjected to amplification by culture as a first pre-treatment step.

The pathogen may be viral, bacterial, fungal or parasitic. The pathogen may be a RNA virus or a DNA virus.

The virus may be an attenuated form of the virus, for example a vaccine.

The nucleotide identities of a plurality of polymorphic positions in the pathogenic genomic material may be determined in step e) and step f).

The mutation at the at least one polymorphic position may be a non-synonymous mutation or a synonymous mutation. The pathogen-specific polynucleotides may comprise ribopolynucleotides or deoxyribopolynucleotides.

The at least one set of pathogen-specific polynucleotides may comprise a plurality of overlapping polynucleotides spanning a pathogenic genomic region of interest. The at least one set of vaccine-specific polynucleotides may comprise a plurality of overlapping polynucleotides complementary to a vaccine genomic region of interest.

A plurality of sets of pathogen-specific polynucleotides may be provided.

The plurality of sets of pathogen-specific polynucleotides may be specific for the same pathogen. Each of the plurality of sets of pathogen-specific polynucleotides may be specific for a different pathogen.

The immobilization tag may comprise biotin and the binding partner may comprise streptavidin.

The solid surface may comprise magnetic beads. According to the second aspect of the present invention there is alternatively provided a method of determining evolution of a pathogen, the method comprising: a) providing at least one set of pathogen-specific polynucleotides each comprising an immobilization tag; b) contacting under hybridising conditions the at least one set of pathogen- specific polynucleotides with a sample obtained from an individual infected with the pathogen; c) exposing the mixture from b) to a solid surface provided with a binding partner specific to the immobilization tag to isolate pathogenic genomic material hybridised to the pathogen-specific polynucleotides; d) sequencing at least part of the pathogenic genomic material; e) determining the nucleotide identity of at least one polymorphic position in the pathogenic genomic material; wherein a mutation in the at least one polymorphic position in the pathogenic genomic material relative to at least one reference sequence for that pathogen is indicative that the pathogen has evolved; or wherein a mutation in the at least one polymorphic position in the pathogenic genomic material relative to at least one reference sequence for that pathogen is indicative that a variant of the pathogen has been selected for.

The reference sequence may comprise a known sequence of the pathogen. Alternatively, the reference sequence may be determined from a first sample obtained at a first timepoint from an individual infected with the pathogen, the first sample also being subjected to steps a) to e) of the method. In this embodiment, the sample referred to in step b) may be a second sample obtained at a second timepoint from the individual infected with the pathogen. According to a third aspect of the invention there is provided a kit-of-parts for use in a method of determining evolution of a pathogen, the kit comprising: at least one set of pathogen-specific polynucleotides each comprising an immobilization tag; and a solid surface provided with a binding partner specific to the immobilization tag.

According to a fourth aspect of the invention there is provided a kit-of-parts for use in a method of quality controlling a batch of vaccine, the kit comprising: at least one set of vaccine-specific polynucleotides each comprising an immobilization tag; and a solid surface provided with a binding partner specific to the immobilization tag.

The kit-of-parts of the third or fourth aspects of the invention may comprise an instruction manual for carrying out the method. Any one or more features described for any aspect of the present invention or preferred embodiments or examples thereof, described herein, may be used in conjunction with any one or more other features described for any other aspect of the present invention or preferred embodiments or examples therefore described herein. The fact that a feature may only be described in relation to one aspect or embodiment or example does not limit its relevance to only that aspect or embodiment or example if it is technically relevant to one or more other aspect or embodiment or example.

Detailed Description of the Invention

The present invention uses target capture technology and whole genome sequencing to separate and enrich for pathogenic nucleic acid at various timepoints, thereby permitting monitoring for evolution of the pathogen. The present invention also uses the same target capture technology and whole genome sequencing to monitor batches of vaccine for quality assurance.

Sample

The sample may be a biological sample obtained from a patient or an individual. The sample may include whole blood, blood serum, semen, peritoneal fluid, saliva, stool, urine, synovial fluid, wound fluid, vesicle fluid, cerebrospinal fluid, tissue from eyes, intestine, kidney, brain, skin, heart, prostate, lung, breast, liver muscle or connective tissue and tumour cell lines. The sample may be referred to as a clinical sample.

The sample may comprise nucleic acid extracted from a biological sample obtained from an individual. Methods for extracting nucleic acid from patient samples to obtain a mixture of patient nucleic acid and pathogen nucleic acid are well known in the art and generally known as total DNA extraction methods.

The sample may be obtained from the same individual or from a different individual. The sample may be obtained from an individual who has been inoculated with the vaccine. In an alternative embodiment, the sample is not obtained from an individual but is instead obtained from a batch of vaccine. The sample may be obtained from a viral vaccine, for example from a varicella vaccine. The sample may be obtained from a bacterial vaccine, for example the BCG vaccine against tuberculosis or the Ty21a vaccine against typhoid fever. In one embodiment, the nucleic acid extracted from the sample may be used in the methods of the invention without pre-amplification by culture or PCR. In one embodiment, the sample may comprise less than 3 starting nucleic acid, for example less than 2 starting nucleic acid, less than 1 μg starting nucleic acid. In one embodiment, the sample may comprise less than 900 ng starting nucleic acid, for example less than 800 ng starting nucleic acid, less than 700 ng starting nucleic acid, less than 600 ng starting nucleic acid. In one embodiment, the sample may comprise 500 ng starting nucleic acid or less.

In order to monitor for evolution of a virus, other pathogen or to monitor a viral or bacterial vaccine for selection of a variant, samples are obtained at different timepoints. In one embodiment, a second timepoint may be up to two weeks from the first timepoint when the first or initial sample is obtained. In one embodiment, a second timepoint may be at least two weeks after the first timepoint when the first or initial sample is obtained, for example at least four weeks or at least eight weeks. Alternatively the second timepoint may be more than two months after the first timepoint, for example more than three months or more than four months, more than six months. In one embodiment, the sample is obtained up to two weeks after inoculation of a patient with a vaccine. In one embodiment, the sample is obtained at least two weeks after inoculation of a patient with a vaccine, for example at least four weeks or at least eight weeks. Alternatively the sample may be obtained more than two months after inoculation, for example more than three months or more than four months, more than six months.

The length of time between the first and second timepoints may depend on the extent of the selective pressures being exerted on the virus, other pathogen or viral or bacterial vaccine. A batch of vaccine in a sealed vial will be subjected to fewer local environmental evolutionary pressures than a vaccine or other pathogen present in a human or animal body. Thus, relative to the first timepoint, the second timepoint for testing a batch of vaccine may be significantly later in time than the second timepoint for testing evolution of a vaccine or other pathogen in a patient or group of patients.

Pathogens

The method of the invention is suited to isolating or fishing out any foreign or invader genomic material from the biological sample containing pathogenic genomic material and host genomic material. For example, the pathogenic genome(s) of interest may be viral and/or bacterial. The pathogenic genome of interest may be fungal or parasitic. In one embodiment, the method of the invention may isolate a single pathogen from a biological sample. In one embodiment, the method of the invention may isolate multiple, i.e. two or more different pathogens from one biological sample. Vaccines

The method of the invention is suited to isolating or fishing out any vaccine genomic material from the biological sample comprising vaccine genomic material and host genomic material. For example, the vaccine genome(s) of interest may be viral and/or bacterial. In one embodiment, the method of the invention may isolate a single vaccine from a biological sample. In one embodiment, the method of the invention may isolate multiple isoforms of a single vaccine from a biological sample. In one embodiment, the method of the invention may isolate multiple, i.e. two or more different vaccines from one biological sample.

Pre-treatment Before contacting the sample under hybridising conditions with the set of pathogen- specific polynucleotides or vaccine-specific nucleotides, the method may comprise the step of subjecting the sample to a pre-treatment step.

The sample may contain sufficient pathogenic DNA or RNA that no pre-amplification is required. The sample may contain sufficient vaccine DNA or RNA that no pre- amplification is required. The sample may be amplified using whole genome amplification (WGA) as a pre-treatment step.

In one embodiment, the pre-treatment step may comprise isolation of the total DNA contained within the biological sample by any known method.

In one embodiment, the sample may be fragmented by biological, chemical or mechanical means. In one embodiment, the sample may be mechanically fragmented by shearing, nebulisation or sonication. In an alternative embodiment the sample may be biologically fragmented by a nuclease treatment.

In a yet further embodiment the sample may be pre-treated by addition of standard primers and/or other attachments for later use in a sequencing protocol. Polynucleotide Bait

The bait or polynucleotide bait comprises a set of polynucleotides specific to the pathogenic genome of interest, a viral or bacterial vaccine genome of interest or a host gene of interest. For example, the set of polynucleotides are complementary to one strand of the genomic region of interest. The polynucleotide may be a ribopolynucleotide or a deoxyribopolynucleotide. The polynucleotide is preferably more than about 50 bases in length, for example more than about 100 bases in length, for example more than about 150 bases in length. In one embodiment the polynucleotide bait is more than about 200 bases in length, for example more than about 500 bases in length, for example more than about 1000 bases in length. In another embodiment, the polynucleotide is less than about 200 bases in length, for example less than about 150 bases in length. In one embodiment the polynucleotide is about 120 bases in length, for example from about 110 bases to about 130 bases in length. In one embodiment the polynucleotide is about 150 bases in length, for example from about 140 bases to about 160 bases in length. In one embodiment the polynucleotide is about 170 bases in length, for example from about 160 bases to about 180 bases in length.

The bait may comprise one or more immobilization tags bonded to the polynucleotide to facilitate immobilization of the target-bait hybrid to a solid surface.

In one embodiment, the second set of baits used on the sample obtained at a second timepoint comprises the same set of baits used on the sample obtained at the first timepoint. In an alternative embodiment, the second set of baits may be a different set of baits to the set used on the sample obtained at the first timepoint. This different set of baits may comprise one or more changes in nucleotide sequence to positively fish out suspected variants.

In one embodiment, the polynucleotide may comprise one or more modifications, for example the presence of one or more modified nucleotides or unnatural nucleotides. For example, the bait may comprise 5-substituted pyrimidine derivatives to which the immobilization tag may be connected. In an alternative embodiment, the bait may comprise 7-substituted purine derivatives to which the immobilization tag may be connected. Preferably, the bait comprises a set of polynucleotides, for example a plurality of polynucleotides. In one embodiment, the bait comprises a plurality of overlapping polynucleotides spanning a pathogenic or vaccine genomic region of interest.

