WO2004081178A2 - Methods and composition for producing infectious hpv stocks - Google Patents

Abstract

A purified, infectious stock of human papillomavirus (HPV) type 6a is useful for testing and developing vaccines and other therapeutic agens for HPV related diseases and conditions. Methods for selecting biological specimens for production of infectious HPV stocks are also provided. A neutralization test for vaccines uses HPV 6a.

Description

METHODS AND COMPOSITIONS FOR PRODUCING INFECTIOUS HPV

STOCKS

BACKGROUND OF THE INVENTION

[0001] Human papillomaviruses (HPVs) are small (50-60 nm), nonenveloped, icosahedral DNA viruses with genes that encode for up to eight early and two late proteins. The open reading frames (ORFs) of the virus genomes are designated El to E7 and LI and L2, where "E" denotes early and "L" denotes late. LI and L2 code for virus capsid proteins. The LI protein is the major capsid protein and has a molecular weight of 55-60 kDa. The LI ORF is highly conserved among different HPVs. The L2 proteins are less conserved among different HPVs. The early (E) genes are associated with functions such as viral replication and cellular transformation.

[0002] HPVs are assigned type designations based on DNA sequence homologies.

Subtypes may also exist within a type.

[0003] Human papillomaviruses (HPVs) cause condylomata acuminata, or genital warts, a condition characterized by an infection of genital epithelial surfaces. Of the at least 90 known HPV types, approximately 30 infect the genital tract, causing a range of manifestations from asymptomatic, latent infection, to the typical exophytic cauliflower-like growths known as condylomata acuminata, to dysplasia and invasive carcinoma.

[0004] The majority of condylomata acuminata, a benign condition, is associated with

HPV type 6 (HPV 6) or HPV 11 viruses. HPV 6 has been detected approximately three times as often as HPV 11 in these lesions. Together, HPV types 6 and 11 have been detected in up to 86% of condylomata acuminata. These HPV types are thought to be of low malignant potential, although some studies have demonstrated the presence of HPV 6 or HPV 11 in malignant genital lesions, especially of the vulva. Other genital HPVs such as HPV type 16, 18, and 31 are associated with dysplasic cervical lesions and they constitute high risk sub types. [0005] HPV 6 has been subtyped on the basis of restriction digestion patterns of DNA visualized in Southern blots. There may be functional differences between the HPV 6 subtypes, which have been designated HPV 6a through HPV 6f. In addition, knowledge of the specific subtypes and their infectivity may be useful in epidemiologic studies. HPV 6b was previously considered to be the "prototype" of HPV 6, because it was the first to be cloned and sequenced. However, HPV 6a is the most commonly detected subtype of HPV 6. The entire genome of HPV 6a has been cloned and sequenced. HPV 6a causes up to 75% of all genital warts.

[0006] Prophylactic vaccines are being developed to reduce the frequency of clinical lesions due to HPV infection. Therapeutic vaccines are also contemplated. Successful vaccines would reduce the frequency of genital warts and cancer. Two of the problems in developing HPV vaccines are producing infectious stocks of HPV for determining antigenic components that are needed for vaccines, and testing the efficacy of the vaccines.

[0007] In order to approve vaccines before releasing them for clinical use, regulatory agencies require proof of efficacy. One proof is that antiserum or cervical secretions from persons inoculated with the vaccine neutralizes infectious HPV of the types (subtypes) targeted by the vaccine. Neutralization assays are used for this purpose. There are a few neutralization assays that use pseudovirions rather than infectious virus that attach to human cells in culture, but these assays lack specificity and the results are difficult to interpret.

[0008] The development of vaccines against human papillomaviruses (HPV) has been hampered by the lack of a conventional viral culture system because HPV completes its life cycle only in fully differentiated human tissue. To overcome this obstacle, the athymic mouse xenograft system provides an important model for testing the ability of antibodies to protect against HPV infection and has been used to study the pathogenesis of HPV. An organotypic raft culture may also be used, where human keratinocytes transfected with HPV DNA are grown at an air/medium interface to induce differentiation. Antiserum or monoclonal antibodies raised against human papillomavirus type 11 (HPV 11) virions or virus-like particles (VLPs) have been shown to neutralize HPV 11 viral infection of human foreskin implants grown in athymic mice. To determine if neutralization of HPV 11 in the mouse xenograft system correlated with presence of antibodies to native virus, several neutralization experiments were performed using one of two polyclonal rabbit sera raised against purified HPV 11 virions. The concentration of the HPV 11 major capsid protein (LI protein) VLP-specific IgG in the anti-HPV 11 sera was established by enzyme-linked immunosorbent assay (ELISA). Anti-HPV 11 sera were used in neutralization experiments in the mouse xenograft system, using known, standardized quantities of purified HPV 11 virions. The concentration of HPV 11 LI VLP-specific IgG for each serum was then correlated with the neutralizing titer of the sera. Coupling the athymic mouse system with other infectious virions would benefit vaccine development. It is clear that live, infectious stocks of a variety of types of human papillomavirus are needed in order to produce and test broad spectrum vaccines against these viruses. It is particularly important that such a vaccine is targeted to commonly found HPV types. At present, only a small number of HPV types, HPV 11, HPV 59 and HPV 83, are readily available as infectious stocks. It is important to have a stock of infectious HPV 6a, for example, for development of vaccines against HPV and testing the efficacy of the vaccine. No such stock of HPV 6a was available prior to this invention.

Despite the fact that HPV 6a causes up to 75% of all genital warts and the entire sequence of HPV 6a genome is known since 1995, a highly infectious stock of HPV 6a is not available. This illustrates the difficulty in generating infectious stocks of HPV 6a from its biological source. In addition, there is a strong need among researchers to generate infectious stock of HPV 6a to better understand HPV infectivity in order to develop therapeutic cures for HPV 6a infection. However, methods that are useful in generating infectious stocks of other HPV types do not work effectively for HPV 6a. This is an especially important problem because many HPV types and subtypes show high level of similarity in their genomes, but display varied infectivity levels. Therefore, despite some existing methods for obtaining infectious stocks of certain HPV types, developing an effective method that consistently generates infectious stocks of HPV 6a is not trivial. The present disclosure provides methods and compositions that overcome many of the problems associated with the earlier methods of viral production, e.g. low infectivity, inconsistent infection, infectivity results that are difficult to interpret and lack of reliable propagation of virus.

SUMMARY OF THE INVENTION

[00011] The present disclosure relates to the field of virology and control of viral disease. Improved methods are provided for producing a purified, infectious stock of human papillomavirus (HPV). Methods for selecting biological specimens for production of infectious HPV stocks are also provided. An infectious stock of HPV 6a useful for testing and developing vaccines and other therapeutic agents for HPV related diseases and conditions is disclosed.

