WO2014062060A1

WO2014062060A1 - New method for producing high titer cell-free vzv vaccine virus

Info

Publication number: WO2014062060A1
Application number: PCT/NL2013/050737
Authority: WO
Inventors: Georges VERJANS; Albertus Dominicus Marcellinus Erasmus Osterhaus
Original assignee: Erasmus University Medical Center Rotterdam
Priority date: 2012-10-18
Filing date: 2013-10-18
Publication date: 2014-04-24

Abstract

The invention comprises a method for obtaining an attenuated Varicella Zoster Virus (VZV) vOka-derived clone comprising: a. isolating a VZV from vesicular fluid from VZV vOka vaccinees; b. testing said isolated virus for the presence of the 6 SNPs as indicated in Table 1, and c. optionally testing said isolated virus for minimal replication in the human skin xenograft SCID mouse model as compared to the pOka strain; d. passaging said VZV virus strain in a suitable in-vitro cell culture system; and e. selecting said virus if it is able to generate at least 10,000 PFU/mL cell-free infectious virus. The invention further comprises an attenuated vOka strain produced according to said method, a vaccine comprising said vOka strain and its use in preventing VZV infection or diseases caused by reactivation of VZV.

Description

Title: New method for producing high titer cell -free VZV vaccine virus

FIELD OF THE INVENTION

The invention relates to the field of cell culture and production of vaccines, especially vaccines of Varicella Zoster Virus (VZV).

BACKGROUND

Several countries, including the USA, Japan, Australia, Greece and Germany have issued to vaccinate children for Varicella (chickenpox), and in the case of the USA, to vaccinate individuals older than 60 years of age for herpes zoster (shingles). Varicella zoster virus (VZV) reactivation of latent endogenous VZV, which is causing the herpes zoster (HZ) disorder, correlates with a decline in VZV-specific cell-mediated immunity, which occurs in the elderly or those who are immunocompromised (Weinberg et al., 2009. J Infect Dis. 200: 1068-77). In some patients, pain associated with HZ can persist for months or even years after the HZ rash has healed, a complication referred to as post-herpetic neuralgia (PHN).

The currently available commercial vaccine for both chickenpox and HZ is based on the live-attenuated VZV Oka vaccine strain (vOka) generated at the Biken Institute (Osaka, Japan). The manufacturing of the current, worldwide approved VZV vaccines for children (Varilrix™,

GlaxoSmithKline Biologicals, Rixensart, Belgium and Varivax™ Sanofi Pasteur MSD, Lyon, France) and for the new combination measles, mumps, rubella, varicella (MMRV) vaccines Priorix-Tetra™, GlaxoSmithKline Biologicals, Rixensart, Belgium and ProQuad™, Sanofi Pasteur MSD, Lyon, France) and especially the high-dose VZV shingles vaccine for the elderly (Zostavax™, MSD) has faced serious manufacturing issues resulting in short supplies for several years. Both companies have generated their own formulations of the vOka strain: the vOka/Merck and vOka/GSK strains. vOka-based VZV vaccines are safe and well-tolerated in healthy individuals. However, the main adverse effect following vaccination with low-dose VZV vaccine (e.g., Varilrix™ and Varivax™) is a moderate varicella-like rash, which can be a generalized rash or solely at the injection site, which occurs within 1-6 weeks post-vaccination. The incidence thereof is 5% in children and 8-10% in adults (M.L. Quinlivan et al., 2011, Expert Rev. Vaccines 10: 1321-1336).

The main difficulty in manufacturing the vaccines is the inability to generate sufficient amounts of high titer cell-free vaccine virus in vitro in VZV susceptible cells. Currently, VZV vaccine producing companies use MRC-5 cells, which are FDA approved for vaccine production.

SUMMARY OF THE INVENTION

The inventors now have found a method for obtaining an attenuated Varicella Zoster Virus (VZV) vOka-derived clone comprising a. isolating a VZV from vesicular fluid from VZV vOka vaccinees; b. testing said isolated virus for the presence of the 6 SNPs as indicated in Table 1, and

c. optionally testing said isolated virus for minimal replication in the human skin xenograft SCID mouse model as compared to the pOka strain

d. passaging said VZV virus strain in a suitable in-vitro cell

culture system; and

e. selecting said virus if it is able to generate at least 10,000

PFU/mL cell -free infectious virus.

Preferably, in this method the VZV vaccinees are individuals or patients that have developed a vaccine-induced varicella as a result of vaccination with a vOka quasispecies.