The method of the present invention is suited to multiplexing in which a plurality of sets of polynucleotides are provided, each set being specific to a different genome of interest. In an alternative embodiment, a plurality of sets of polynucleotides are provided, wherein at least one set of polynucleotides are specific to a host genomic region of interest. Each set of polynucleotides may be provided with a different immobilization tag specific to a different binding partner provided on the solid surface. By providing each set of polynucleotides with different immobilization tags specific to different binding partners, the method of the invention is able to selectively fish out of the sample as many different pathogenic, vaccine or host genomes as different immobilization tags are used.

In one embodiment, the bait may comprise further tags or labels as may be required. For example, in one embodiment, the bait may comprise one or more fluorescent labels. In the embodiment in which the bait comprises a plurality of sets of polynucleotides and each set is specific for a different pathogen or vaccine, each set of polynucleotides may comprise a different fluorescent label. Examples of suitable fluorescent labels include but are not limited to Cy-dyes, fluorescein, Alexa dyes, rhodamine dyes.

Immobilization tag and binding partner

The bait may comprise one or more immobilization tags bonded to the polynucleotide to facilitate immobilization of the target-bait hybrid to a solid surface. The solid surface may be provided with a binding partner with a high specificity for the immobilization tag. In one embodiment, the immobilization tag and the binding partner bind reversibly, i.e. in a non-covalent manner. For example, in one embodiment, the immobilization tag comprises biotin and the binding partner comprises streptavidin. Examples of other such non-covalent immobilization tags known in the art include antibodies, monoclonal antibodies and tags typically used in protein purification such as FLAG tag or His-tag. In one embodiment, the immobilization tag and binding partner may bind irreversibly, i.e. in a covalent manner. In this embodiment, the reaction between the immobilization tag and binding partner preferably proceeds in a near stoichiometric manner. In one embodiment, the immobilization tag may comprise a terminal alkyne and the binding partner may comprise an azido moiety. In this embodiment, the terminal alkyne and the binding partner may undergo a copper(l) catalysed cycloaddition ("Click chemistry") to form a triazole. Other high efficiency reactions which are compatible with the polynucleotide backbone may be suitable and are known in the art.

Solid surface The solid surface may be any suitable material which can be surface modified to incorporate the binding partner to the immobilization tag. The solid surface may comprise beads of glass or plastic, for example polystyrene. In another embodiment, the solid surface may comprise magnetic beads which facilitate removal of bait and captured target of interest. Reference Sequences

The reference sequence may be a known sequence of the pathogen or batch of vaccine. Such sequences may be readily available in public depositories, or may be readily determined using a sample of the pathogen or vaccine and any known sequencing protocol. Alternatively, the reference sequence may be a sequence isolated from a sample obtained from a patient. The sample may be obtained from a patient infected with the pathogen. The sample may be obtained from a patient inoculated with the vaccine. The reference sequence may be a sequence isolated from a sample obtained at a first timepoint from a patient infected with the pathogen. The reference sequence may be a sequence isolated from a sample obtained at a first timepoint from a patient inoculated with the vaccine.

Multiplexed isolation of multiple pathogenic genomes

The method of the invention enables the simultaneous isolation of multiple pathogenic genomes of interest from a biological sample. Thus, in one embodiment, the biological sample may be contacted with a plurality of sets of pathogen-specific polynucleotides. In one embodiment, at least one set of baits may comprise polyribonucleotides and at least one set of baits may comprise polydeoxyribonucleotides. Thus, in one embodiment, the biological sample may be contacted with a plurality of sets of pathogen-specific polyribonucleotides and a plurality of sets of pathogen-specific polydeoxyribonucleotides. Each set of pathogen-specific polynucleotides may be provided with a different immobilization tag.

In one embodiment, each set of pathogen-specific polynucleotides may facilitate isolation of a different target pathogenic genome onto a different solid surface. In this embodiment, each solid surface is provided with a binding partner specific to one immobilization tag present on only one set of pathogen-specific polynucleotides. Thus, through binding of each different immobilization tag to its specific binding partner the different pathogenic genomes of interest can be isolated onto different solid surfaces.

For example, if a first pathogenic genome of interest is isolated onto a set of magnetic beads and a second pathogenic genome of interest is isolated onto a set of polystyrene or glass beads, a simple magnetic separation can remove the magnetic beads from the polystyrene or glass beads thereby isolating two different pathogenic genomes. However, it is also possible to isolate multiple different targets on the same solid surface and rely on the sequencing and mapping protocols to separate and identify the different targets.

Multiplexed Vaccine Isolation

The method of the invention may be used to simultaneously identify in a sample a viral or bacterial vaccine and one or more minority variants of the viral or bacterial vaccine. In this embodiment, a set of baits corresponding to the vaccine are provided with a first binding partner specific to one immobilization tag, and at least a second set of baits corresponding to at least a second variant of the vaccine are provided with a second binding partner specific to a second immobilization tag.

In an alternative embodiment, the method of the invention may be used to simultaneously identify in a sample a plurality of different viral or bacterial vaccines. In this embodiment, a set of baits corresponding to the first vaccine are provided with a first binding partner specific to one immobilization tag, and at least a second set of baits corresponding to the second vaccine are provided with a second binding partner specific to a second immobilization tag.

Thus, through binding of each different immobilization tag to its specific binding partner the different genomes of interest can be isolated onto different solid surfaces. It will be readily understood that the number of different variants that may be isolated is limited only be the number of specific binding partner/immobilization tags available. However, it is also possible to isolate multiple different targets on the same solid surface and rely on the sequencing and mapping protocols to separate and identify the different variants. Multiplexed Host/Pathogen Genome Isolation

The method of the invention may be used to simultaneously identify in a sample a particular pathogen and a host genetic marker which is useful in predicting a patient's response to a particular treatment for the pathogen in question. The method of the invention may be used to simultaneously isolate and sequence an entire host genome and a pathogenic genome.

In this aspect, a set of host-specific polynucleotide baits are provided along with the set of pathogen-specific polynucleotide baits. In this way, the host gene or genomic region of interest is isolated along with the genome of the pathogen of interest. Sequencing of the host gene or genomic region of interest allows determination of the presence or absence of an SNP of interest, which can be used as a guide to selecting an appropriate treatment regime for the pathogen of interest.

In one embodiment of this aspect of the invention, the set of host-specific polynucleotide baits may comprise a set of polyribonucleotide baits and the set of the pathogen-specific polynucleotide baits may comprise a set of polydeoxyribonucleotides. Alternatively, the set of host-specific polynucleotide baits may comprise a set of polydeoxyribonucleotide baits and the set of the pathogen- specific polynucleotide baits may comprise a set of polyribonucleotides. Methods of the Invention

The methods of the invention make use of two specific binding interactions to isolate a genome of interest. Firstly, by providing a bait in the form of a set of polynucleotides which are complementary to one strand of the genome of interest, a strong interaction occurs through hybridization of the two strands to each other.

Secondly, the hybridized bait/target complex can be immobilized on the solid surface due to the presence of the immobilization tag on the bait and of the binding partner on the solid surface.

The set of polynucleotides may be designed to span an entire genome or a region of interest using software known in the art, for example the eArray software provided by Agilent Technologies. Preferably, the set of polynucleotides comprises a plurality of overlapping polynucleotides. In one embodiment, the set of polynucleotides provides 2x coverage of the genomic region of interest. Preferably, the set of polynucleotides provides at least 2x coverage, for example at least 5x coverage of the genomic region of interest. In one embodiment, the set of polynucleotides provides at least 10x coverage, for example at least 100x coverage, for example 1000x coverage of the genomic region of interest.

A sample may undergo one or more pre-treatment steps as outlined previously. It will be understood that these do not necessarily fall within the scope of the invention but may provide advantages for later manipulation of the isolated genome of interest.

The sample is then hybridised with the set of pathogen-specific polynucleotides or vaccine-specific polynucleotides and/or the set of host gene-specific polynucleotides under conditions suitable to promote hybridisation.

The hybridised target-bait complex is then contacted with the solid surface and becomes immobilized on that solid surface due to the specificity of the binding between the immobilization tag and the binding partner.

A simple wash then removes all other material in the sample, for example unwanted host DNA, leaving the target pathogenic or vaccine DNA bound to the solid surface. Thus, the methods of the invention advantageously allow the isolation and enrichment of a pathogenic or viral or bacterial vaccine genome of interest and/or simultaneous isolation of a host marker directly from a sample comprising a complex mixture of host genomic material and pathogenic or vaccine genomic material, even when the host genomic material is in vast excess. Preferably, the sets of polynucleotide baits are ribopolynucleotides. In this embodiment, the RNA bait can be selectively digested by any known means to leave only the target DNA present in the sample.

If the amount of pathogenic or vaccine genomic material present in the sample is high, the enriched target DNA isolated in this manner can be directly used in a sequencing protocol. In an alternative embodiment in which the amount of initial target DNA was low, the isolated and enriched target DNA may be subjected to a few rounds of PCR amplification in order to provide sufficient material for a particular sequencing protocol.

The number of rounds of PCR amplification (if required) necessary for this step is dictated by the required starting amounts for a given sequencing protocol. Prior art methods of amplifying viral nucleic acid for sequencing require a minimum of at least thirty cycles. In contrast, far fewer rounds of amplification are required following the method of the invention. For example, the enriched nucleic acid may be subjected to less than 16 rounds of PCR, for example less than 10 rounds of PCR. It is expected that as sequencing technologies evolve and improve, smaller and smaller amounts of starting nucleic acid will be required for each sequencing run. As such, it will be readily recognised that this amplification step post-enrichment will not always be required, even if the starting amount of pathogen or vaccine nucleic acid in the sample is low.

However, as has previously been stated, PCR amplification, or amplification by cell culture can result in errors or mutations appearing in the viral genome, such that single nucleotide variances are not detectable during the deep sequencing. As an alternative, whole genome amplification methodologies are used in place of PCR amplification or cell culture. Whole genome amplification of sample nucleic acid may use the Phi29 polymerase. Kits for performing the methods

The kit for performing the methods according to the invention may comprise one or more sets of pathogen-specific or viral or bacterial vaccine-specific polynucleotides provided with immobilization tags as previously described. The kit may comprise a set of host-specific polynucleotides. The kit may comprise at least one solid phase provided with a binding partner specific to the immobilization tag. The kit may comprise an instruction manual for carrying out the method.