[00012] The present disclosure provides compositions of live infectious stock of human papillomavirus (HPV) type 6a and provides methods for producing infectious stocks of other specific HPV types. A method for selecting a suitable biological specimen from a plurality of specimens, where specimens are from lesions known to be associated with HPV infection, is used to generate a highly infectious stock of a specific HPV. The selection method is an improvement over previous methods, e.g. by taking a single shot "rifle" approach rather than a "shotgun approach." A single source, rather than pooled specimens from different patients are used. This method includes the steps of:

(a) selecting the biological specimen that exhibits the highest HPV DNA copy number (at least 10-100 copies per cell); suitable specimens include tissue biopsies of lesions related to HPV infection;

(b) determining whether the LI major capsid protein from the specific virus is in the specimen, indicating the likely presence of infectious virions; DNA copies alone may not signify infectious virus, for that determination proteins must be present also, as determined by the detection of LI major capsid protein; (c) combining data from the DNA copy numbers and the presence of capsid proteins to select the most suitable specimen or specimens to yield an infectious extract containing a specific HPV type. [00013] To determine the DNA copy number and presence of LI protein in a specimen, a biologic specimen such as a condylomata acuminata lesion was examined by several tests. First, LI immunohistochemistry was performed, yielding a positive result. A positive result in this assay indicates that virions are present in the lesion, as opposed to just viral DNA alone. Previous results indicate that approximately 30 to 100 viral copies per cell are needed to yield a positive result in the LI assay, but the results also demonstrate the presence of virions. PCR/reverse blot strip assay was performed on DNA extracted from the lesion. This test was to indicate that at least 10 viral copies per cell were present. This assay reaches a signal plateau at approximately 10 copies per cell of input DNA. [00014] A method for producing an infectious stock of HPV includes the steps of:

(a) specifically selecting an HPV-infected biological specimen by methods disclosed herein rather than processing pools of specimens;

(b) extracting HPV virions from the biological specimen;

(c) infecting human foreskin tissue with the extract containing HPV virions;

(d) implanting the infected tissue into an athymic animal host, such as a mouse, as xenografts;

(e) allowing the xenografts to grow in the animal host; and

(f) isolating an infectious stock of HPV from tumors arising from the xenografts. In particular, HPV 6a stocks were produced.

[00015] A method for testing a candidate vaccine that includes HPV epitopes, for the vaccine's ability to elicit a HPV 6a-neutralizing immune response in a mammal, includes the steps of:

(a) providing serum or secretions from a mammal immunized with the candidate vaccine that include epitopes from HPV 6a; (b) providing an infectious stock of HPV 6a isolated by methods disclosed herein;

(c) contacting an implantable tissue e.g. human foreskin, with the infectious HPV 6a, in the presence and absence of the serum or secretions;

(d) implanting the tissue into an athymic animal host;

(e) comparing the frequency of HPV 6a infection in the implanted tissue contacted with the infectious HPV 6a in the presence of the serum or secretions, with HPV 6a infection in the implanted tissue contacted with the infectious HPV 6a in the absence of the serum or secretions, the infection frequency is expressed by the number of tumors produced in the mouse; and

(f) inferring from (e) the ability of the candidate vaccine to elicit a HPV 6a-neutralizing immune response in the mammal. No tumors due to HPV type 6a infection should be detected if a neutralizing immune response was produced by the vaccine. ] A method for testing candidate antiviral agents directed against HPV 6a, the method includes the step of:

(a) providing an infectious stock of HPV 6a isolated by the methods disclosed herein;

(b) contacting human foreskin tissue with the infectious HPV 6a, in the presence and absence of the candidate antiviral agent;

(d) implanting the infectious tissue into an athymic animal host;

(e) comparing the frequency of HPV 6a infection in the implanted tissue contacted with the infectious HPV 6a in the presence of the candidate antiviral agent, with HPV 6a infection in the implanted tissue contacted with the infectious HPV in the absence of the candidate antiviral agent; and

(f) inferring from (e) the ability of the candidate antiviral agent to reduce or inhibit HPV 6a infection or replication. There should be no or significantly fewer, HPV 6a-infected tumors in the presence of an antiviral agent than in its absence. [00017] The present invention provides a purified infectious stock of HPV type 6a.

This virus stock is substantially free of other HPV types and has been deposited in the ATCC.

DEFINITIONS

Athymic Animal = a thymus-less mouse suitable for accepting foreign implantable tissue such as human foreskin.

Biological Specimen = tissue, serum, blood, skin, and bodily secretions.

DNA Copy Number = relates to the DNA content of a particular HPV in a lesion.

HPV Extract = virions and cells from a biological specimen infected with HPV, usually the cells are epithelial.

Infectious Stock = the biological material capable of replicating and infecting a suitable host under proper conditions e.g., an infectious stock of HPV6a means that the HPV6a is capable of infecting human foreskin tissue implanted in an athymic mouse, thereby causing tumors.

Pseudovirions = non-infectious, non-replicating viral capsids that enclose non- viral genetic material.

Purified = free of HPV types other than the one selected for production.

Virions = virus particle including capsid protein and viral genome.

VLP = virus like particles produced recombinantly.

Xenograft = a tissue from one species transplanted into another species.

DETAILED DESCRIPTION OF THE INVENTION [00018] Improved methods for producing infectious HPV virus, and a stock of infectious HPV 6a virus, are provided. [00019] Several aspects of the present invention use the athymic mouse model including production of infectious HPV stock, and a neutralization assay to see if a vaccine including specific HPV epitopes successfully neutralizes the HPV in organisms innoculated with the vaccine. [00020] Production of Infectious Stocks of HPV (Table I). An infectious stock of

HPV 6a, a genital human papillomavirus, is provided. The preparation of this infectious stock is summarized in this section and described in detail in Example 1. The successful propagation of a substantially pure stock of infectious HPV 6a using the mouse xenograft system is surprising and unexpected, in view of the failure of others to provide infectious stocks in amounts suitable for vaccine production and testing. The inventor's success in propagating a substantially pure HPV 6a type resulted from repeated screenings of samples from patients, until a particular sample was identified that was characterized by a sufficiently high titer of the virus as determined by DNA copy number and the presence of the LI protein, to enable successful propagation of the HPV 6a in the athymic mouse xenograft system. An improvement of the invention is to use a single clinical source rather than a mixture of sources, i.e. a significant improvement was found if a single source, rather than pooled sources, was used. This system is also useful to prepare highly infectious stocks of other HPV types.

[00021] To identify and isolate additional genital HPV types for use in development of vaccines and elucidation of HPV biology, condylomata acuminata lesions containing a high copy number (at least 10 copies of HPV type specific DNA) of HPV and detectable LI major capsid protein were used to prepare infectious virus stocks. Human foreskin fragments were infected with the virus preparations and implanted under the renal capsules of athymic mice. After five months of growth, implant tissue was removed and processed for studies to detect HPV infection. Evidence of HPV infection was noted in some of the implants, but in contrast to HPV 6a-infected epithelium, the implants derived from the new virus preparations contained a lesser degree of acanthosis; less developed koilocytosis; and a reduced number of preserved nuclei in the hyperkeratotic material within the cyst lining.

[00022] The presence of virions of the isolate, HPV 6a, in the implants, rather than non-infectious DNA alone, was verified by immunohistochemical detection of LI major capsid protein. A second extract was made from one of the new implants and used to successfully propagate HPV 6a.