In another embodiment, the following nucleotides are located within the VZV genes: 560T (ORFO), 69,349G (ORF38), 105,705C (ORF62), 106,262C (ORF62), 107,252C (ORF62) and 108, 111C (ORF62). In a further preferred embodiment the virus produced in step e) is stocked in frozen condition and the amount of PFU/mL is detected after thawing of an aliquoted frozen virus stock. In this aliquoted frozen virus stock, when thawed, the amount of PFU/mL is at least 50,000, more preferably at least 100,000.

In a further embodiment the cells are passaged on human retinal epithelial like cells, preferably Per.C6 cells

Further part of the invention is an attenuated VZV Oka strain obtained with a method according to the invention. Also part of the invention is said attenuated VZV Oka strain obtained with a method according to any of claims 1-6 for use in therapy.

Further, the invention comprises a vaccine composition comprising the attenuated VZV Oka strain according to the invention and an acceptable excipient. Preferably said vaccine composition further comprises an adjuvant.

The present invention also comprises a method for preventing or treating a primary infection with VZV by administering a vaccine

composition according to the invention.

Also comprised in the invention is a method for preventing, ameliorating, abrogating or reducing the likelihood of reactivation of VZV, for preventing or reducing the likelihood of developing herpes zoster, for reducing the severity or duration of herpes zoster, or for preventing or reducing the likelihood of developing post-herpetic neuralgia associated with reactivation of VZV by administering a vaccine composition according to the invention.

DETAILED DESCRIPTION

In the following description and examples a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given to such terms, the following definitions are provided. Unless otherwise defined herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

As used herein, the term "treatment" refers to both a therapeutic treatment itself, but also to prophylactic or preventative measures, such as vaccination. Individuals in need of treatment include those already infected with the virus as well as those prone to become infected with the virus or those in which infection is to be prevented. Treatment of a patient with the cell -free VZV vaccine strain of the invention includes one or more of the following: inducing/increasing an immune response against VZV in the patient; preventing infection with wild-type VZV, especially in children; preventing, ameliorating, abrogating, or reducing the likelihood of

reactivation of VZV in patients who have been infected with VZV or received a live VZV vaccine;, preventing or reducing the likelihood of developing herpes zoster and/or other disease or complication associated with VZV reactivation such as PHN; and reducing the severity or duration of HZ and/or other disease or complication associated with the reactivation of VZV, such as PHN.

The term "therapeutically effective amount" means a sufficient composition of the vaccine to produce a desired effect, including, but not limited to: inducing/increasing an immune response against VZV in a patient, preventing, ameliorating or abrogating reactivation of VZV in patients who have been infected with VZV or received a live VZV vaccine preventing HZ and/or PHN, reducing the severity or duration of HZ and/or PHN. One skilled in the art recognizes that this level may vary.

The term "immune response" refers to a cell-mediated (T-cell) immune response and/or an antibody (B-cell) response.

The term "patient" refers to any human being that is to receive the VZV vaccine, or pharmaceutical compositions, described herein, including both immunocompetent and immunocompromised individuals. As defined herein, a "patient" includes persons who have not been infected yet with VZV, also called VZV-naive patients, and those already infected with VZV, either through natural infection or vaccination.

The term "bulk" refers to a liquid formulation or composition comprising more than one dose of vaccine.

The term "undetectable levels," in reference to the infectivity of a particular vaccine formulation or composition, means that the formulation or composition comprises <0.050 infectious units or plaque-forming units ("PFU's") of infectious VZV virus per mL of sample, preferably O.040 PFU's/mL, <0.030 PFU's/mL, O.020 PFU's/mL, O.015 PFU's/mL, O.010 PFU's/mL, < 0.009 PFU's/mL, or < 0.008 PFU's/mL, more preferably < 0.007 PFU's/mL, < 0.006 PFU's/mL, < 0.005 PFU's/mL, < 0.004 PFU's/mL < 0.003 PFU's/mL, more preferably < 0.002 PFU's/mL, or O.001 PFU's/mL. The PFU's of a particular sample may be determined using, for example, a VZV plaque assay such as the assay described in Krah et al. (J. Virol. Methods (1990) 27: 319-26). The infectivity of a sample may also be confirmed by VZV-specific immunostaining and/or PCR method. A vaccine that is able to evoke an immune reaction or to be otherwise useful in the present invention (e.g. to reduce the disease effects caused by VZV reactivation) preferably comprises more than 10,000 PFU/ml of infectious, attenuated virus.