For performing a multiplexed method of the invention, the kits may comprise a plurality of different solid phases with each solid phase provided with a different binding partner specific for a particular immobilization tag. For example, the kit may comprise one solid phase comprising magnetic beads provided with a first binding partner and a second solid phase comprising controlled pore glass beads provided with a second binding partner.

Sequencing Sequencing of the enriched genomic material, for example the isolated pathogenic genome or viral or bacterial vaccine genomic region of interest may be carried out by any method known in the art.

In one embodiment, the pathogenic genome, viral vaccine genome, bacterial vaccine genome or host genomic region of interest may be sequenced by a paired-end sequencing method. In this embodiment the sample may be subjected to a pre- treatment step in which standard primers are ligated to each end of a fragment of the sample.

Definitions

As used herein, the term "prepared or isolated from" when used in reference to a nucleic acid "prepared or isolated from" a pathogen or vaccine refers to both nucleic acid isolated from a virus or other pathogen, and to nucleic acid that is copied from a virus, e.g., by a process of reverse-transcription or DNA polymerization using the viral nucleic acid as a template. The nucleic acid of the pathogen may be isolated from a sample in conjunction with host nucleic acid. An "isolated" or "purified" sequence may be in a cell free solution or placed in a different cellular environment. The terms "isolated" or "purified" do not imply that the sequence is the only nucleotide present, but that it is essentially free (about 90-95%, up to 99-100% pure) of non-nucleotide or non-polynucleotide material naturally associated with it.

As used herein the terms "host", "patient" and "individual" are used interchangeably and refer to any organism which has been infected with a pathogen. A host may be a vertebrate, for example a mammal, including but not limited to a human.

As used herein the terms "host gene of interest" or "host genomic region of interest" refer to any genetic marker which provides information regarding susceptibility to a particular disease state. This may be a variation such as a mutation or alteration in the genomic loci that can be observed. For example, this may be a short DNA sequence, such as a sequence surrounding a single base-pair change (single nucleotide polymorphism, SNP), or a long sequence such as a minisatellite. As used herein the term "pathogen" refers to an organism, including a microorganism, which causes disease in another organism (e.g., animals and plants) by directly infecting the other organism, or by producing agents that causes disease in another organism (e.g., bacteria that produce pathogenic toxins and the like). As used herein, pathogens include, but are not limited to bacteria, protozoa, fungi, nematodes, viroids and viruses, or any combination thereof, wherein each pathogen is capable, either by itself or in concert with another pathogen, of eliciting disease in vertebrates including but not limited to mammals, and including but not limited to humans. As used herein, the term "pathogen" also encompasses microorganisms which may not ordinarily be pathogenic in a non-immunocompromised host. Specific non-limiting examples of viral pathogens include Varicella Zoster Virus (VZV), Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpes virus (KSHV), HSV1 , HSV2, CMV, HHV6, HHV7, hepatitis B, hepatitis C, adenovirus, JVC and BKV.

"Bacteria", or "Eubacteria", refers to a domain of prokaryotic organisms. Bacteria include at least 11 distinct groups as follows: (1) Gram-positive (gram+) bacteria, of which there are two major subdivisions: (i) high G+C group (Actinomycetes, Mycobacteria, Micrococcus, others) (ii) low G+C group (Bacillus, Clostridia, Lactobacillus, Staphylococci, Streptococci, Mycoplasmas); (2) Proteobacteria, e.g., Purple photosynthetic+non-photosynthetic Gram-negative bacteria (includes most "common" Gram-negative bacteria); (3) Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes and related species; (5) Planctomyces; (6) Bacteroides, Flavobacteria; (7) Chlamydia; (8) Green sulfur bacteria; (9) Green nonsulfur bacteria (also anaerobic phototrophs); (10) Radioresistant Inicrococci and relatives; (11) Thermotoga and Thermosipho thermophiles.

"Gram-negative bacteria" include cocci, nonenteric rods, and enteric rods. The genera of Gram-negative bacteria include, for example, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium;

"Gram-positive bacteria" include cocci, nonsporulating rods, and sporulating rods. The genera of Gram-positive bacteria include, for example, Actinomyces, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, and Streptomyces.

As used herein, the terms "vaccine" refers to a biological preparation comprising weakened or completely inactivated forms of a disease-causing microorganism used to inoculate an individual to prevent later infection with that disease. The vaccine may be a virus, for example the varicella vaccine against chickenpox, or the vaccine may be bacterial, for example the BCG vaccine against tuberculosis. The vaccine may be a monoclonal vaccine, or the vaccine may comprise multiple isoforms of the vaccine. The vaccine may be prophylactic or therapeutic. As used herein, the term "adverse-vaccine reaction" refers to a situation in which an individual is inoculated with a vaccine and subsequently displays some form of adverse reaction to the vaccine, for example a vaccine rash, due to an "adverse-vaccine reaction event".

As used herein, the term "adverse-vaccine reaction event" refers to that event which results in an adverse-vaccine reaction. For example, an "adverse-vaccine reaction event" may refer to the selection of a minority isoform present in the original vaccine due to evolutionary pressures such that that minority variant establishes itself in the individual. Alternatively, the "adverse-vaccine reaction event" may be a denovo mutation in the vaccine genomic material, post-inoculation, or in the genomic background of the individual. As used herein, the term "sample" refers to a biological material which is isolated from its natural environment and contains a polynucleotide. A sample according to the methods described here, may consist of purified or isolated polynucleotide, or it may comprise a biological sample or clinical sample such as a tissue sample, a biological fluid sample, or a cell sample comprising a polynucleotide. A biological fluid includes, but is not limited to, blood, plasma, sputum, urine, cerebrospinal fluid, lavages, and leukophoresis samples, for example.

As used herein, the term "bait" refers to a polynucleotide which is complementary to one strand of the pathogenic genome of interest. The term "bait" may also refer to a polynucleotide which is complementary to one strand of a host genomic region of interest. The polynucleotide may be a ribopolynucleotide or a deoxyribopolynucleotide. The polynucleotide will have sufficient complementarity to one strand of the pathogenic genome or host gene of interest such that the bait is able to hybridise with that strand to form a duplex. The polynucleotide may not have 100% complementarity so long as it is able to hybridise to the target. "Hybridisation conditions" as used herein are the conditions that allow two complementary strands of nucleic acid to anneal together to form a double stranded nucleic acid. It is understood that this can be effected under a range of conditions (e.g., nucleic acid concentrations, temperatures, buffer concentrations). It is also understood that multiple temperatures may be required. Conditions that promote hybridisation need not be identical for all baits and targets in a mix, and hybridisation may still occur under suboptimal conditions.

Primer pair "capable of mediating amplification" is understood as a primer pair that is specific to the target, has an appropriate melting temperature, and does not include excessive secondary structure. The design of primer pairs capable of mediating amplification is within the ability of those skilled in the art. "Conditions that promote amplification" as used herein are the conditions for amplification provided by the manufacturer for the enzyme used for amplification. It is understood that an enzyme may work under a range of conditions (e.g., ion concentrations, temperatures, enzyme concentrations). It is also understood that multiple temperatures may be required for amplification (e.g., in PCR). Conditions that promote amplification need not be identical for all primers and targets in a reaction, and reactions may be carried out under suboptimal conditions where amplification is still possible.

As used herein, the term "amplified product" refers to polynucleotides that are copies of a particular polynucleotide, produced in an amplification reaction. An "amplified product," according to the invention, may be DNA or RNA, and it may be double- stranded or single-stranded. An amplified product is also referred to herein as an "amplicon".

As used herein, the term "amplification" or "amplification reaction" refers to a reaction for generating a copy of a particular polynucleotide sequence or increasing the copy number or amount of a particular polynucleotide sequence. For example, polynucleotide amplification may be a process using a polymerase and a pair of oligonucleotide primers for producing any particular polynucleotide sequence, i.e., the whole or a portion of a target polynucleotide sequence, in an amount that is greater than that initially present. Amplification may be accomplished by the in vitro methods of the polymerase chain reaction (PCR). See generally, PCR Technology: Principles and Applications for DNA Amplification (R. A. Erlich, Ed.) Freeman Press, NY, NY (1992); PCR Protocols: A Guide to Methods and Applications (Innis et al., Eds.) Academic Press, San Diego, CA (1990); Mattila et al., Nucleic Acids Res. 19: 4967 (1991); Eckert et al., PCR Methods and Applications 1 : 17 (1991); PCR (McPherson et ai. Ed.), IRL Press, Oxford; and U. S. Patent Nos. 4,683,202 and 4,683, 195, each of which is incorporated by reference in its entirety.

Other amplification methods include, but are not limited to: (a) ligase chain reaction (LCR) (see Wu and Wallace, Genomics 4: 560 (1989) and Landegren et al., Science 241 : 1077 (1988); (b) transcription amplification (Kwoh et al., Proc. Nati. Acad. Sci. USA 86: 1 173 (1989); (c) self-sustained sequence replication (Guatelli et al., Proc. Nati. Acad. Sci. USA, 87: 1874 (1990); and (d) nucleic acid based sequence amplification (NABSA) (see, Sooknanan, R. and Malek, L, Bio Technology 13: 563-65 (1995), each of which is incorporated by reference in its entirety.

As used herein, a "target polynucleotide" (including, e.g., a target RNA or target DNA) is a polynucleotide to be analyzed. A target polynucleotide may be isolated or amplified before being analyzed using methods of the present invention. For example, the target polynucleotide may be a fragment of a whole genome of interest. A target polynucleotide may be RNA or DNA (including, e.g., cDNA). A target polynucleotide sequence generally exists as part of a larger "template" sequence; however, in some cases, a target sequence and the template are the same. As used herein, a "target-specific polynucleotide", for example a "pathogen-specific polynucleotide", a "host-specific polynucleotide" or "vaccine-specific polynucleotide" means specific to that particular target, as opposed to an alternative target, for example a different pathogen, host or vaccine. The target-specific polynucleotide may be fully complementary to the target polynucleotide, or it may be only partially complementary to the target polynucleotide. It will be understood that if the target-specific polynucleotide is only partially complementary, that it will still have sufficient complementarity in order for it to bind to and isolate the target polynucleotide.