[00023] Several lines of evidence indicate that the HPV 6a-infected implants were substantially, if not completely, free of other HPV types. The low stringency Southern blot using probes of HPV types 6, 11, 16, and 18 yielded bands adding up to the approximate size of an HPV genome. These bands matched the Pstl pattern of HPV6a. Third, the high stringency Southern blot using the HPV 6b whole genomic probe yielded a Pstl restriction pattern identical to that of the low stringency blot. Fourth, the DNA in situ assay was negative for HPV types. PCR/reverse blot strip assay showed that HPV 6a was the only type in the specimens.

[00024] Live, infectious stocks of HPV 6a are prepared by grinding the infected mouse tumor tissue in an appropriate buffer, e.g., phosphate-buffered saline, then freezing the tissue suspension. The live, infectious HPV 6a stock of the present invention is used to produce and/or test a vaccine, or a component of a multi-target vaccine, to elicit neutralizing antibodies against different HPV types. Such vaccines are expected to elicit an immune response against HPV 6a and variants, and other variants which have not yet been identified.

[00025] Infective stocks of HPV 6a can be used in any standard methodology to test candidate vaccines directed to HPV. In an embodiment, the neutralization assay in athymic mouse xenografts as described is used. The steps are as follows. First, HPV 6a virions are purified by cesium gradient centrifugation from the HPV-infected foreskin tissue described herein. The protein content of the HPV 6a preparation is determined by measuring the total protein content of the preparation, then estimating the percentage of the major capsid (LI) protein in the preparation using SDS polyacrylamide gel electrophoresis and immunoblotting with anti-Ll serum.

[00026] A neutralization assay is needed for vaccine development. Neutralization of infectious virus by serum or secretions indicates that immunization has been successfully achieved. For HPV vaccines, there are few HPV types available to test neutralization. The neutralization assay will determine if serum from a mammal vaccinated with an vaccine HPV 6a produces antibodies that can neutralize infectious HPV 6a.

[00027] An assay was developed to determine whether a serum specimen or other biological specimen e.g., cervical lavage, could neutralize a specific type of HPV, such as HPV 6a. This is the most common HPV type found in condylomata acuminata (genital warts). The assay involves infecting a piece of human foreskin tissue with the HPV 6a virus stock in the presence of the biological sample (serum or other biological specimen, such as a cervical lavage) and then implanting the tissue under the renal capsule of an athymic mouse. To perform the neutralization assay, foreskin tissue is obtained from routine circumcision, the dermis is removed, and the remaining epidermal tissue is cut into small fragments. Tissue fragments are added to a tube containing medium in which is dispersed a known quantity of HPV 6a virions, together with various dilutions of the antiserum (including a control in which no antiserum is added). Following a short incubation at 37°C, the fragments are implanted under the renal capsules of athymic mice. To ensure HPV infection adequate for detection, mice are not killed until 10-12 weeks after implantation. Implants are then removed from the mice, measured and placed in formalin solution for preparation of paraffin-embedded sections. If HPV is present in the tissue obtained from mouse tumor, then the serum is not neutralizing. If no infection is detected, then there is evidence that the serum contains neutralizing antibodies.

)28] The assay is useful to test for neutralizing antibodies in subjects immunized with HPV vaccines that have been developed by any group or any company. The assay is functional and can be used to test serum or other secretions (cervical) for neutralizing antibodies against HPV 6a.

Ϊ29] For the DNA in situ assay to detect the presence or absence of HPV 6a infection, the sections of foreskin implants are deparaffinized and processed with the Digene Tissue Hybridization Kit (Digene Diagnostics, Beltsville Md.) according to the manufacturer's instructions. Following deparaffinization, sections are hybridized with a biotinylated HPV 6a DNA probe. Detection of the hybridized probe is performed by incubation of slides with a streptavidin-alkaline phosphatase conjugate, followed by reaction with a colored product-forming substrate. Cells positive for HPV DNA are identified as purple nuclear-staining of differentiated epithelial cells in the foreskin implants by light microscopy. Neutralization of HPV 6a is demonstrated by an absence of HPV 6a DNA in the DNA in situ hybridization assay. [00030] HPV 6a virions are also used to screen for antiviral agents. This is accomplished using the mouse xenograft system in a manner similar to that described herein for identifying neutralizing antibodies. Infection and implantation are carried out in the presence or absence of a candidate antiviral agent, and the effect of the agent on subsequent infectivity and replication of the virus in the graft tissue is observed.

EXAMPLE 1: Identification and Characterization of HPV 6a and Propagation in Athymic Mouse Xenograft System [00031] To identify and characterize genital HPV types for elucidation of HPV pathogenesis and development of vaccines, an extract was prepared from a biopsy from one patient selected because of the presence of HPV 6a. This example describes the identification of an HPV isolate (HPV 6a) from this source, and its successful propagation in the athymic mouse xenograft system. [00032] Preparation of virus extract from single tissue specimens. Excision biopsies of exophytic condylomata acuminata lesions were performed as previously described (Brown et al, 1994b). All of the biopsy samples were analyzed by Southern blot analysis or hybrid capture for detection of HPV DNA (Brown et al, 1993).

PCR/reverse blot strip assay and immunohistochemistry were performed to determine the copy number and the presence of LI protein as described in the Materials and Methods. [00033] Infection and implantation of tissue into athymie mice. Infection of human foreskin fragments and growth in athymic mice was performed as described by Kreider et al. (1987) with modifications as previously described (Brown et al., 1995). Experiments were performed using crude extracts and using eight athymic mice. Additional experiments were performed using extracts from tumors resulting from the first experiments to demonstrate propagation of virus in the athymic mouse xenograft system. Mice were sacrificed 150 days after implantation of foreskin fragments. [00034] Light microscopy, immunohistochemistry, and electron microscopy. A portion of each implant was placed in zinc formalin, and paraffin-embedded sections were prepared. One section was stained with hematoxylin and eosin. Histologic evaluation of sections was performed, using uninfected implant tissue and HPV 6a- infected human foreskin implants grown in athymic mice for 150 days for comparison (Brown et al, 1994b). Additional sections were deparaffinized and used to detect LI protein with a rabbit polyclonal antiserum raised against a bacterially expressed HPV 11 trpE/Ll fusion protein, as previously described (Brown et al, 1994b). This antiserum has been shown to react with LI protein from several HPV types. Preimmune rabbit serum was used as control serum. Antibody binding was detected using the Vectastain ABC Kit (Novocastra, Newcastle upon Tyne, UK). Positive cells for LI protein were identified by brown staining of nuclei in differentiated keratinocytes. [00035] Extraction of DNA and Southern blot hybridization to confirm the presence of

HPV 6a were done as described in Materials and Methods.

RESULTS

[00036] Propagation of HPV 6a. To verify that HPV 6a was propagated in the athymic mouse xenograft system, an extract was made from an implant from the initial experiment and used to infect a second human foreskin. Fragments of this foreskin were implanted into 16 athymic mice. Mice were sacrificed after 150 days of growth and implants were removed. Histological analyses of these implants was similar to that of the original group of HPV 6a implants.