The term "vOka" refers to the original VZV vOka/Biken vaccine strain generated at the Biken Institute (Osaka, Japan), derivatives thereof generated by companies like MSD and GSK. The varicella vaccine strain manufactured by MSD was derived from pOka by growth at low

temperature (32°C) and passaged 11 times in human embryonic lung cells, 12 times in guinea pig embryo fibroblasts, once in WI-38 cells, and 9 times in MRC-5 cells. The Biken vOka strain or rather clinical quasi-species thereof obtained from vOka vaccinees with vaccine rashes is used for producing the vaccines that have been mentioned in the background section above. This vaccine strain is denominated as vOka (vaccine-Oka) to distinguish it from the parental Oka (pOka) strain that was isolated initially. The vOka strain as it is currently used, consists of a mixture of genetically related viruses, in which mixture a multitude of genetically related VZV genotypes is present. This is called a quasi-species in contrast to a virus strain that consists of viruses with one and the same genotype, which is also referred to as 'clone'. It was now found that the reason that the current system fails to produce the high titers cell-free virus of the live-attenuated VZV vaccine strain vOka that are needed for vaccine production is to be found in the inability of the commonly used in-vitro culture systems of laboratory strains of the virus, including the vOka strain used by all VZV vaccine companies, to generate sufficiently high titers of cell-free virus to be used in the vaccine. Even if the viruses are cultured in retina pigmented epithelial (RPE) cells, which have been indicated to be very suitable for passaging VZV (J.

Schmidt-Chanasit et al., 2008, J. Clin. Microbiol. 46:2122-2124; J.C. Milikan et al., 2009, Invest. Ophthalmol. Vis. Sci. 50:743-751), the titers of cell-free virus remain low. Our preliminary data show that these laboratory strains, including the VZV vOka vaccine and Dumas strain do not facilitate the generation of high-titer cell -free VZV stocks when cultured on human MRC- 5 (American Tissue Culture Collection, ATCC171) and ARPE-19 cells

(ATCC2302): an average cell-free VZV titer of l-5xl0³ pfu/ml was found. The reason for this low titer may be that in vitro adaptation to cell lines of non- RPE background, including the MRC-5 cell line currently used for the VZV vaccine virus preparation, in the past and frequent passaging of VZV on these cell lines has most likely selected out the ability of the virus strain to replicate at high cell-free titers. Further, it has probably negatively affected the extra-cellular stability of the virus particles upon secretion or rupturing of VZV-infected cells.

The solution for this lack of sufficient generation of high titer cell- free VZV is to find virus variants from the VZV vaccine vOka strain that are able to replicate with a high cell -free titer, while still being both low- pathogenic and immunogenic as the current commercial vOka vaccine strains. Such vOka variants are preferably obtained from vesicular fluid from patients that have suffered from a VZV vOka vaccine-mediated vesicular skin rash, which can be generalized rash or at the vaccine injection site, resembling mild chickenpox after vaccination with the currently commercially available VZV vOka-based vaccines. Advantageously in such a case, limiting dilutions of the first cell -free VZV passage should be obtained and selected for genuine low-pathogenic attenuated VZV vOka variants based on the virus genotype and phenotype. Genotypic criteria for such a selection can be derived from the mutations that have been proposed as causing the attenuated VZV vOka virus phenotype (M.L. Quinlivan et al., 2011, Expert Rev. Vaccines 10: 1321-1336; R.K. Kanda et al. 2011. Vaccine. 29:3293-3298; M.L. Kanda et al. 2012. J Clin Microbiol. 50: 1533-1538 and G.A. Peters et al. 2012. J Virol. 86: 10695-10703). However, it should be kept in mind that the vOka vaccine product consists of a mixture of genetically related vOka genotypes: i.e., vOka quasi-species. It is currently agreed upon that vOka attenuation requires a set of single nucleotide polymorphisms (SNPs) within several viral genes. More particular, the following 6 SNPs are currently proposed in the literature to be associated with attenuation of the vOka vaccine strain: Table 1. SNPs in the VZV genome that indicate attenuation.

Nucleotide Nucleotide change in vOka compared position SNP in VZV genome¹ VZV gene to pOka strain

560 ORF0 T→C

69,349 ORF38 G=G²

105,705 ORF62 T→C

106,262 ORF62 T→C

107,252 ORF62 T→C

108, 111 ORF62 T→C

Nucleotide positions refer to nucleotide position in the VZV reference strain

Dumas (Genbank entry number S i ^'. 001■ ;H).

²The ORF38 SNP is essential to define the VZV strain as Clade 2 VZV, being the origin of the vOka vaccine strain. Different clades of wild-type VZV predominate in Europe and the US. Notably, both the pOka and vOka strains have a "G" at nucleotide position 69,349.