As used herein, an "oligonucleotide primer" refers to a polynucleotide molecule (i.e., DNA or RNA) capable of annealing to a polynucleotide template and providing a 3' end to produce an extension product that is complementary to the polynucleotide template. The conditions for initiation and extension usually include the presence of four different deoxyribonucleoside triphosphates (dNTPs) and a polymerization-inducing agent such as a DNA polymerase or reverse transcriptase activity, in a suitable buffer ("buffer" includes substituents which are cofactors, or which affect pH, ionic strength, etc.) and at a suitable temperature. The primer as described herein may be single- or double- stranded. The primer is preferably single-stranded for maximum efficiency in amplification.

"Primers" may be less than or equal to 100 nucleotides in length, e.g., less than or equal to 90, or 80, or 70, or 60, or 50, or 40, or 30, or 20, or 15, but preferably longer than 10 nucleotides in length. The term "nucleotide" or "nucleic acid" as used herein, refers to a phosphate ester of a nucleoside, e.g., mono, di, tri, and tetraphosphate esters, wherein the most common site of esterification is the hydroxyl group attached to the C-5 position of the pentose (or equivalent position of a non-pentose "sugar moiety"). The term "nucleotide" includes both a conventional nucleotide and a non-conventional nucleotide which includes, but is not limited to, phosphorothioate, phosphite, ring atom modified derivatives, and the like, e.g., an intrinsically fluorescent nucleotide.

As used herein, the term "conventional nucleotide" refers to one of the "naturally occurring" deoxynucleotides (dNTPs), including dATP, dTTP, dCTP, dGTP, dUTP, and dITP.

As used herein, the term "non-conventional nucleotide" or "unnatural nucleotide" refers to a nucleotide which is not a naturally occurring nucleotide. The term "naturally occurring" refers to a nucleotide that exists in nature without human intervention. In contradistinction, the term "non-conventional nucleotide" refers to a nucleotide that exists only with human intervention. A "non-conventional nucleotide" may include a nucleotide in which the pentose sugar and/or one or more of the phosphate esters is replaced with a respective analog. Exemplary pentose sugar analogs are those previously described in conjunction with nucleoside analogs.

Exemplary phosphate ester analogs include, but are not limited to, alkylphosphonates, methylphosphonates, phosphoramidates, phosphotriesters, phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoroanilidates, phosphoroamidates, boronophosphates, etc., including any associated counterions, if present. A non-conventional nucleotide may show a preference of base pairing with another artificial nucleotide over a conventional nucleotide (e.g., as described in Ohtsuki et al. 2001 , Proc. Nat!. Acad. Sci., 98 : 4922-4925, hereby incorporated by reference). The base pairing ability may be measured by the T7 transcription assay as described in Ohtsuki et al. (supra). Other non-limiting examples of "artificial nucleotides" may be found in Lutz et al. (1998) Bioorg. Med. Chern. Lett., 8 : 1 1491152); Voegel and Benner (1996) Helv. Chim. Acta 76, 1863-1880; Horlacher et al. (1995) Proc. Natl. Acad. Sci., 92: 6329-6333; Switzer ef al. (1993), Biochemistry 32: 10489-10496; Tor and Dervan (1993) J. Am. Chem. Soc. 1 15: 4461-4467; Piccirilli et al. (1991) Biochemistry 30: 10350-10356; Switzer et al. (1989) J. Am. Chem. Soc. 11 1 : 8322-8323, all of which hereby incorporated by reference. A "non-conventional nucleotide" may also be a degenerate nucleotide or an intrinsically fluorescent nucleotide.

A "non-conventional nucleotide" or "unnatural nucleotide" may refer to a nucleotide in which the nucleobase has been modified so that substituents can be incorporated into the polynucleotide. Typical nucleobase modifications include substitutions at the 5- position of the naturally occurring pyrimidines uracil, thymine and cytosine, or at the 7- or 8-positions of the naturally occurring purines adenine and guanine.

As used herein, a "polynucleotide" or "nucleic acid" generally refers to any polyribonucleotide or poly-deoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotides" include, without limitation, single- and double-stranded polynucleotides. The term "polynucleotides" as it is used herein embraces chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including for example, simple and complex cells. A polynucleotide useful for the present invention may be an isolated or purified polynucleotide or it may be an amplified polynucleotide in an amplification reaction.

As used herein, the term "set" refers to a group of at least two. Thus, a "set" of polynucleotide baits comprises at least two polynucleotide baits. In one aspect, a "set" of polynucleotide baits refers to a group of baits sufficient to span a genomic region of interest.

As used herein, "a plurality of" or "a set of" refers to more than two, for example, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more 10 or more etc.

As used herein, the term "cDNA" refers to complementary or copy polynucleotide produced from an RNA template by the action of an RNA-dependent DNA polymerase activity (e.g., reverse transcriptase).

As used herein, "complementary" refers to the ability of a single strand of a polynucleotide (or portion thereof) to hybridize to an anti-parallel polynucleotide strand (or portion thereof) by contiguous base-pairing between the nucleotides (that is not interrupted by any unpaired nucleotides) of the anti-parallel polynucleotide single strands, thereby forming a double-stranded polynucleotide between the complementary strands. A first polynucleotide is said to be "completely complementary" to a second polynucleotide strand if each and every nucleotide of the first polynucleotide forms base-pairing with nucleotides within the complementary region of the second polynucleotide.

A first polynucleotide is not completely complementary (i.e., partially complementary) to the second polynucleotide if one nucleotide in the first polynucleotide does not base pair with the corresponding nucleotide in the second polynucleotide. The degree of complementarity between polynucleotide strands has significant effects on the efficiency and strength of annealing or hybridization between polynucleotide strands.

Brief Description of the Figures

The present invention will now be described, by way of example only and without limitation, with reference to the following Figures and Examples, in which: Figure 1 depicts a map of the VZV genome. Arrows denote SNPs under natural selection that have been identified using the methods of the present invention, colour denotes presence in zoster- or varicella-like rashes, direction indicates whether selection is for parental or vaccine allele;

Figure 2 includes Table 1 , summarising in tabulated form the data shown in Figure 1 ; Figure 3 shows comparative analyses of allele frequencies at polymorphic sites between sequencing samples prepared using different methodologies from the same source DNA. Comparisons are as follows: (A) Direct sequencing (no enrichment) vs. SureSelect (enrichment); (B) Whole genome amplification and SureSelect vs. SureSelect; (C) Whole genome amplification followed by direct sequencing vs. SureSelect; and (D) SureSelect vs. SureSelect (repeat);

Figure 4 shows scatter plots comparing vaccine allele frequencies at 233 polymorphic sites (panels A-C) with high correlation scores. Similarly, the mean vaccine allele frequency in vaccine batches correlates well with the mean vaccine allele frequency derived by averaging across all sequenced rashes (panel D). Figure 5 shows that culturing of vaccine rash strains reduces the number of polymorphic sites, while vaccine batches have significantly more polymorphic sites than associated rashes. Data points labelled in grey indicate samples that have been sequenced on both platforms. Horizontal lines indicate the mean of each subset. Error bars represent 1 S.E. * p < 0.001. Data taken from (A) GAIIx sequencing dataset and (B) MiSeq sequencing dataset.

Examples

Example 1

Materials and Methods Clinical Sample collection

Clinical samples in the form of vesicle fluid were obtained from patients who had received the Varivax vaccine and subsequently developed a vaccine rash. Written consent was obtained in all cases. Samples were taken from four individuals who had been vaccinated using the vaccine and subsequently developed Varicella-like vaccine rashes ("Var 1", "Var 2" and "Var 3"), and from five individuals who had been vaccinated using the vaccine and subsequently developed Zoster-like vaccine rashes ("Zos 1", "Zos 2", "Zos 3", "Zos 4" and "Zos 5"). All clinical samples contained a mixture of patient genomic material and vaccine rash genomic material.

Vaccine batches Three different batches (Batch numbers NL131 10, G007544 and 1526X, termed "Vacc 1", "Vacc 2" and "Vacc 3" respectively) of the Varivax vaccine from Merck were used in the study as reference sequences. The vaccine sequences were determined by known methods in the art.

Sample preparation Total DNA was extracted from clinical samples using a QiaAmp DNA mini kit (Qiagen) according to manufacturer's instructions. Total DNA quantities were determined by NanoDrop and those with a 260/280 ratio outside the range 1.9 - 2.1 were further purified using the Zymoclean Genomic DNA Clean & Concentrator™ (Zymo Research Corp.).

Whole genome amplification

All clinical samples were amplified (10ng starting DNA) using Genomiphi V2 (GE Healthcare) and purified using Zymoclean Genomic DNA Clean & Concentrator™ (Zymo Research Corp.), both according to manufacturer's instructions.

RNA bait design

Overlapping 120-mer RNA baits (generating a 2x coverage for VZV) spanning the length of the positive strand of the reference genomes were designed using in house Perl scripts for VZV and Agilent eArray software (https://earray.chem. agilent.5 com/earray/). A further 552 control baits were designed against a 16 kbp region of the Salmo trutta trutta mitochondrion (NC_010007). The specificity of all baits was verified by BLASTn searches against the Human Genomic + Transcript database. Bait libraries for VZV were uploaded to E-array and synthesised by Agilent Biotechnologies. Library preparation, hybridisation and enrichment

DNA preparations of 2-3 μg were sheared for 6 x 60 seconds using a Covaris E210 (duty cycle 10%, intensity 5 and 200 cycles per burst using frequency sweeping). Prior to enrichment for viral DNA (by hybridisation) all samples were prepped for paired-end lllumina sequencing. Here, the samples were pre-treated by an end repair, addition of 3'polyA, and adaptor ligation, according to the Agilent Technologies SureSelect lllumina Paired-End Sequencing Library protocol (Version 1.0) http://www.genomics.agilent.com/files/Manual/G4458-

90000_SureSelect_DNACapture.pdf; or available from Agilent Technologies) observing all recommended quality control steps. Hybridisation to the bait libraries, enrichment PCR and all post-reaction cleanup steps were performed according to the same protocol.