[00037] Overall Infectivity. Infection of foreskin xenografts was performed using an

HPV 6a extract. Four separate experiments have been performed with the HPV 6a extract, using different dilutions. A total of 20 xenografts have been recovered from 24 mice, 17 were positive for HPV 6a (57%).

[00038] Neutralization assay (Table II).The serum samples used were from an

African Green Monkey immunized with HPV 6a VLPs (Merck). Immune serum is from vaccinated organisms. The combined data from two individual neutralization experiments is as follows:

MATERIALS AND METHODS

A. Biopsy and preparation of tissue sections

[00039] Patients were evaluated for condylomata acuminata who had lesions clinically consistent with the diagnosis of condylomata acuminata, either on gross examination in the case of exophytic lesions, or following dilute acetic acid staining in the case of lesions of the uterine cervix. A condylomata acuminata lesion was removed from the genital region of a male subject. A fragment of the lesion was placed in zinc formalin (Z-Fix, Anatech, Battle Creek, MI), and tissue was embedded in paraffin on the same day as fixation. Serial sections were prepared (4 um). One section from each tissue was stained with hematoxylin and eosin for confirmation of histology consistent with papillomavirus infection. Some of the tissue was frozen in liquid nitrogen.

B. Extraction of DNA and Southern blot

[00040] A portion of the tissue frozen in liquid nitrogen was dismembrated with a

Braun mikro-dismembrator II (B. Braun Instruments, Melsungen, Germany). The resulting material was solubilized in 0.1% SDS and 10 mM EDTA, treated with proteinase K at 60°C, and extracted with phenol/chloroform/isoamyl alcohol. DNA was precipitated with sodium acetate and ethanol and quantitated by spectrophotometry. The presence of high molecular weight DNA was established by agarose gel electrophoresis followed by staining with ethidium bromide. Southern blots were performed under stringency conditions (Tm-15°C) with 10 ug of DNA digested with Pstl for two hours at 37°C. Following electrophoresis, DNA was transferred to a nylon Hybond-N+ membrane (Amersham) by the method of Southern. After transfer, the membrane was prehybridized for 16 hours, then hybridized in a solution containing 5 million counts per minute per ml of heat denatured, P-labelled genomic HPV 6b DNA purified away from the pBR 322 vector. The nylon membranes were then washed in decreasing salt concentrations, and autoradiography was performed. HPV type and subtype was determined by analyzing the molecular weights of Pstl restriction fragments in the autoradiograph. C. PCR reverse blot strip assay

[00041] The PCR reverse blot strip assay (Roche) was performed on DNA from the condylomata acuminata lesion and numerous other lesions to compare HPV 6a viral copy number. To determine specimen adequacy for DNA extracted from each lesion, the GH20 PC04 human β-globin target was co-amplified with HPV sequences. Each primer was labeled with a 5' biotin molecule. Reactions were amplified in a Perkin Elmer TC9600 Thermal Cycler (Perkin Elmer Corp., Foster City, CA) using the following profile: 95°C for 9 minutes (AmpliTaq GOLD activation); 40 cycles of 95°C for 1 minute (denaturation), 55°C for 1 minute (annealing), 72°C for 1 minute (extension); 72°C for 5 minutes (final extension).

[00042] Each amplification reaction contained 500 ng of specimen DNA. Known positive and negative (no DNA) specimens were included in each assay as controls. A reverse blot strip assay was used that contains 29 probe lines plus one reference line, detecting 27 individual HPV genotypes and two concentrations of the β-globin control probe. BSA-conjugated probes for each HPV type were deposited in a single line for each of HPV types. The high and low risk HPV types are visually separated by the β- globin control lines, such that all types between the reference and β-globin control lines are associated with high cancer risk, and all types beyond the control lines are associated with low cancer risk.

[00043] Hybridization and detection of PCR products bound to immobilized probes was then performed. Developed strips were photographed at a standard magnification using an A2000 digital imaging system (Alpha Innotech, San Leandro, CA). The image of the group of strips was scanned using the one-dimensional, multiple lane scanning module of the Alphaease^® software that is an integrated component of the imaging system. The measured density of each band (peak height) was used as the basis of a semi-quantitative, five-point (1 through 5) scoring system for positive bands. The low and high concentration β-globin bands served as reference points for this scoring system. The low positive β-globin band was assigned a value of 2 and the high positive band a value of 4. Bands that were clearly visible with sharp margins extending the full width of the strip but had a peak height that was less than that of the low positive were assigned a value of 1 while bands with a height greater than the high positive were given a value of 5. The other values were interpolated between those extremes.

D. Detection of LI major capsid protein

[00044] Immunohistochemistry was performed to determine if the LI major capsid protein was present in the condylomata acuminata lesion. A positive signal for LI would indicate an increased likelihood of infectious virus in the lesion. Additional sections were deparaffinized and incubated overnight in either preimmune rabbit serum or anti-Ll serum produced by the inventor at 1 :500 dilution. The Vectastain ABC kit (Vector Laboratories, Burlingame, CA) was used according to the manufacturer's instructions to detect antibody binding to LI protein in tissue sections.

E. Preparation and testing of the HPV 6a extract

[00045] Tissue frozen in liquid nitrogen was crushed with a mortar and pestle in sterile

KGM (Keratinocyte growth medium, Clonetics, San Diego, CA). This patient-derived extract was used to initially propagate the HPV 6a virus in the athymic mouse xenograft system. Human foreskin fragments were incubated at 37°C in 400 μl of the patient-derived extract, and then implanted under the renal capsules of eight athymic mice. Implants were removed after twelve weeks of growth. Briefly, the mice are euthanized by overdose with inhalant anesthesia (Halothane or Isoflurane), in a closed bell jar, in a fume hood. A dorsal incision is made to expose the kidney. The implants, identified by a tumor-like fragment under the renal capsule are carefully dissected away from the kidney tissue and weights of each implant were determined. After weight measurements, the implants were divided, and portions were reserved for DNA extraction, preparations of paraffin-embedded sections, and for preparation of a new virus extract. As indicated herein, implants that were infected with HPV 6a were used to prepare a large amount of infectious HPV 6a extract.

F. Histology of implants

[00046] Histologic examination of the condylomata acuminata lesion revealed typical features of condylomata acuminata: koilocytosis, acanthosis, and parakeratosis. The implants removed from mice displayed similar features.