In the present application a VZV virus that encompasses all 6 of the above mentioned SNPs is held to be an attenuated vOka strain. The attenuated status of such a vOka derived strain can be confirmed in an animal test model, which can optionally be performed in the method of the present invention.

The humanized SCID mouse model (SCID-hu) of VZV

pathogenesis provides the only animal model that allows an assessment of VZV virulence in intact human tissues infected in vivo. For example, infection of human fetal skin xenografts with vOka leads to minimal

replication compared to infection with the parental strain pOka (AM Arvin, 2006, Herpes 13:75-80; J.F. Moffat et al., 1998. J Virol. 72:965-974).

Minimal replication in this respect means a replication frequency in the SCIDhu mouse model that is about equal to the replication frequency of known vOka strains and which is less, preferably much less, than the replication of the pOka strain. The protective immune response elicited by a vOka variant and a control sample may be measured first in an appropriate animal model like the simian varicella virus (SW) non-human primate model, e.g. vOka vaccination and subsequent challenge with pathogenic SW (Felsenfeld A.D. et al., 1979. J Gen Virol. 42: 171-178), and subsequently in the human population in a clinical trial, for example, by measuring one or more of the following parameters: (1) the VZV-specific responder T-cell frequency (as described in Calandra et al., WO 94/002596, the frequency of (2) anti-VZV cytotoxic T-cells (CTLs), or VZV specific CD8<+> cells, (3) the anti-VZV helper T-cells, or VZV specific CD4<+> cells, (4) the level of anti-VZV specific antibodies, or (5) the level of cytokines such as interferons (e.g., interferon-gamma) or interleukins (e.g., interleukin-2).

Techniques that are useful for measuring the VZV-specific immune response are known in the art and include, but are not limited to: intra-cellular T-cell cytokine assay, T-cell proliferation assay, cytokine ELISPOT assay, ELISA assays and virus-neutralizing antibody assay.

To determine the sufficiency of the immune response elicited by the vaccines/ pharmaceutical compositions of the present invention and made by the methods described herein, and also the efficacy of the

inactivated vaccine, it may be useful to measure clinical outcomes in a clinical trial of human volunteers/subjects to measure, for example, a reduction of the duration or severity of HZ and/or a reduction in the duration of PHN in an individual to a period of less than one month following development of herpes zoster, reduction in the incidence of HZ in the patient population, on a statistical level, below the incidence found in the general population or of similarly at risk or immunecompromised individuals.

In such a way a virus can be isolated that is sufficiently immunogenic, i.e. equal to or more than the currently used vOka VZV strains, which is still attenuated (as defined by the presence of the 6 indicated SNPs and confirmed by minimal replication in the human skin xenograft SCID-hu mouse model as compared to pOka) and which gives rise to a desired VZV-specific immunogenic effect. Preferably, such a virus strain is composed of viruses of one single genotype, i.e. is a vOka-derived clone.

The person of skill in the art can readily determine a proper amount of VZV antigen or attenuated virus to be used as a therapeutically effective amount of VZV. Such amount will vary according to intended use, or other factors such as the adjuvant used in the vaccine, the route of administration, the particular patient population and/or the need for long- term storage of the vaccine. In some embodiments of the vaccine

compositions described herein, the amount of VZV is more than 10,000 PFU's per dose, preferably more than 15,000 PFU's per dose, more preferably more than 16,000 PFU's per dose, more preferably more than 17,000 PFU's per dose, more preferably more than 18,000 PFU's per dose, more preferably more than 19,000 PFU's per dose, more preferably more than 20,000 PFU's per dose, more preferably more than 21,000 PFU's per dose, more preferably more than 22,000 PFU's per dose, more preferably more than 23,000 PFU's per dose, more preferably more than 24,000 PFU's per dose, more preferably more than 25,000 PFU's per dose, more preferably more than 30,000 PFU's per dose, more preferably more than 35,000 PFU's per dose, more preferably more than 40,000 PFU's per dose, more preferably more than 45,000 PFU's per dose, more preferably more than 50,000 PFU's per dose. The amount of virus can be determined with an appropriate assay, for example, a VZV plaque assay such as the assay described in Krah et al. (J. Virol. Methods (1990) 27: 319-26). One skilled in the art will realize that other methods are also useful for determining infectivity of the VZV sample or composition and may be used in place of the VZV plaque assay

Also provided herein is a pharmaceutical composition comprising a therapeutically effective amount of the attenuated VZV described above and a pharmaceutically acceptable carrier, excipient or diluent.