Whole genome sequencing

Sample multiplexing (2 - 7 samples per lane on an 8 lane flow cell) cluster generation and sequencing was conducted using an lllumina Genome Analyzer llx and lllumina HiSeq (lllumina Inc.) at UCL Genomics (UCL, London, UK) or Wellcome Trust Sanger Institute (Hinxton, UK). Base calling and sample demultiplexing were performed using the standard lllumina pipeline (CASAVA 1.7) producing paired FASTQ files for each sample. For each data set, all read-pairs were subject to quality control using the QUASR pipeline (http://sourceforge.net/projects/quasr/) to first trim the 3' end of reads (to ensure the median Phred quality score of the last 15 bases exceeded 30) and subsequently to remove read-pairs if either read had a median Phred quality score below 30 or were less than 50 bp in length. Duplicate read-pairs were also removed. All remaining read-pairs were mapped to the reference genome (pOka) using the Burrows-Wheeler Aligner (maximum insert 50 bases, 25 maximum distance between paired ends 500) generating SAM files containing all mapped and unmapped reads. SAM files were subsequently processed using SAMTools to produce pileup files for consensus sequence generation and SNP calling using VarScan v2.2.8 (~min- coverage 3, ~min-reads2 3, -p-value 5e-02).

Identification of segregating sites

Using a dataset comprising the 3 vaccines and 12 vaccine rashes, segregating sites were identified under the following conditions.

1 : The variant must be present in 2 or more samples 2: The variant must appear on two independent reads

3: The minimum total read depth at a segregating site should be > 50

4: The variant allele (if present) should be the same in all samples.

Modelling selection and drift

The Balding-Nichols model is a population genetics model that describes the distribution of allele frequencies in sub-divided populations - given a background allele frequency p the allele frequencies in the population is assumed to be drawn from a beta distribution:

Where q is 1-p and F is a parameter related to genetic drift. To identify those sites where a change in the variant allele frequency from the vaccine to the vaccine rash may have occurred under natural selection, we applied a beta regression of the vaccine rash allele frequencies on the mean vaccine allele frequencies (with a logit link function and the mean vaccine as an offset). Outliers from the regression were identified by plotting the standardised weighted residuals ("sweighted2") recommended by Espinheira et al (Journal of Applied Statistics, 2008 35(4), 407-419) and identifying those sites where exceeded +1-2. The beta regression was then repeated for each individual vaccine rash with the outliers removed and the magnitude of genetic drift inferred from the variance around the mean of the fitted distribution. All beta regressions were carried out using the R betareg package (Cribari-Neto et al Journal of Statistical Software, 2010 34(2), 1-24.).

The samples were treated according to the protocols set out above and subsequently analysed using the Balding-Nichols model. The present invention allows the identification of patterns of natural selection and genetic drift that are associated with adverse reactions to vaccines. Such patterns have not previously been identifiable with existing methods. Indeed, using the methods of the invention in this Example, it has been possible to identify a further nine SNPs in vaccine rash samples that have not been reported previously. While many alleles have changed substantially but inconsistently across rash groups, the methods of the present invention identified sites showing consistent changes in all varicella-like and/or zoster-like rashes. Figures 1 and 2 indicate sites that have been identified across the different rash samples. These include:

1) Fixation of the vaccine allele at 58595 in ORF31 (gB) is consistent across all rashes; 2) Independent non-synonymous coding changes in ORF 55 (function) for both rash types;

3) Additional non-synonymous coding changes observed in ORFs 7 (function), 20 (function) and 31 (gB) for varicella-like rashes; 4) Additional non-synonymous coding changes observed in ORFs 13 (function), 50 (function) and 59(function) for zoster-like rashes;

5) Of note is the relative lack of sites in ORF 62 (function) that appear under natural selection. Example 2

Sample collection and ethics

Diagnostic samples from patients with confirmed vaccine-associated rashes (both Varicella- (n=8) and Zoster-like (n=12)) collected in the UK and USA between 1988 and 2010, were retrieved from the Breuer lab cryobank. The clinical specimens, collected as part of standard clinical practice to genotype vaccine rashes occurring in recipients of vOka vaccine, were independently obtained from patients who developed skin rashes following immunization with the Merck vOka vaccine. All rashes were confirmed as vOka strain using previously published methods [8,9]. Rashes occurring within 42 days of vaccination were classified as varicella-like. The exception was one case, T61 , which was reported as a varicella-like rash 90 days post-vaccination. While this was thought to be more likely to be disseminated zoster, the case was included as varicella-like. Cases presenting with the classical dermatomal rash were diagnosed clinically as herpes zoster. Samples were stripped of all personal identifiers other than details of vaccination and the type of rash, no patient information was available. The use of these specimens for research was approved by the East London and City Health Authority Research Ethics Committee (P/96/046: Molecular typing of cases of varicella zoster virus).

In addition, three batches of the Varivax live-attenuated VZV Vaccine preparation were collected in 2010 (VV10 (UK) and WAG (USA)) and 2012 (VV12) and batch and lot numbers used to verify them as independent preparations (Table 2).

Cell culture

Cultured isolates (Table 2) were passaged in melanoma cells (MeWo) that were propagated in culture media (MEM (Sigma) supplemented with 10% FBS and 1 % nonessential amino acids). Viruses were harvested for DNA extraction 3-4 days post inoculation when syncytia was visible.

Library construction, targeted enrichment and sequencing

Total DNA was extracted from each sample using the QiaAMP DNA mini kit (QIAGEN) according to manufacturer's instructions. DNA quantification was performed using a NanoDrop spectrophotometer and those with 260/280 ratios outside the range 1.7 - 2.1 and 260/230 ratios out the range 1.8 - 2.2 were further purified using the Zymoclean Genomic DNA Clean & Concentrator™ (Zymo Research Corp.). Whole-genome amplification using GenomiPhi V2 (GE Healthcare) was performed using 10ng of starting material where < 50ng total DNA was available (Table 2). Libraries were constructed as per Example 1 using the standard SureSelect XT v1.3 (Agilent) and NEBNext (New England Biolabs) protocols, the latter modified to include a second PCR amplification step (6 cycles) to enable multiplexing using standard lllumina barcodes. Enrichment for VZV sequences was performed as described in Example 1. Samples were sequenced across several lllumina platforms (GAIIx, HiSeq and MiSeq) according to availability. Specific sample preparation and sequencing metrics are shown in Table 2.

Genome assembly and variant calling Each dataset was parsed through QUASR [10] for duplicate removal and read-trimming (-q 30, -I 50) and subsequently aligned against pOka (AB097933.1) using BWA [11]. Resulting alignments were processed using SAMTools [12] to generate pileup files for each sample. A consensus sequence for each dataset was called with the QUASR module 'pileupConsensus' and a 50% frequency threshold (i.e. no ambiguities were included). Variant profiling for each dataset was performed using VarScan v2.2.1 1 [13] with the following parameters: basecall quality≥ 20, read depth ≥ 50, independent reads supporting minor allele ≥ 2 per strand. In addition, variant calls showing directional strand bias ≥ 0.85 were excluded from further analyses. Consensus sequences were generated for each rash sample but iterative repeat regions (ORIS R1 , R2, R3, R4 and R5) and the terminal repeat region were trimmed prior to analysis. Consensus Sequence Analyses

DNA sequences were aligned using the program Mafft, v6 [14] with alignments checked manually; no insertions or deletions were inferred from the alignment. Prior to further analysis the VZV repeat regions R1 , R2, R3, R4, R5 and OriS [15, 16] were removed as these could not be accurately mapped given the limited DNA available. Bayesian phylogenetic trees were inferred using the program Beast, v1.7.2 [17]. Two independent Monte Carlo-Markov chains were run for 50,000,000 iterations for the whole genome (minus repeat regions) with a thinning of 1 ,000. The most appropriate model of nucleotide substitution was selected using the program jModelTest [18], which identified a general-time-reversible model of nucleotide substitution with a gamma- shaped rate distribution and a proportion of invariable sites (GTR + γ + I) as being the best site model. The phylogeny was reconstructed under a number of different tree priors including the Bayesian skyline, constant and exponential growth models. Runs were checked for convergence and that an effective sample size of at least 200 had been achieved for all parameters. Runs were combined using LogCombiner, v1.6.1 and TreeAnnotator, v1.6.1 , used to obtain the highest clade credibility tree and posterior probabilities per node. Neighbour-joined phylogenies were also inferred using Mega, v5.05 [19], under the same site model (GTR + γ + I). All trees were visualised and drawn using Figtree v1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/). The program BaTS [20] was used to test for an association between the bayes-inferred phylogeny and the vaccine rash phenotype (Varicella-like or Zoster-like). BaTS estimates the association using the association index [21 , parsimony score [22] and maximum monophyletic clade statistics.

Statistical Analyses To compensate for intrinsic differences in the sequencing technologies used [2-4], their respective error profiles and fold differences between the mean read depths of individual samples, we identified a minimal criterion for defining bi-allelic sites that segregate our samples (1) total read depth≥ 50, (2) the vaccine allele must appear≥ 2 independent reads mapping to each strand, (3) the vaccine frequency must be≥ 1 % and (4) the segregating site must appear in≥ 2 vaccines and/or vaccine rash samples at the above criteria. These criteria were applied only to data obtained using the MiSeq and HiSeq platforms and yielded a total of 368 sites that were segregating across the vaccines and vaccine rashes. The allele frequencies at these sites in the GAIIx derived data were subsequently included if the vaccine allele was a match (i.e. not a sequencing error). The frequency of the vaccine allele in vaccine rash samples at the majority of these sites was effectively binary, either 0% or 100%. To look for significant differences in the vaccine allele frequency between the vaccine and the vaccine rashes, i.e., sites where the change in allele frequency cannot be explained by random drift alone, we first identified the set of vaccine polymorphic sites. There were 224 sites that were polymorphic in two or more vaccines and where the vaccine allele occurred in≥2 reads on opposite strands with a read depth≥ 50. Sites that were polymorphic in just the one vaccine were excluded from further analysis. Subsequently, we calculated the mean vaccine allele frequency for each of these polymorphic sites in the three sequenced vaccines, VV10, VV12 and WAG. As all of these sites in the vaccine rashes were fixed for either the vaccine or the wildtype allele we counted the number of rashes with the vaccine allele. We estimated the effect size (based upon the mean vaccine allele frequency in the vaccines and the proportion of vaccine rashes fixed for the vaccine allele) and power [7], and identified those sites for which there was≥80% power to compare the vaccine rashes with the vaccine. Finally, we applied two-tailed binomial exact tests to those sites with≥80% power with the probability of "success" equal to the mean vaccine allele frequency in the vaccines. We corrected for multiple analyses using FDR [25].