G. Southern blot and PCR/reverse blot strip assay results

[00047] Southern blot analysis demonstrated the presence of HPV 6a. The

PCR/reverse blot strip assay revealed 5+ HPV 6, indicating a high viral copy number. The PCR reverse blot strip assay does not discriminate between HPV 6 subtypes. H. Infectivity of the HPV 6a extract

[00048] Athymic mouse foreskin xenografts (implants) were analyzed for evidence of

HPV 6a infection. A portion of each HPV 6a-positive implant was used to prepare the extract. Tissue was frozen in liquid nitrogen was crushed with a mortar and pestle in sterile KGM (Keratinocyte Growth Medium) (Clonetics, San Diego, CA). To further propagate the HPV 6a virus, additional human foreskin fragments were incubated at 37°C in 80 μL of the new implant-derived virus extract plus 320 μL of KGM (1 :5 dilution of extract), then implanted under the renal capsules of eight additional athymic mice. Implants were removed after twelve weeks of growth. Implants were divided, and portions were reserved for DNA extraction and for preparations of paraffin-embedded sections. Fifty percent of the new implants were positive for HPV 6a, demonstrating the infectivity of the new HPV 6a extract. I. RT-PCR

[00049] The RT-PCR for HPV 6a is a validating tool for the infectivity of the HPV 6a extract. HPV 6a is applied to cells in culture, then after 4 days, RNA is harvested. An RT-PCR is done to show that transcripts of HPV 6a were present, thus verifying that infection had occurred. Primers for the RT-PCRare designed by methods known to those of skill in the art. Standard Operating Protocol (SOP) for Human Papillomavirus Type 6a (HPV 6a) Neutralization Assay in the Athymic Mouse Xenograft System

Table of Contents

A. Neutralization Assay

B. Implantation in athymic nude mice

C. Sacrifice of athymic mice

D. Processing of formalin fixed implants

E. Hematoxylin and eosin staining for histology

F. DNA in situ hybridization

G. Interpretation of results

A. Neutralization Assay

Introduction Antiserum or monoclonal antibodies raised against HPV 6a virions or viruslike particles (VLPs) have been shown to neutralize HPV 6a infections of human foreskin implants grown in athymic mice. Because HPV cannot be grown by standard culture methods, the mouse xenograft system provides an important model for testing the ability of antibodies to protect against HPV infection. All reagents will be recorded for each experiment in the Neutralization Assay Record

Equipment List

Disposable petri dishes

Disposable polypropylene tubes

Microcentrifuge tubes

Laminar flow hood with germicidal ultraviolet lamp

Parafilm

Sterile needles, 20 gauge

Scalpel blades, #10

Scalpel, #10 Adson forceps (Thomas Scientific, 3863-F15)

Surgical scissors (Thomas Scientific, 3867-Y60)

Styrofoam square, Vz inch thick

Heat block capable of maintaining 37oC

Microcentrifuge

Sharps disposal container

Pipette

Pipette tips

Bench towel

Sterile gloves

Autoclips (Thomas Scientific, 1111-C15)

Autoclip (Thomas Scientific, 1111-C40)

Autoclip remover (Thomas Scientific, 1111-C45)

Curved dissecting forceps (Thomas Scientific, 3865-J50)

1 cc tuberculin syringes/25 gauge needle

Gauze pads

Reagent List

Minimal Essential Medium Eagle (Sigma, M-5650)

Antibiotic/Antimycotic Solution (Sigma, A-5955) (contains 10 u/nil penicillin, 10 mg/ml streptomycin, and 25 mg/ml amphotericin B)

HPV 6a virus

Human serum samples to be tested

70% ethanol (Aaper Alcohol and Chemical Co.)

Pentobarbital sodium injection, 50 mg/ml, diluted 1:10 in sterile H2O (Abbott Laboratories)

Controls S700 or S696 African Green Monkey sera (Week 28); received from Merck

Research Laboratories 7-13-96. One of these sera is used as a positive neutralization control at a dilution of 1 :50. Each serum sample has been shown to neutralize in multiple experiments at dilutions up to 1:320

Purified HPV 6a virions in MEM with S700 or S696 pre-immune serum at a dilution of 1 :50 are used as a positive growth control. Two mice are used for the growth control and two mice are used for the neutralization control. Nomenclature System for Neutralization Assay [00052] The first experiment performed with a batch of serum samples is designated with a number from 1 through 45 without a letter A following the number. The designated number for the experiment is recorded on the Mouse Sacrifice Worksheet as the "Batch Number". If an experiment is found to be invalid (see criteria below), the repeat experiment has the same number as the invalid one, followed by the letter B (for example, experiment IB). Subsequent experiments with the same serum samples are performed if the follow-up experiment is also found to be invalid, and are given letters C, D, E, etc. as needed for example, experiment IC. Explanation of sterile technique [00053] Sterile technique is used for all implantation procedures. Specific steps include use of a laminar flow hood in preparation of tissue fragments, infection of tissue with HPV 6a, and addition of serum in sterile microfuge tubes. Gloves are worn at all times. Tissue fragments are prepared in sterile petri dishes using instruments sterilized by autoclaving. The opening of the Sigma MEM is flamed using a Bunsen burner. In the athymic mouse facility, tissue fragments are implanted into mice under a laminar flow hood using sterile instruments. Investigators wash hands prior to surgery and wear surgical hair cover, masks, gowns, shoe coverings, and gloves.

B. Implantation in athymic nude mice

Preparation of tissue [00054] Human foreskin tissue is obtained from the Wishard Memorial Hospital nursery from routine circumcisions. The tissue is placed immediately into Minimal Essential Medium Eagle (Sigma M-5650) containing Antibiotic/Antimycotic Solution (Sigma A5955) at 1:10,000 dilution. It is transported to the HPV laboratory, where it is prepared using sterile technique in the vertical laminar flow hood.

[00055] The foreskin is transferred to a sterile petri dish containing excess medium to rinse away any remaining traces of betadine left from the circumcision procedure. The foreskin is then staked out using 20 gauge sterile needles on a piece styrofoam covered with a piece of Parafilm. The vascular dermis is removed using sterile scalpel and scissors. When only epidermis remains, the tissue is placed in a clean petri dish with MEM. The foreskin is scored several times on each side with a sterile scalpel blade. The foreskin is then cut into small pieces, approximately l x l mm. Labeled sterile microcentrifuge tubes are prepared with MEM, HPV 6a virus, and serum according to the current experiment. Foreskin fragments are added to each tube, two pieces per mouse to be implanted. The tubes containing foreskin, virus, MEM, and serum are incubated in a 37oC heat block for 90 minutes. Implantation

[00056] The tubes containing tissue, virus, and sera are transported to the Laboratory

Animal Research Center in the heat block. The implantation procedure is performed using sterile technique in a vertical laminar flow hood. Sterile technique includes hand washing, wearing of gowns, head and shoe covers, and sterile surgical gloves. In addition, all instruments are autoclaved prior to use. Athymic nude (nu/nu on a balb/c background) mice are anesthetized with pentobarbital, 50 mg/kg, via intraperitoneal injection. A dorsa-lateral incision is made to expose the kidney. A 2 mm linear transverse incision is made through the renal capsule and a piece of infected foreskin tissue is implanted under the renal capsule with forceps. The incision is closed with autoclip staples, and the procedure is repeated on the opposite side. The mice recover from the anesthesia within approximately 20 minutes of implantation and are observed and housed for a ten week period. Daily care of the mice is resumed by the Indiana University Laboratory Animal Research Center staff during this ten week period.

C. Sacrifice of athymic mice Introduction [00057] After ten weeks of growth, the athymic mice are sacrificed for the recovery of implants to determine neutralization activity. Implants are examined by three methods: physical evidence (weight, size, and geometric diameter), histological examination, and DNA in situ hybridization.