Pharmaceutically acceptable carriers useful in the compositions of the invention include any compatible agent that is non-toxic to patients at the dosages and concentrations employed, such as water, saline, dextrose, glycerol, ethanol, buffers, and the like, and combinations thereof. The carrier may also contain additional components such as a stabilizer, a solubilizer, a tonicity modifier, such as NaCl, MgC , or CaC etc., a surfactant, and mixtures thereof.

The vaccine or pharmaceutical invention may further include an adjuvant or any agent that is capable of boosting an immune reaction.

"Adjuvant" refers to compounds that, when administered to an individual or tested in vitro, increase the immune response to an antigen in the

individual or test system to which the antigen is administered. In another embodiment, an immune adjuvant enhances an immune response to an antigen that is weakly immunogenic when administered alone, i.e., inducing no or weak VZV-specific antibody titers or T-cell immune response. In another embodiment, the adjuvant increases antibody titers to the antigen. In another embodiment, the adjuvant lowers the dose of the antigen effective to achieve an immune response in the individual. Adjuvants are known in the art and include, but are not limited to, oil-in-water emulsions, water-in oil emulsions, alum (aluminum salts), liposomes and microp articles including but not limited to, polystyrene, starch, polyphosphazene and polylactide/polyglycosides. Other suitable adjuvants also include, but are not limited to, MF59, DETOX(TM) (Ribi), squalene mixtures (SAF-1), muramyl peptide, saponin derivatives, mycobacterium cell wall preparations, monophosphoryl lipid A, mycolic acid derivatives, nonionic block copolymer surfactants, Quil A, cholera toxin B subunit, polyphosphazene and

derivatives, and immunostimulating complexes (ISCOMs) such as those described by Takahashi et al. (1990) Nature 344:873-875, as well as, lipid- based adjuvants and others described herein. The invention also provides multi-dose compositions which contain more than one dose of the vaccine containing the attenuated VZV vOka clone of the invention, an anti-microbial preservative, which prevents inadvertent microbial contamination upon introduction of a syringe to the vial comprising the composition, and a pharmaceutically acceptable carrier. Such multi-dose compositions can be provided to a patient at several occasions, after a predetermined amount of time has passed or to more than one patient.

To that end, the invention provides attenuated, live VZV and pharmaceutical compositions/vaccines comprising said VZV particles, wherein the pathogenity of the virus is virtually absent, while still being immunogenic. Such VZV compositions are as effective as the previously disclosed, commercially available vaccines for the treatment and/or prevention of varicella, HZ or other diseases associated with VZV

reactivation, where said earlier disclosed vaccine compositions contain the vOka strain of VZV. The present VZV is able to replicate at high cell -free titers and thus is suitable to a more efficient manufacturing process.

In principle, any VZV strain can be used in the compositions and methods described herein, including a wild-type VZV strain, but preferably the strains of the invention are derived from vesicle fluid of vOka vaccinees that developed a post-vaccine varicella treated with an attenuated strain such as the vOka strain, as described in U.S. Patent 3,985,615. The parental Oka (pOka) strain was originally obtained from the vesicle fluid of an infant named Oka who had typical chickenpox and passaged 11-times in human embryonic lung cells at 34°C (M. Takahashi et al., 1974. Lancet. 2: 1288- 1290) at the Biken Institute (Osaka, Japan). It is attenuated at the Biken Institute by adaption to grow in guinea pig embryo cell cultures and human diploid lung fibroblast cell cultures (e. g., MRC-5 cells). In preferred embodiments of the compositions and methods described herein, the VZV is the original Biken vOka strain or a vOka strain derivative, such as the vOka/Merck or vOka/GSK VZV strain. An "vOka strain derivative," as used herein, is a strain that is derived from a vOka strain and which is able to replicate with a high cell-free titer in-vitro, while still being both low- pathogenic and immunogenic as compared top the current commercially used vOka vaccine strains. Such a vOka strain derivative or "Oka variant" is preferably obtained from vesicular fluid from patients that have suffered from a vOka-induced varicella after vaccination with the currently commercially available vOka/Merck- and/or vOka/GSK-based VZV vaccines and further culturing, limiting diluting and subsequent passaging the appropriate attenuated vOka clone, identified after validation by the genotypic and phenotypic assays as indicated earlier herein, in an

appropriate cell type. In the methods and compositions of the invention described herein, a live attenuated VZV, such as the vOka strain or derivative thereof, may be used directly in a vaccine composition or a strain that can be inactivated by heat-treatment or with gamma-irradiation prior to administration to a patient may be used.