We also used the power analyses to identify a set of the vaccine polymorphic sites that could be analyzed with SNP typing of an additional 36 vaccine rashes, which were readily available and/or the inclusion of previously published data [2,4,23]. Using binomial exact tests we compared the proportion of the vaccine allele at these sites in all of the vaccine rashes (sequenced, SNP typed and previously published) with the mean frequency in the sequenced vaccines (with a FDR correction for multiple analyses). In addition we re-analyzed those sites that we showed to have sufficient power from the sequencing (see above) with the addition of SNP typed and previously published data. Finally, we used generalized linear modeling with binomial error to identify any differences between varicella and zoster vaccine rashes at the 224 vaccine polymorphic sites. All binomial tests and the generalized linear modeling were performed in R (http://www.r-project.org/index.html). Effect size and power calculations were done using the "pwr" package in R. Selection of low-level de novo variants

Mutations acquired post-inoculation (i.e. de novo) were identified in datasets derived from the MiSeq under the following criteria; the variant allele occurred in≥2 reads on opposite strands with a read depth≥ 50 and a frequency exceeding 1 %. To identify if there was any selection operating upon the new mutations in the vaccine rashes we used the Kolmogorov-Smirnov test to compare the total number of non-synonymous mutations with the sum of the synonymous and non-coding mutations in all of the rashes.

Results Viral DNA was enriched and sequenced from all vaccine and vaccine-rash samples with a median of 87.3% on-target reads (Table 2). To determine whether any bias was associated with these methods, we prepared five sequence libraries (WGA only, SS only, an SS only replicate, WGA+SS and direct sequencing without WGA or SS - Table 4) from a single batch of VZV vaccine. The consensus sequences for each sample were identical, while the correlation of allele frequencies between libraries was high (Pearson's product moment R² > 0.93) at sites where total read depth was over 50 reads per base and where the variant allele was present on two or more independent reads mapping to opposite strands (Figure 3). Deviations in the variant allele frequency of up to 3.2% were observed but these were limited to comparisons between enriched and non-enriched libraries (Figure 3). This is explained by the fact that read depths in non-enriched libraries are -20 fold. A comparison of replicate samples that were independently enriched showed the standard deviation from the mean allele frequency i.e. the error of the method to be 0.654%.

Conservation of polymorphic sites in vaccine All three sequenced vaccine batches were heterogeneous with between 235 and 336 polymorphic sites being identified. Of these, 224 were present in two or more vaccine preparations (data not included) and 207 were shared between all three vaccine preparations, of which 36 had been previously identified by Sanger or pyro-sequencing [2-5,24]. Five sites (12694, 12779, 31732, 82225 and 106710) previously reported as polymorphic in Merck vaccine batches were fixed for the wild type allele in all batches sequenced here [1 ,2,3,24]. The vaccine allele frequencies at each of the 224 sites shared between vaccines were highly conserved (Pearson's product moment R² > 0.98), indicating minimal batch to batch variation at vaccine loci despite each having been issued in different years and in one case a different country (Table 2 and Figure 4). Therefore we infer that the vaccines sequenced in this study are genetically comparable to the batches used to inoculate the subjects whose rash viruses were analyzed. This is also supported by the high correlation (Pearson's product moment R²=0.83) between the mean allele frequencies in the vaccines and rash samples (Figure 4D).

Vaccine rashes are mono- or oligomorphic Between 32 and 1 12 polymorphisms were identified in the uncultured vaccine rashes, as compared with 235 - 336 in the vaccine batches. The lower genetic diversity of rashes can also be quantified by the proportion of polymorphic sites, being 0.19% for the vaccine, which is 2-4 times greater than rash diversity (0.03- 0.09%). \n-vitro tissue culture further reduced rash virus diversity even at≤ 3 passages (Figure 5). Of the polymorphisms in the vaccine rashes, 37% - 93% (mean 55%) were at the sites of 224 'vaccine SNPs' (loci polymorphic in all three vaccine samples). Vaccine-allele frequencies at the majority of these sites were lower than in the original vOka vaccines (data not shown).

These data are consistent with the conclusion that there exist one or more bottlenecks following inoculation of vaccine and the formation of rashes. There is a low level of polymorphism at novel loci which can be attributed to mutation since the bottleneck. The rashes sequenced here were fixed for the vaccine allele at four amino acid coding positions in ORF 62, 105705, 106262, 107252 and 10811 1 , and their presence may explain the mild presentation of most vaccine rashes compared with wild type rashes [6-7]. In addition to the 224 vaccine SNPs, we identified a further 39 polymorphic sites present in at least one vaccine and one vaccine rash and two present in two or more vaccine rashes (Table 5). Since we know that none of the rashes arose from the same vaccine batch (Table 2), the most likely explanation is that for these 41 sites, the vaccine allele is present in the original vaccine at frequencies that are at the limit of detection by deep sequencing and therefore are not always detected as polymorphic. De novo mutations occurring post-inoculation

Sixty-seven percent (258) of polymorphic sites in the rashes were due to new mutations that were not detected in the deep sequenced vaccine preparations (data not shown). These included 33 fixed substitutions of which 16 were non-synonymous and occurred in eight rashes (VR1 , VR2, A182B, L53, 027, T25, T17 and T61 - Table 3). The remaining new mutations were largely present at allele frequencies of 10% or less. The mutations in rash A182B, a highly passaged isolate, for which plenty of DNA was available, were confirmed by Sanger sequencing. In addition, there was sufficient material to re-sequence three uncultured blister samples of rashes, K1 1 , L53 and N13. Overall there was good correlation between the replicates with confirmation of 62/68 (91.2%) of the new mutations in these three rashes.

Selection for rash-forming alleles

We have shown the segregating allele frequencies in the Merck vOka vaccine are sufficiently non-variable between batches, that most of the vaccine alleles can be considered to have been present in the particular vaccine batch inoculated into patients (Figure 5). We carried out analyses to evaluate whether there was any consistent change in allele frequencies between vaccine and rashes. We used a generalized linear mixed model which showed a significant trend for the ancestral allele to increase in frequency at the expense of the novel vaccine allele, at non-synonymous sites (pO.001).

A second analysis was used to identify SNPs with the clearest evidence for selection: those at which the vaccine allele repeatedly decreased in frequency, which could indicate loci with a role in rash formation. Of the 224 vaccine polymorphic sites there was statistical power (≥80%) to perform a binomial analysis for seven: 59591 , 94167, 102002, 105356, 106001 , 107797 and 108838. At six of these sites, five non- synonymous and one non-coding, the vaccine allele decreased significantly in the rashes. The vaccine allele increased significantly at the remaining synonymous site (Table 6). Using rash genotyping data from the literature and results from SNP typing of material from 36 additional rashes, we confirmed there was selection of the ancestral allele for three of the five non-synonymous sites, 105356 and 107797 which have previously been reported [2], and 106001 , and the non-coding site 102002 in the ORF 60 promoter. We analyzed a further seven non-synonymous, four non-coding and one synonymous sites (560, 58595, 87306, 89734, 90318, 97479, 97748, 107599, 109137 and 109200) for which SNP typing data from the literature and/or genotyping of a further 36 rashes provided enough power to confirm a significant change in allele frequencies (Table 6). Of these, there was a significant decrease in the frequency of the vaccine allele at five non-synonymous sites: 560 (ORF 0), 58595 (ORF 31), 97748 (ORF 55), 97479 (ORF 55) and 105599 (ORF 62), two non-coding: 109137 (ORF 62 promoter) and 105169 (61/62 intergenic) and a significant increase in the vaccine allele at one synonymous site: 89734 in ORF 51 (Table 6). In addition to positions 105169, 105356 and 107797 which have previously been reported [2], three of the loci under selection, sites 106001 (K1045E) in ORF 62 domain IV, 102002 in the ORF60 promoter and 97479 (V495A) in ORF 55 have not previously been identified as polymorphic in the vaccine. None of the non-synonymous loci were associated with changes in silico in known or predicted HLA class 1 epitopes (data not shown). The availability of material from vOka varicella and zoster rashes together with the vaccine strain ancestral to both provided a unique opportunity to determine whether vOka rashes caused by viruses reactivating from latency differed genetically from those causing skin rashes directly, ie within 42 days following inoculation. We found no significant differences in allele frequencies between viruses directly causing rash post- vaccination and those which established latency before reactivating to cause a zoster- rash.

The present invention enables recovery of sufficient VZV DNA from rashes caused by the vOka vaccine for whole viral genome sequencing, so that variation levels can be accurately quantified to assess genomic populations. The present invention was used to investigate vOka rash formation and the results shed light on the pathogenesis of VZV and the live attenuated vaccine strain vOka.

The present invention enabled the identification of at least eight non-synonymous residues in four ORFs across the VZV genome that are likely important for replication of vOka and other VZV strains in the major target organ, skin. From our data we find no evidence for population bottlenecks or viral selection related to latency, or reactivation and no evidence that vOka evolves as a quasispecies. Taken together, we predict that while vOka I variation plays some role in the development of varicella-like rashes after inoculation, it plays little or no part in the pathogenesis of herpes zoster and related reactivation illnesses The failure to identify neurotropic vOka strains provides important data for current efforts to develop vaccines that do not establish latency.

Industrial Applicability These data demonstrate the suitability and accuracy of target capture technology for enriching very low quantities of viral vaccine nucleic acid from complex DNA populations where the host genome is in vast excess such that mutational events in the viral vaccine can be monitored at different time points.

The utility of the method is demonstrated by directly sequencing samples from vaccine rashes. These data provide a useful reference point for quality assurance of vaccine production lots.

The foregoing broadly describes the present invention without limitation to particular embodiments. Variations and modifications as will be readily apparent to those skilled in the art are intended to be within the scope of the invention as defined by the following claims.