Equipment List

Teri wipers

Surgical scissors

Adson forceps

Scalpel blades

Small disposable weigh boats

Digital balance (Sartorius 1264MP)

Specimen bottle, 4 ml (Wheaton, #225532)

Bio-freeze vials, 2 ml (Costar, #2128)

Biohazard disposal bags

Autoclave

Neutralization Assay Record

Reagent List

IX Zinc Formalin (Anatech Ltd., Battle Creek, Michigan)

Removal of Implants [00058] After the ten week period, the mice are euthanized by overdose with inhalant anesthesia (Halothane or Isoflurane), in a closed bell jar, in a fume hood. Mice are then transported to the HPV laboratory for dissection. A dorsal incision is made to expose the kidney. The implant, identified by a tumor-like tissue fragment under the renal capsule is carefully dissected away from the kidney tissue and placed in a labeled weigh boat. The implants are numbered chronologically, using an A or B to designate those implanted under the right and left kidneys of each mouse. Each implant is weighed on the Sartorius 1264MP balance to +/- 10 mg. They are measured in three dimensions in millimeters and the mean geometric diameter is determined for each implant (the cubed root of the volume). These three measurements are recorded on the Mouse Sacrifice Worksheet generated for each experiment. The implant is then divided with a sterile scalpel blade. Based on the size, all or part of the implant is fixed in 2 ml Zinc-Formalin for histological sectioning. If the implant is of a significant size, a portion is quick frozen and stored at -70oC. Frozen tissue is noted on the Mouse Sacrifice Worksheet under the "comments" column. In cases no implant can be found in the mouse, a "0" is recorded in the Mouse Sacrifice Worksheet for that implant. Remaining kidney tissue and mouse carcass are autoclaved and disposed of according to university guidelines.

D. Processing of formalin fixed implants Introduction The processing of tissue fixed in Zinc-Formalin is done by the

Department of Anatomy, Indiana University School of Medicine. Equipment List

Shandon Hypercenter XP

Tissue Tek embedding center

Reichert-Jung Microtome 820

Water Bath glass slides

Reagent List

100% ethanol

95% ethanol

Xylenes

Paraplast

Protocol

70% ethanol— hold 70%) ethanol — 30 minutes 80%) ethanol — 30-45 minutes 80% ethanol— 30-45 minutes 95% ethanol — 30-45 minutes 95% ethanol — 30-45 minutes 100% ethanol— 30-45 minutes 8. 100% ethanol— 30-45 minutes

9. Xylenes — 30-45 minutes

10. Xylenes — 30-45 minutes

11. Paraffin — 45-60 minutes

12. Paraffin — 45-60 minutes

[00060] All times depend on the size, density and number of blocks being processed.

Large samples are processed for the maximum time as noted above, as are numerous blocks being processed at one time. When the blocks have cooled, they are cut on the Reichert-Jung microtome at a thickness of 5 microns. Cut sections are floated on a tissue water bath and picked up on a positively charged slide. Eight slides are cut for each tissue section. The first and eighth section are stained with hematoxylin and eosin (see below).

E. Hematoxylin and eosin staining for histology

Introduction [00061] The cytoplasm and nucleus reflect many of the dynamic processes of the cell.

Hematoxylin is the preferred nuclear stain for routine histology sections because it increases nuclear detail and contrast within the cell. HPV 6a-infected human foreskin implants grown in athymic mice or uninfected control implants, are examined for evidence of HPV 6a infection.

Equipment List staining dishes paraffin embedded tissue glass coverslips

Reagent List

Xylenes

100% Ethanol

Harris hematoxylin non-acidified

Eosin Y alcoholic stain

Ammonia solution (28% ammonium hydroxide 3:1000 in dH2O) Acid alcohol (HC1 1 :200 in dH2O)

Mounting medium

Protocol

Deparaffinize slides in 2 changes of xylenes

Hydrate sections through graded ethanol changes to water.

Place slides in Harris hematoxylin for 6a minutes. This is a regressive stain and is used to stain the nuclei blue.

Gently agitate the slides in running water for 20 seconds.

Differentiate the slides in acid alcohol solution 1-5 seconds.

Rinse slides well in running tap water for 5 minutes to remove excess acid. Acid will cause the differentiating action to continue and result in a poor nuclear stain.

Blue section for 30-90 seconds in ammonia solution.

Wash slides in running tap water for 5 minutes.

Place slides in alcoholic eosin solution for 1-3 minutes to stain the cytoplasm pink.

Dehydrate the slides through reverse graded ethanol changes to xylenes.

Add a drop of mounting medium and coverslip.

Interpretation of results Hematoxylin and eosin stained sections are examined under low and high power. If the nuclei are not clearly visible, or if the color differentiation is not apparent (blue nuclear staining and pink cytoplasmic staining), more slides are cut and restained. Sections are examined initially for epithelial tissue. If none is present, additional sections are cut. If there is still no epithelium present, the histology is recorded on the Mouse Sacrifice Worksheet as NE for "no epithelium". The changes in epithelium that are regarded as suggestive of HPV 6a infection include thickening (acanthosis) compared to control epithelium, koilocytosis, hyperkeratosis, and the presence of nuclei in the corneal layers of cells. Absence of these characteristics is recorded as negative for histological evidence of HPV 6a infection.

F. DNA in situ hybridization Introduction The ENZO in situ Hybridization System is used to localize specific DNA sequences within the cells of tissue specimens. This system utilizes streptavidin as a link between a biotinylated probe and biotinylated reporter molecules of alkaline phosphatase (AP). A record of the probe used for each assay is kept on the DNA in situ Assay Probe Record. Following hybridization of the biotinylated probe to the cellular target sequences, streptavidin is bound to the probe via one of the four binding sites on the streptavidin molecule. The biotinylated AP is then allowed to saturate one or more of the remaining binding sites on the streptavidin. The AP enzyme produces a colored reaction product as the result of hydrolysis of its substrate, BCIP (5-bromo-4- chloro-3-indolyl phosphate) and the concomitant reduction of NBT (nitro-blue tetrazolium). The blue-purple reaction product of this reaction is insoluble and thus is deposited directly at the site of hybridization.

Equipment List

Slide warmer or oven capable of maintaining temperature of 95oC

Oven capable of maintaining temperature of 6O-8O0C

Incubator capable of maintaining temperature of 37oC

Humidity chamber or box lined with wet paper towels

Metal slide tray

Staining dishes and slide rack

Hybri-slips (Research Products International, Catalog No. 247455)

Glass coverslips, 18 x 18 mm and 22 x 40 mm

Light microscope

Pipettes

Stirbars and magnetic stirplate

Kimwipes

Fume hood

Aluminum foil

Reagent list ENZO Pathogene HPV in situ Typing Assay for Tissue Sections Kit (Catalog No. 32895)

Xylenes

Absolute ethanol (Aaper Alcohol and Chemical Company) 90% ethanol 70% ethanol 50% ethanol

Deionized distilled water

Accu-Mount 60 Mounting Medium (Baxter Scientific Products, Catalog No. M7630-2)

Controls [00064] A histologically-positive HPV 6a mouse implant is included in each DNA in situ hybridization assay as a positive control. A paraffin-embedded human foreskin is used as a negative control. Reagent Preparation [00065] Signasure Wash Buffer: Dissolve one packet in 1 L dH2O and adjust pH to

8.0 with 5 M NaOH. Store at 4°C between uses. Warm to room temperature before use. [00066] 100X Proteinase K Stock solution: Resuspend Proteinase K in 2.0 ml

Signasure Wash Buffer. Aliquot 0.1 ml (2.5 mg/ml stock) into separate tubes and store at -20°C. Working solution of Proteinase K has final concentration of 25 mg/ml. Use Signasure wash buffer for dilutions. Make fresh working solution for each experiment. [00067] Dilute eosin counterstain to a final volume of 200 ml with absolute ethanol.