The VZV vaccine viruses should preferably be cultured on human primary retina pigmented epithelial (RPE) or RPE-like cells to maintain a high titer of cell -free infectivity after thawing of aliquoted virus stocks. An especially preferred human retinablast cell culture with epithelial cell morphology is the PER.C6® cells, which are adenovirus-transformed human embryonic retinoblast cells (F.J. Fallaux et al. 1998. Human Gene Therapy. 9: 1909-1917). The ability of PER.C6® cells to grow to exceptionally high densities means that much more biological product can be harvested from much smaller bioreactors. For example, PER.C6® cells infected with the VZV vOka clone, generated for manufacturing purposes, is expected to produce at least 10- to 50-times more cell-free infectious virus per milliliter than any other cell line does. The same yield can therefore be obtained from a 500-liter bioreactor using PER.C6® as a 5,000-liter or larger bioreactor using another cell line. This translates into significant savings in capital expenditure and production costs, as well as shorter production times

Also provided herein is a method of preparing an inactivated VZV comprising heat-killing or gamma-irradiation of a sample comprising a VZV by incubating the virus preparation at 50°C for about 30 to about 60 minutes or gamma irradiation using about 5 to about 50 kGy, respectively.

The invention also provides an attenuated VZV produced or obtainable by the methods described herein, including a VZV having the 6 SNPs and demonstrating minimal replication in the human skin xenograft SCID-hu mouse model as compared to pOka, as indicated earlier herein. Additionally provided is a vaccine comprising a therapeutically effective amount of the attenuated VZV produced by the methods described herein and a pharmaceutically acceptable carrier.

The invention also provides, in one aspect, a method for the treatment, prevention of, immunization against, or reduction in the likelihood of varicella, herpes zoster and/or any other disease or

complication associated with the infection with or reactivation of VZV, e.g., post-herpetic neuralgia, in a patient, the method comprising administering to the patient a therapeutically effective amount of a vaccine or

pharmaceutical composition comprising an attenuated VZV strain according to the invention and a pharmaceutically acceptable carrier, wherein the VZV is obtained from vesicular fluid from a subject that developed varicella due to a commercially available VZV vaccine and passaged on human RPE- like cells. In such methods of treatment/immunization described above, the pharmaceutical composition comprising the attenuated VZV vOka-derived clone may be administered to the patient through any suitable route including, but not limited to: subcutaneous injection, intra-dermal introduction, impression though the skin, or other modes of administration such as, intravenous, intramuscular or inhalation delivery. In preferred embodiments of the methods described herein, the mode of administration is subcutaneous or intramuscular.

The methods and compositions described above are useful for preventing varicella, HZ and/or PHN, or reducing the incidence, severity or duration thereof in a patient. In some embodiments of the methods described herein, the attenuated VZV vOka vaccine is administered to a patient who is at greater risk for varicella or HZ due to disease, for example, autoimmune diseases, blood cancers; solid tumor malignancies; rheumatoid arthritis (RA), systemic lupus (SLE), Crohn's disease, psoriasis, and multiple sclerosis or treatment for disease (such as for hematologic malignancies, solid tumor malignancies or chemotherapy, or treatment for autoimmune diseases).

Patients may be infants or children or otherwise VZV-naive patients in which the vaccine is administered to prevent infection. Patients may also be adults of any age. In additional embodiments of the methods described herein, the vaccine or pharmaceutical composition may be administered concomitantly with other commonly administered 'standard of care' therapies; or with other vaccines for targeted patient populations.

In specific embodiments of the methods of treatment/prevention provided herein, the method further comprises allowing an appropriate predetermined amount of time to pass and administering to the patient one or more additional doses of the pharmaceutical composition. In said embodiments, one additional dose may be administered to the patient after an appropriate amount of time has passed, alternatively, two, three or four additional doses, each being administered after an appropriate amount of time has passed. In an exemplary embodiment, 3 or 4 doses are

administered to the patient as part of a dosing regimen that is properly separated over a course of time. One skilled in the art will realize that the amount of time between doses may vary depending on the patient

population, dosage of the vaccine and/or patient compliance. In an exemplary embodiment, a time period of about 3 weeks, about 1 month, about 6 weeks, about 2 months, or about 3 months or more is allowed to pass between administrations of each dose to the patient.

The invention will be explained in more detail in the following, non-limiting examples.

EXAMPLES

The invention involves 5 key stages in preparing an attenuated VZV vOka-derived clone.