_h V_i e rasaccos z % Country and Days to

%

ID Type Platform Coverage year of rash

OTR

at 50x vaccination formation

N13^' 83.7 97.6 USA-1998 unknown

VR1 89.4 99.9 Europe-2007 14

MiSeq

VR2 86.4 98.3 UK-2006 16 c

Ό VR3 uncultured 81.1 99.9 UK-2006 14 o

> VR4 71.0 99.9 UK-2010 unknown

TO 027^* GAiix 92.7 90.5 USA-1999 16 ω

o N13^* 92.3 96.8 USA-1998 unknown

'__

re

> A182B^* high- 93.4 99.6 USA-1988 16

HiSeq

A185B^* passage 97.2 99.5 USA-1988 21 ω

c VV10 HiSeq 66.8 99.9 UK-2010 N/A

Varivax

VV12 89.5 100.0 UK-2012 N/A batches MiSeq

WAG^* 88.1 99.9 USA-2009 N/A

K11 79.7 99.8 USA-1997 74

L53^' MiSeq 70.5 98.4 USA-1997 130

ZR1 91.0 99.3 UK-2006 330

T61 94.8 99.0 USA-2001 90

(A

re K11 uncultured 93.4 98.9 USA-1997 74 ω L53^*

c 97.2 99.4 USA-1997 130 o

o U14^* 73.1 98.2 USA-1997 > 1500

> K48^* 18.1 83.8 USA-1995 603

R73^* GAiix 9.6 98.0 USA-? unknown

R3^* 57.3 98.0 USA-1999 243

R52^* 94.8 98.7 USA-1999 489 low- T17^* passage 95.7 99.5 USA-1999 31 1 T25^* 60.4 99.4 USA-2000 546 v76 79.3 99.5 USA-1998 730

OTR- On-target reads (i.e. reads mapping to pOka reference genome)

* Underwent whole genome amplification prior to SureSelect

Sample in italics were sequenced on both MiSeq and GAiix platforms

Table 2 Position Wild Amino

Vaccine ORF Type Rash ID (Dumas) type Acid

89 T C N/A N/A non-coding T61

967 G A N/A N/A non-coding T61

6420 G A ORF 6 A720T non-synonymous T61

10540 C T ORF 8 A43V non-synonymous T25

12588 A G ORF 10 P143 non-synonymous T17

21595 C A ORF 15 T295K non-synonymous A182

23756 G T ORF 17 M13I non-synonymous A182

37959 G A ORF 22 D1293N non-synonymous T61

3811 1 A C ORF 22 T1343 non-synonymous T61

41323 G A ORF 22 E2468K non-synonymous L53

44265 T C ORF 25 137V non-synonymous VR1

51 190 G A ORF 29 A1 12T non-synonymous 027

51206 G A ORF 29 R1 17Q non-synonymous T61

53993 G A ORF 29 R1046Q non-synonymous T61

61629 C T ORF 33 S170 synonymous T61

6281 1 T c ORF 34 T273 synonymous K1 1

65074 c T ORF 36 Q90* non-synonymous T61

67603 A G ORF 37 S510 synonymous A185

72395 G A ORF 40 D286N non-synonymous T61

79183 G A ORF 43 V431 I non-synonymous VR2

80298 G T N/A N/A non-coding T17

81940 G A ORF 42 L218 synonymous T61

82225 A G ORF 42 P123 synonymous V76

98893 T C ORF 56 L109P non-synonymous T61

102976 c T N/A N/A non-coding R73

106497 T c ORF 62 G879 synonymous A182

109139 T c N/A N/A non-coding A182

109749 T G N/A N/A non-coding A185

109751 T G N/A N/A non-coding A185

109752 T G N/A N/A non-coding A185

109754 A G N/A N/A non-coding A185

109927 A G N/A N/A non-coding A182

1 17055 C A ORF 68 T416 synonymous R52

Table 3

Table 4

Vaccine pOka Variant

Position Coverage Frequency Vaccine Rash allele allele

VIP66 1053 A G 4102 99.88% VV12

K1 1 5437 C T 1532 1.76% WAG

K1 1 6488 C T 1305 2.76% WAG

K1 1 6904 C T 1675 1.01 % WAG

K1 1 7066 G A 1488 3.49% WAG

VIP70 14085 C A 417 4.66% VV12

N13 14676 C T 2904 1.79% VV10

VIP58 20624 C A 1814 3.47% VV10

VIP66 20624 C A 870 4.02% VV10

N13 20624 C A 1731 2.83% VV10

VIP58 20632 T A 1665 4.74% VV12

VIP66 20632 T A 805 5.09% VV12

N13 20632 T A 1506 7.17% VV12

K1 1 20632 T A 1867 5.03% VV12

N13 20644 T C 1348 2.08% VV10

K1 1 20750 A C 445 1.12% VV10

VIP66 20842 A T 506 2.37% VV10

VIP70 20842 A T 386 2.33% VV10

N13 22050 G A 3669 2.13% VV10

VIP58 23487 C T 1618 1.05% WAG

VIP66 24619 C T 3575 1.51 % VV10

N13 31061 G A 3601 1.17% VV12

K1 1 41394 T C 293 1.72% VV12

N13 50350 G A 886 6.54% VV10

VIP66 65904 C T 532 4.89% WAG

VIP70 71896 G A 723 1.94% WAG

N13 73648 G A 2912 4.19% VV10

K1 1 74177 C T 3699 1.11 % WAG

N13 74593 C A 1904 1 % WAG

L53 76498 G A 256 1.56% VV12

VIP66 77277 C T 632 1.11 % VV10

L53 83748 G A 194 98.45% VV10

VIP66 91402 C T 562 1.07% VV10

VIP66 102812 G A 300 98.67% VV10

K1 1 106012 G A 619 1.13% VV12

K1 1 108572 G A 929 1.29% VV12

K1 1 109225 G A 686 1.17% VV12

K1 1 1 10318 T G 28 24.14% VV12

K1 1 1 10320 T G 18 73.68% VV12

Table 5(a) Sites appearing in just one rash and one vaccine batch Vaccine pOka Variant

Position Coverage Frequency Rash allele allele

VIP70 1255 1.67%

15902 C T

L53 247 2.43%

N13 1606 1.49%

61891 G A

VIP66 653 1.68%

Table 5(b): Sites appearing in just two rashes but no vaccine batches

Sites Significant from the whole genome sequencing of Vaccine rashes

Table 6(a)

Table 6(b)

Table 6(c)

References

1. Loparev VN, Rubtcova E, Seward JF, Levin MJ, Schmid DS (2007) DNA sequence variability in isolates recovered from patients with postvaccination rash or herpes zoster caused by Oka varicella vaccine. The Journal of infectious diseases 195: 502-510. Available: http://www.ncbi.nlm.nih.gov/pubmed/17230409.

2. Quinlivan ML, Gershon A a, Al Bassam MM, Steinberg SP, LaRussa P, et al. (2007) Natural selection for rash-forming genotypes of the varicella-zoster vaccine virus detected within immunized human hosts. Proceedings of the National Academy of Sciences of the United States of America 104: 208-212. Available: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1765436&tool=pmcentrez&r endertype=abstract.

3. Tillieux SL, Halsey WS, Thomas ES, Voycik JJ, Sathe GM, et al. (2008) Complete DNA sequences of two oka strain varicella-zoster virus genomes. Journal of virology 82: 11023-11044. Available: http://www.pubmedcentral.nih. gov/articlerender.fcgi?artid=2573284&tool=pmcentrez&r endertype=abstract. Accessed 30 August 2011.

4. Loparev VN, Rubtcova E, Seward JF, Levin MJ, Schmid DS (2007) DNA sequence variability in isolates recovered from patients with postvaccination rash or herpes zoster caused by Oka varicella vaccine. The Journal of infectious diseases 195: 502-510. Available: http://www.ncbi.nlm.nih.gov/pubmed/17230409.

5. Kanda RK, Quinlivan ML, Gershon a a, Nichols R a, Breuer J (2011) Population diversity in batches of the varicella Oka vaccine. Vaccine 29: 3293-3298. Available: http://www.ncbi.nlm.nih.gov/pubmed/21349363. Accessed 14 December 2012.

6. Tsolia M, Gershon A a, Steinberg SP, Gelb L (1990) Live attenuated varicella vaccine: evidence that the virus is attenuated and the importance of skin lesions in transmission of varicella-zoster virus. National Institute of Allergy and Infectious Diseases Varicella Vaccine Collaborative Study Group. Journal of Pediatrics 116: 184- 189. 7. Cohen J I (2007) Varicella-zoster vaccine virus: evolution in action. Proceedings of the National Academy of Sciences of the United States of America 104: 7-8. Available:

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1765479&tool=pmcentrez&r endertype=abstract.

8. Larussa P, Steinberg S, Arvin A, Dwyer D, Burgess M, et al. (1998) Polymerase Chain Reaction and Restriction Fragment Length Polymorphism Analysis of Varicella- Zoster Virus Isolates from the United States and Other Parts of the World. Journal of Infectious Diseases 178: 64-66. 9. Parker SP, Quinlivan M, Taha Y, Breuer J (2006) Genotyping of Varicella-Zoster Virus and the Discrimination of Oka Vaccine Strains by TaqMan Real-Time PCR Genotyping of Varicella-Zoster Virus and the Discrimination of Oka Vaccine Strains by TaqMan Real-Time PCR. 44. doi: 10.1 128/JCM.00346-06.

10. Watson SJ, Welkers MRA, Depledge DP, Coulter E, Breuer JM, et al. (2013) Viral population analysis and minority-variant detection using short read next-generation sequencing. Philosophical Transactions B.

1 1. Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows- Wheeler transform. Bioinformatics (Oxford, England) 25: 1754-1760. Available: http://www.pubmedcentral.nih. gov/articlerender.fcgi?artid=2705234&tool=pmcentrez&r endertype=abstract.

12. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, et al. (2009) The Sequence Alignment/Map format and SAMtools. Bioinformatics (Oxford, England) 25: 2078-2079. Available:

http://www.pubmedcentral.nih. gov/articlerender.fcgi?artid=2723002&tool=pmcentrez&r endertype=abstract. Accessed 1 November 2012.

13. Koboldt DC, Zhang Q, Larson DE, Shen D, McLellan MD, et al. (2012) VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing. Genome research 22: 568-576. Available: http://www.pubmedcentral.nih. gov/articlerender.fcgi?artid=3290792&tool=pmcentrez&r endertype=abstract. Accessed 26 October 2012. 14. Katoh K (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Research 30: 3059-3066. doi: 10.1093/nar/gkf436.

15. Davison AJ, Scott JE (1986) The Complete DNA Sequence of Varicella-Zoster Virus. Journal of general virology: 1759-1816.