Can be stored at room temperature in tightly capped bottle. Day 1 Assay Protocol Preparation

Pre-heat vacuum oven (without use of vacuum) to 80°C. Pre-heat incubator to 37°C. Make a humidity chamber by lining an airtight plastic container with paper towels and saturate them with dH2O.

Allow probe and other reagents to come to room temperature.

Pre-treatment of Sections

Place slides (maximum of 50) in slide holder.

Heat slides 15 minutes at 80oC

Place in dish containing:

Xylene x 10 minutes

Xylene x 10 minutes

100% Ethanol x 1 minute

100%) Ethanol x 1 minute

90%) Ethanol x 1 minute

70% Ethanol x 1 minute

50% Ethanol x 1 minute dH2O x 1 minute

Wipe off excess water and dry slides at 37°C for 10 minutes.

Cover tissue section with 25 mg/ml Proteinase K solution (-0.05 ml) and incubate at 37°C for 5 minutes for 4-5 micron tissues sections.

Tap off Proteinase K and soak slides at room temperature in Signasure Wash Buffer for 1 minute.

Dehydration

Place in dish containing: dH2O x 1 minute

50%) Ethanol x 1 minute

70% Ethanol x 1 minute

90% Ethanol x 1 minute

Dry slides at 37°C for 10 minutes.

Hybridization

Lay the slides flat and add one drop of probe to each section.

Place a Hybrislip over each section and place the slides on a metal tray. Place the tray of slides in vacuum oven at 95°C for 10 minutes.

Transfer the slides to a humidity chamber and incubate at 37°C for 3 hours or overnight.

Day 2 Assay Protocol

Washes

Remove coverslips by soaking in room temperature Signasure Wash buffer.

Soak slides in Signasure Wash buffer for an additional 5 minutes with gentle agitation.

Wipe off excess buffer and add 2-3 drops of Hybridization Wash Reagent. Incubate for 10 minutes at 37°C.

Soak slides for 1 minute in Signasure Wash Buffer 3 times with gentle agitation.

Remove the slides individually from Wash Buffer; do not allow the slides to dry.

Place the slides flat and blot around each section with a Kimwipe.

Apply enough AP Detection Reagent to completely cover the specimen and incubate at 37°C for 30 minutes.

Rinse off with Signasure Wash Buffer from a wash bottle and place in coplin jar of Signasure. Continue incubating 1 additional minute after slides are all in Signasure. Soak slides 1 minute in Signasure two more times.

Detection

Prepare NBT/BCIP Reaction Mixture: To each 1.0 ml of AP Reaction Buffer, add 2.5 1 NBT Reagent and 10 1 BCIP Reagent. This solution should be protected from direct bright light.

Place the slides flat and blot around each section with a Kimwipe. Apply enough NBT/BCIP reagent to each slide to completely cover the specimen and incubate at 37 oC for 20 minutes protected from direct bright light.

Tap off the substrate solution from each slide and immerse the slides in Signasure Wash Buffer. After all slides are placed in buffer, transfer them to dH2O for up to 2 hours.

Counter staining and coverslip mounting

Place in dish containing: dH2O x 10 minutes

Counterstain with Eosin x 3 minutes

95% ethanol x 1 minute

100% ethanol x 1 minute

100% ethanol x 1 minute

Xylenes x 1 minute

Add a drop of mounting media and coverslip to each slide.

Allow the slides to dry in the fume hood.

Interpretation of DNA in situ assay results

[00068] The presence of target DNA is indicated by a purplish blue precipitate. Dark staining is visible in the nuclei of cells in the outer epithelial layer. Non-specific staining may occur, such as staining of mouse kidney tissue. Therefore, correlation with tissue histology is critical for accurate interpretation of the DNA in situ assay. Tissue which does not contain target DNA should appear pink as a result of the counterstain. Results are expressed as either positive or negative for HPV DNA. A single positive cell in a section, assuming that a correlation with histology has been made appropriately, provides evidence that the implant is infected with HPV 6a.

G. Interpretation of results Validity of neutralization experiment

[000S9] Neutralization Control: The assay is considered valid if 100%) neutralization is achieved by the neutralization control serum (at least two interpretable implants). If any neutralization control implant is positive, the assay is declared invalid and must be repeated.

[00070] Growth Control: The assay is considered valid if >50% of the positive growth control implants are determined positive (4/4, 3/3, 2/2, 3/4, or 2/3). In addition, if 50% of the positive growth control implants are detemiined positive (2/4 or 1/2), and one patient serum sample in the experiment yields at least 4/6 positive implants, the assay is considered valid. If <50% of the positive growth control implants are determined positive (1/3 or 1/4) the assay is declared invalid and must be repeated. [00071] Following completion of all assays, each implant is given a final grade based on the accumulated data. If both histology and DNA in situ are negative, the implant will be recorded as negative. If both histology and DNA in situ are positive, the implant is recorded as positive. If an implant is negative by histology and positive by DNA in situ, the implant is considered positive by DNA in situ and positive for the final grade. If an implant is positive by histology and negative by DNA in situ assay, the DNA in situ assay is repeated for those slides. If the same results are found on the second assay, they are recorded as valid, and the implant is recorded negative for DNA in situ and positive for the final grade.

Interpretation of neutralization assay for a serum sample

[00072] For a given serum sample, a decision is made on whether neutralization has occurred based on results of a valid neutralization assay. A sample is considered neutralizing if all implants are histologically negative for evidence of HPV infection and the DNA in situ assay is negative for all implants. At times not all implants will contain epithelium. The particular serum is designated neutralizing if at least three implants can be shown to contain epithelium and are negative in both histology and for HPV DNA. In most cases however between 4 and 6 implants are sufficient for each serum sample. A sample is called not neutralizing if any implant shows evidence of HPV infection by either histology or in the DNA in situ assay. A specific serum sample is re-tested under the following circumstances: first, if the entire assay is deemed invalid, then each serum sample in that assay needs to be repeated. Second, if less than three implants contains sufficient epithelium for a careful assessment of histology or DNA in situ assay then that particular serum is added to a subsequent assay. Lastly, if there is strong evidence that an error was made in the preparation of the particular neutralization assay using a given serum then that serum is added to a subsequent study to verify whether it is neutralizing or not neutralizing. Any variations from the protocal will be recorded on the Protocol Variance Record. TABLE I:

Preparation of Infectious HPV Stock condylomata lesion tissue from a patient

1 frozen

I crushed with mortar & pestle in KGM media

I patient-derived extract incubated with human foreskin fragments i

implanted under renal capsules of athymic mice

4

12 weeks i

HPV-positive implant i tissue frozen

I crushed with mortar & pestle in KGM media i incubated with foreskin fragments (implant-derived extract) i propagation

4

HPV stock of infectious virus

¹When DNA is being prepared, liquid N₂ is suitable.