First, fresh vesicle fluid from a VZV naive patient, preferable an adult, who developed a moderate varicella-like skin rash with

distinguishable vesicles (i.e., fluid-filled blister), which presented as a generalized rash or a blisters solely at the injection site within 1-6 weeks post-vaccination with a vOka-based low-dose VZV vaccine (e.g., Varilrix™ and Varivax™), is obtained with a sterile needled syringe punctured in a non-crusted vesicle to obtain vesicular fluid and subsequently rubbing the base of the same bhster with a sterile polyester swab to collect VZV-infected epithelial cells without causing bleeding. Both the vesicular fluid and swab are put in the same sterile tube containing 1 mL commercial virus transport medium. Multiple vesicles, clearly anatomically separated, are sampled from the same patient and regarded as separate clinical isolates. The tubes with the inoculated commercial virus transport medium are cooled and transported at 2-8°C and used to infect RPE-like cell cultures within 3 days after clinical specimen collection.

Second, the inoculated commercial virus transport medium, 1 tube/blister, is divided into two equal aliquots and mixed with 1 volume of the appropriate culture medium (hereafter referred to as RPE medium) of the human RPE-like cell line used: e.g. in case of primary human adherent RPE cells the medium may consist of a 1: 1 ratio (vol/vol) of Dulbecco's modified Eagle's medium (DMEM) and Ham F12 nutrient mixture (both Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum and antibiotics as described (J. Schmidt-Chanasit et al., 2008, J. Clin. Microbiol. 46:2122-2124; J.C. Milikan et al., 2009, Invest. Ophthalmol. Vis. Sci. 50:743- 751). Two 25 cm² flasks of washed human RPE-like cells are resuspended (in case of single cell suspensions) or overlayed (in case of semi-confluent monolayers) in the aforementioned 1 mL medium aliquot and incubated at 37°C in a CO2 incubator. After 2 hours, 4 mL pre-warmed (37°C) RPE medium is added to the cultures and incubated for 3-7 days at 37°C in a CO2 incubator. The cultures are examined regularly, by an inverted microscope, for typical VZV cytopathic effect (CPE) characterized as enlarged cells with increased granularity and the formation of multinucleated syncytia. To confirm the presence of VZV-infected cells immunocytology or Uowcytometry using VZV-specific antibodies or VZV-specific PCR can be performed on an aliquot of inoculated cell cultures as described (J. Schmidt-Chanasit et al., 2008, J. Clin. Microbiol. 46:2122-2124; J.C. Milikan et al., 2009, Invest. Ophthalmol. Vis. Sci. 50:743-751; L. Remeijer et al., 2009, J. Infect. Dis. 200: 11-19). Cell cultures with confirmed VZV infection, and 30-70% CPE, are cryopreserved in aliquots with dimethylsulfoxide as viable cells, referred hereafter as cell-associated VZV preparations, in liquid N2.

Third, the aforementioned primary VZV passage is cloned by limiting dilution on the same human RPE-like cell line as used for the generation of the first VZV passage. In case of adherent human RPE-like cells semiconfluent cell monolayers cultured in 6-well plates in RPE medium are inoculated in quadruplicate with 10-fold serial dilutions of the VZV- infected cells of the first passage in lmL RPE medium at 37°C in a CO2 incubator. After 2 hours, medium is discarded and 4 mL pre-warmed (37°C) RPE medium supplemented with 0.05% methylcellulose is added and incubated for 5-8 days at 37°C in a CO2 incubator. The cultures are examined regularly, by an inverted microscope, for small areas of radial disruption of the monolayer due to VZV CPE referred to hereafter as VZV plaques. One skilled in the art will recognizes that such a VZV plaque consists of one VZV strain. Human RPE-like cells, exponentially growing in suspension, are co-incubated in quadruplicate with 10-fold serial dilutions of the VZV-infected cells of the first passage in 1 mL RPE medium in 24-well plates, with 5-10xl0⁵ uninfected human RPE-like cells/well, for 5-8 days at 37°C. One skilled in the art will recognize that the clonality of a VZV culture, i.e. one VZV strain/well, is based on the dilution used and the number of positive wells with VZV CPE. Independent VZV plaques, and cell suspensions showing VZV CPE, are used to inoculated the same human

RPE-like cell line as used for the generation of the first VZV passage, grown in 25 cm² flasks in the appropriate RPE medium, to generate the third passage VZV as described (J. Schmidt-Chanasit et al., 2008, J. Clin.