16. Hondo R, Yogo Y (1988) Strain variation of R5 direct repeats in the right-hand portion of the long unique segment of varicella-zoster virus DNA. 62.

17. Drummond AJ, Suchard MA, Xie D, Rambaut A (2012) Bayesian phylogenetics with BEAUti and the BEAST 1.7. Molecular biology and evolution. doi: 10.1093/molbev/mss075.

18. Posada D (2008) jModelTest: phylogenetic model averaging. Molecular biology and evolution 25: 1253-1256. doi: 10.1093/molbev/msn083.

19. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, et al. (201 1) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular biology and evolution 28: 2731-2739. doi: 10.1093/molbev/msr121.

20. Parker J, Rambaut A, Pybus OG (2008) Correlating viral phenotypes with phylogeny: accounting for phylogenetic uncertainty. Infection Genetics and Evolution 8: 239-246. 21. Wang TH, Donaldson YK, Brettle RP, Bell JE, Simmonds P (2001) Identification of shared populations of Human immunodeficiency Virus Type 1 infecting microglia and tissue macrophages outside the central nervous system. Journal of virology 75: 11686- 1 1699.

22. Slatkin M, Maddison WP (1989) A cladistic measure of gene flow measured from the phylogenies of alleles. Genetics 123: 603-613.

23. Thiele S, Borschewski A, Kuchler J, Bieberbach M, Voigt S, et al. (201 1) Molecular analysis of varicella vaccines and varicella-zoster virus from vaccine- related skin lesions. Clinical and vaccine immunology : CVI. Available: http://www.ncbi.nlm.nih.gov/pubmed/21562115. Accessed 20 June 2011.

24. Gomi Y, Sunamachi H, Mori Y, Nagaike K, Takahashi M, et al. (2002) Comparison of the Complete DNA Sequences of the Oka Varicella Vaccine and Its Parental Virus. Society 76: 1 1447-1 1459. doi: 10.1128/JVI.76.22.1 1447.

25. Hochberg Y, Benjamini Y. More powerful procedures for multiple significance testing; Stat Med. 1990 Jul;9(7):81 1-8.

Claims

Claims

1. A method of quality controlling a batch of vaccine, the method comprising: a) providing at least one set of vaccine-specific polynucleotides each comprising an immobilization tag; b) contacting under hybridising conditions the at least one set of vaccine- specific polynucleotides with a sample obtained from a patient who has been inoculated with the batch of vaccine; c) exposing the mixture from b) to a solid surface provided with a binding partner specific to the immobilization tag to isolate vaccine genomic material hybridised to the vaccine-specific polynucleotides; d) sequencing at least part of the vaccine genomic material from step c); e) determining the identity of at least one polymorphic position in the vaccine genomic material from step c); wherein a mutation in the at least one polymorphic position in the vaccine genomic material relative to at least one reference sequence for that batch of vaccine is indicative that a variant of the vaccine has been selected for.

2. The method of claim 1 , wherein the vaccine is a viral vaccine.

3. The method of claim 1 , wherein the sample is obtained from a vaccine rash.

4. The method of any one of claims 1 to 3, wherein the sample is obtained up to two weeks after inoculation.

5. The method of any one of claims 1 to 3, wherein the sample is obtained at least two weeks after inoculation.

6. The method of claim 5, wherein the sample is obtained at least four weeks after inoculation.

7. The method of any one of claims 1 to 6, wherein the variant comprises a minority isoform of the vaccine present in the batch of vaccine.

8. The method of any one of claims 1 to 6, wherein the variant comprises a denovo mutation in the vaccine genomic material relative to the at least one reference sequence.

9. The method of any one of claims 1 to 8, wherein the method further comprises subjecting the sample to a pre-treatment step before contacting it under hybridising conditions with the set of vaccine-specific polynucleotides.

10. The method of claim 9, wherein the pre-treatment step comprises fragmenting the sample.

1 1. The method of any one of claims 1 to 10, wherein the vaccine is a RNA viral vaccine.

12. The method of any one of claims 1 to 11 , wherein the vaccine is a DNA viral vaccine.

13. The method of any one of claims 1 to 12, wherein the vaccine is an attenuated form of the virus.

14. The method of any one of claims 1 to 13, wherein the nucleotide identities of a plurality of polymorphic positions in the vaccine genomic material are determined in step e).

15. The method of any one of the preceding claims, wherein the mutation at the at least one polymorphic position is a non-synonymous mutation.

16. The method of any one of claims 1 to 15, wherein the mutation at the at least one polymorphic position is a synonymous mutation.

17. The method of any one of claims 1 to 16, wherein the vaccine-specific polynucleotides comprise ribopolynucleotides.

18. The method of any one of claims 1 to 16, wherein the vaccine-specific polynucleotides comprise deoxyribopolynucleotides.

19. The method of any one of claims 1 to 18, wherein the at least one set of vaccine- specific polynucleotides comprises a plurality of overlapping polynucleotides spanning a vaccine genomic region of interest.

20. The method of any one of claims 1 to 19, wherein a plurality of sets of vaccine- specific polynucleotides is provided.

21. The method of claim 20, wherein the plurality of sets of vaccine-specific polynucleotides are specific for the same vaccine.

22. The method of claim 21 , wherein each set of the plurality of sets of vaccine- specific polynucleotides is specific to a particular isoform of the vaccine.

23. The method of any one of claims 1 to 20, wherein each set of the plurality of sets of vaccine-specific polynucleotides is specific for a different vaccine.

24. The method of any one of claims 1 to 23, wherein the immobilization tag comprises biotin and the binding partner comprises streptavidin.

25. The method of any one of claims 1 to 24, wherein the solid surface comprises magnetic beads.

26. The method of any one of claims 1 to 25, wherein the vaccine is the varicella Oka vaccine.

27. The method of claim 26, wherein the at least one polymorphic position is selected from Table 1.

28. The method of any one of the preceding claims, further comprising administering an alternative batch of vaccine to an individual in need thereof, wherein it is known that no variant has been selected for in the alternative batch of vaccine.

29. A method of determining evolution of a pathogen, the method comprising: a) providing at least one set of pathogen-specific polynucleotides each comprising an immobilization tag; b) contacting under hybridising conditions the at least one set of pathogen- specific polynucleotides with a first sample obtained at a first timepoint from an individual infected with the pathogen; c) exposing the mixture from b) to a solid surface provided with a binding partner specific to the immobilization tag to isolate pathogenic genomic material hybridised to the pathogen-specific polynucleotides; d) sequencing at least part of the pathogenic genomic material; e) determining the nucleotide identity of at least one polymorphic position in the pathogenic genomic material; and f) repeating steps a) to e) with at least a second set of pathogen-specific polynucleotides each comprising an immobilization tag and a second sample obtained at a second timepoint from an individual infected with the pathogen; wherein a mutation in the at least one polymorphic position in the pathogenic genomic material is indicative that the pathogen has evolved.

30. The method of claim 29, wherein the first and second samples are obtained from the same individual.

31. The method of claim 29, wherein the first and second samples are obtained from different individuals.

32. The method of claim 29, wherein the sample comprises host genomic material and pathogenic genomic material.

33. The method of any one of claims 29 to 32, wherein the second timepoint is at least two weeks after the first timepoint.

34. The method of claim 33, wherein the second timepoint is at least four weeks after the first timepoint.

35. The method of any one of claims 29 to 34, wherein the method further comprises subjecting the first and/or second sample to a pre-treatment step before contacting it under hybridising conditions with the set of pathogenic-specific polynucleotides.

36. The method of claim 35, wherein the pre-treatment step comprises fragmenting the sample.

37. The method of claim 35 or claim 36, wherein the pre-treatment step comprises whole genome amplification as a first pre-treatment step.

38. The method of any one of claims 35 or 36, wherein the sample is not subjected to amplification by PCR as a first pre-treatment step.

39. The method of any one of claims 35 or 36, wherein the sample is not subjected to amplification by culture as a first pre-treatment step.

40. The method of any one of the preceding claims, wherein the pathogen is viral, bacterial, fungal or parasitic.

41. The method of claim 40, wherein the pathogen is a RNA virus.

42. The method of claim 40, wherein the pathogen is a DNA virus.

43. The method of any one of claims 41 or 42, wherein the virus is an attenuated form of the virus.

44. The method of claim 43, wherein the virus is a vaccine.

45. The method of any one of claims 29 to 44, wherein the nucleotide identities of a plurality of polymorphic positions in the pathogenic genomic material are determined in step e) and step f).

46. The method of any one of claims 29 to 44, wherein the mutation at the at least one polymorphic position is a non-synonymous mutation.

47. The method of any one of claims 28 to 44, wherein the mutation at the at least one polymorphic position is a synonymous mutation.

48. The method of any one of claims 29 to 45, wherein the pathogen-specific polynucleotides comprise ribopolynucleotides.

49. The method of any one of claims 29 to 48, wherein the pathogen-specific polynucleotides comprise deoxyribopolynucleotides.

50. The method of any one of claims 29 to 49, wherein the at least one set of pathogen-specific polynucleotides comprises a plurality of overlapping polynucleotides spanning a pathogenic genomic region of interest.

51. The method of any one of claims 29 to 50, wherein a plurality of sets of pathogen-specific polynucleotides are provided.

52. The method of claim 51 , wherein the plurality of sets of pathogen-specific polynucleotides are specific for the same pathogen.

53. The method of any one of claims 20 to 51 , wherein each of the plurality of sets of pathogen-specific polynucleotides is specific for a different pathogen.

54. The method of any one of claims 29 to 53, wherein the immobilization tag comprises biotin and the binding partner comprises streptavidin.

55. The method of any one of claims 29 to 54, wherein the solid surface comprises magnetic beads.

56. The method of any one of claims 29 to 55, further comprising administering to the individual a medicament to which the pathogen has not developed resistance.

57. A kit-of-parts for use in a method of determining evolution of a pathogen, the kit comprising: at least one set of pathogen-specific polynucleotides each comprising an immobilization tag; and a solid surface provided with a binding partner specific to the immobilization tag.

58. A kit-of-parts for use in a method of quality controlling a batch of vaccine, the kit comprising: at least one set of vaccine-specific polynucleotides each comprising an immobilization tag; and a solid surface provided with a binding partner specific to the immobilization tag.