TABLE II:

HPV Neutralization Assay

HPV crude extract incubated with human foreskin tissue in media (KGM) preimmune or immune serum or secretions

4 implanted under renal capsules of athymic mice

4

4 months (16 weeks)

4 implants analyzed for HPV infection.

4 if no infection, demonstrates presence of neutralizing antibodies in serum or secretions of a vaccinated mammal.

DOCUMENTS

These publications are incorporated by reference to the extent they relate to the present invention.

Bonnez W., Rose R. C, Reichman R. C. (1992 ) J. Infect Dis. 165(2):376-80

Brown D. R., Bryan, J. T., Pratt, L., Handy, V., Fife, K. H., and Stoler, M. H. (1995) Virology 214(1), 259-263.

Brown D. R., Bryan, J. T., Cramer, H., Katz, B. P., Handy, V., and Fife, K. H. (1994a) J. Infectious Diseases 170(4), 759-765.

Brown D. R., Fan, L., Jones, J., and Bryan, J. (1994b) Virology 201(1), 46-54.

Brown D. R., Bryan, J. T., Cramer, H., and Fife, K. H. (1993) J. Clin. Microbiol. 31(10), 2667-2673.

Brown D. R., Schroeder J. M., Bryan J. T., Stoler M. H., Fife K. H. (1999) J. Clin. Microbiol. 37(10):3316-22.

Brown D. R., Bryan J. T., Schroeder J. M., Robinson T. S., Fife K. H., Wheeler C. M., Barr E., Smith P. R., Chiacchierini L., DiCello A., Jansen K. U. (2001) J. Infect. Dis. 184(9): 1183-6.

Bryan et al, 1997. J. Med. Virol. 53:185-188.

Christensen N. D., Kreider J. W., Cladel N. M., Galloway D. A. (1990) Virology 175(l):l-9

Christensen N. D., Kreider J. W., Shah K. V., Rando R. F., (1992) J Gen Virol. 73:1261-7.

Christenseri N. D, Hopfl R, DiAngelo S. L., Cladel N. M., Patrick S. D., Welsh P. A., Budgeon L. R., Reed C. A., Kreider J. W. (1994) J. Gen. Virol. 75: 2271-6

Christensen N. D. and Kreider J. W. (1990) J. Virol. 64(7):3151-6

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Fife K. H., Cramer H. M., Schroeder J. M., Brown D. R. (2001 ) J. Med. Virol. 64(4):550-9.

Hofmann K. J., Cook J. C, Joyce J. G., Brown D. R, Schultz L. D, George H. A, Rosolowsky M, Fife K. H., Jansen K. U. (1995) Virology 209(2):506-518 Koutsky L. A., Ault K. A., Wheeler C. M., Brown D. R., Barr E, Alvarez F. B., Chiacchierini L. M., Jansen K. U. (2002) N. Engl J Med. 347(21):1645-51 ,

Kreider J. W., Howett, M. K., Leure-Dupree A. E., Zaino R. J., Weber J. A. (1987) J. Virol 61(2): 590-593

Kreider J. W., Howett M. K., Wolfe S. A., Bartlett G. L., Zaino R. J., Sedlacek T, Mortel R. (1985) Nature 317(6038):639-41

Lowe R. S., Brown D. R., Bryan J. T., Cook J. C, George H. A., Hofmann K. J., Hurni W. M., Joyce J. G., Lehman E. D., Markus H. Z., Neeper M. P., Schultz L. D., Shaw A. R., Jansen K. U. (1997) J Infect. Dis. 176(5):1141-5.

U. S. Pat. No. 6,290,965

U. S. Pat. No. 6,284,532

Claims

I CLAIM: [C1] 1. A method for selecting an individual biological specimen from a plurality of specimens to generate a highly infectious stock of a human papillomavirus type, the method comprising:

(a) selecting the biological specimen that exhibits the highest human papillomavirus DNA copy number; and

(b) selecting the biological specimen that also has a detectable human papillomavirus LI major capsid protein.

[C2] 2. The method of claim 1 , wherein the DNA copy number is at least 10 copies per cell.

[C3] 3. A method for producing an infectious stock of human papillomavirus, the method comprising:

(a) selecting a human papillomavirus infected biological specimen by the method of claim 1;

(b) extracting human papillomavirus from the biological specimen;

(c) infecting human foreskin tissue with the extract containing human papillomavirus;

(d) implanting the infected human foreskin tissue in an athymic animal host as xenografts;

(e) allowing the xenografts to grow in the animal host; and

(f) isolating an infectious stock of human papillomavirus from tumors resulting from the xenografts.

[C4] 4. The method of claim 3, wherein the human papillomavirus is human papillomavirus 6a.

[C5] 5. The method of claim 3, wherein the animal host is a mouse.

[C6] 6. A method for testing a candidate vaccine that includes human papillomavirus type 6a, for the candidate vaccine's ability to elicit a human papillomavirus 6a- neutralizing immune response in a mammal, comprising:

(a) providing serum or secretions from a mammal immunized with the candidate vaccine; (b) providing an infectious stock of human papillomavirus 6a isolated by the method of claim 4;

(c) contacting an implantable tissue that is capable of being infected by human papillomavirus with the infectious human papillomavirus 6a, in the presence or absence of the serum or secretions;

(d) implanting the infected tissue into an athymic animal host;

(e) comparing the frequency of human papillomavirus 6a infection in the implanted tissue contacted with the infectious human papillomavirus 6a in the presence of the serum or secretions, with human papillomavirus 6a infection in the implanted tissue contacted with the infectious human papillomavirus 6a in the absence of the serum or secretions; and

(f) inferring from (e) the ability of the candidate vaccine to elicit a human papillomavirus 6a-neutralizing immune response in the mammal.

[C7] 7. The method of claim 6, wherein the implanted tissue is human foreskin.

[C8] 8. The method of claim 6 wherein the animal host is a mouse.

[C9] 9. A method for testing candidate antiviral agents directed against human papillomavirus 6a, the method comprising:

(a) providing an infectious stock of human papillomavirus 6a isolated by the method of claim 4;

(b) contacting human foreskin tissue with the infectious human papillomavirus 6a, in the presence or absence of the candidate antiviral agent;

(d) implanting the tissue into an athymic animal host;

(e) comparing the frequency of human papillomavirus 6a infection in the implanted tissue contacted with the infectious human papillomavirus 6a in the presence of the candidate antiviral agent with human papillomavirus 6a infection in the implanted tissue contacted with the infectious human papillomavirus in the absence of the candidate antiviral agent; and

(f) inferring from (e) the ability of the candidate antiviral agent to reduce or inhibit human papillomavirus 6a infection or replication.

[C10] 10. A purified infectious stock of human papillomavirus 6a.