Microbiol. 46:2122-2124; J.C. Milikan et al., 2009, Invest. Ophthalmol. Vis. Sci. 50:743-751). Cell cultures with confirmed VZV infection, as described above, and showing 50-80% CPE after incubation at 37°C in a CO2 incubator are cryopreserved as cell-associated VZV preparations.

Fourth, DNA is isolated from the VZV clones and used to sequence the genomes by next -generation sequencing, and in case of poorly resolved sequences within parts of the genome specifically amplified by PCR and sequenced by standard Sanger sequencing technology, as described (G.A. Peters et al., 2012. J. Virol. 86: 10695-10703). The genomes are aligned to genome sequences of reference and wild-type VZV strains, including vOka and pOka, deposited in Genbank. The sequences are screened for the presence of vOka attenuation-associated mutations 560T (ORF0), 69,349G (ORF38), 105,705C (ORF62), 106,262C (ORF62), 107,252C (ORF62) and 108, 1 l lC (ORF62). Next, the VZV clones, having the vOka genotype and expressing the aforementioned mutations, are subjected to the phenotype assay to demonstrate the attenuated phenotype of the respective vOka- derived VZV clone(s) as described (A.M. Arvin, 2006, Herpes 13:75-80; J.F. Moffat et al., 1998. J Virol. 72:965-974). This phenotype assay is based on the limited virulence of the attenuated vOka strain, compared to pOka, in VZV inoculated fetal skin tissue implants of SCID mice (SCID-hu model). vOka-derived VZV clones are considered attenuated when infectious virus yields and analysis of inoculated human fetal skin implant tissues in SCID- hu mice for VZV DNA and viral protein synthesis resemble that of vOka, but are restricted compared to pOka-infected SCIDhu mice.

Five, the vOka-derived VZV clones that express the vOka genotype and attenuation-associated mutations, and have vOka-resembling pathology in the human skin xenograft SCID-hu mouse model, are considered advantageous for preparing VZV vaccine preparations. The(se) vOka-derived VZV clone(s) are grown to at least 20 passages to large virus batches in the human RPE-like cell line, and validated for genetic stability and the attenuated phenotype, and the ability to generate high titer cell-free infectious VZV, at least 100,000 PFU/mL cell-free infectious VZV after thawing of cell-free virus aliquots. The generation of cell-free VZV from VZV-infected human RPE-like cells, and the determination of the infectious VZV titer thereof, are performed as described (J. Schmidt-Chanasit et al., 2008, J. Clin. Microbiol. 46:2122-2124; J.C. Milikan et al., 2009, Invest. Ophthalmol. Vis. Sci. 50:743-751).

Claims

A method for obtaining an attenuated Varicella Zoster Virus (VZV) vOka-derived clone comprising

a. isolating a VZV from vesicular fluid from VZV vOka vaccinees; b. testing said isolated virus for the presence of the 6 SNPs as indicated in Table 1, and

d. passaging said VZV virus strain in a suitable in-vitro cell

culture system; and

e. selecting said virus if it is able to generate at least 10,000

PFU/mL cell -free infectious virus.

Method according to claim 1, wherein the VZV vaccinees are individuals or patients that have developed a vaccine-induced varicella as a result of vaccination with a vOka quasispecies.

Method according to claim 1 or 2, wherein the following

nucleotides are located within the VZV genes: 560T (ORF0), 69,349G (ORF38), 105,705C (ORF62), 106,262C (ORF62),

107,252C (ORF62) and 108, 111C (ORF62).

Method according to any of the previous claims, wherein the virus produced in step e) is stocked in frozen condition and the amount of PFU/mL is detected after thawing of an aliquoted frozen virus stock Method according to claim 4, wherein the amount of PFU/mL is at least 50,000, more preferably at least 100,000.

A method according to any of the previous claims, wherein the cells are passaged on human retinal epithelial like cells, preferably Per.C6 cells

An attenuated VZV Oka strain obtained with a method according to any of claims 1-6.

An attenuated VZV Oka strain obtained with a method according to any of claims 1-6 for use in therapy.

A vaccine composition comprising the attenuated VZV Oka strain according to claim 7 and an acceptable excipient.

A vaccine composition according to claim 7, further comprising a suitable adjuvant.

A method for preventing or treating a primary infection with VZV by administering a vaccine composition according to any of claims 9 - 10.

A method for preventing, ameliorating, abrogating or reducing the likelihood of reactivation of VZV, for preventing or reducing the likelihood of developing herpes zoster, for reducing the severity or duration of herpes zoster, or for preventing or reducing the likelihood of developing post-herpetic neuralgia associated with reactivation of VZV by administering a vaccine composition according to any of claims 9 - 10.