Oral immunization with papillomavirus virus-like particles

The present invention is directed to a method of expressing the papillomavirus capsid protein coding sequence in a cell using an expression system under conditions facilitating expression of the protein in the cell. In another aspect of the invention, it has been discovered that virus-like particle(s) (VLPs), fragment(s), capsomer(s) or portion(s) thereof are formed from the papillomavirus capsid protein. It was further discovered that the virus-like particle(s) comprises antigenic characteristics similar to those of native infectious papillomavirus particles. In one embodiment of the invention, there is provided a method of expressing the L1 major capsid protein of human papillomavirus type-6 (HPV-6) and type-11 (HPV-11) in Sf-9 insect cells using the baculovirus expression system, and the production of type 6 (HPV-6), type-11 (HPV-11), type-16 (HPV-16) and type-18 (HPV-18) virus-like particles. In yet another embodiment, the invention provides a method of vaccinating a mammal for papillomavirus by administering papillomavirus virus-like particles orally to a mammal in an amount sufficient to induce an immune response to the papillomavirus.

FIELD OF THE INVENTION 
The present invention relates generally to papillomavirus (PV). More 
particularly, the invention relates to a method of expressing the human 
papillomavirus type 6 (HPV-6) and type 11 (HPV-11) capsid protein coding 
sequence using the baculovirus expression system, production of HPV 
virus-like particles (VLPS) and use of these VLPs in production of 
antibodies which recognize epitopes on HPV, and for HPV vaccine 
development, and for development of serologic tests for the detection of 
HPV infection. 
BACKGROUND OF THE INVENTION 
The family Papovaviridae constitutes a group of DNA viruses that induce 
both lytic infections and either benign or malignant tumors. Structurally, 
all are naked icosahedral virions with 72 capsomeres and contain 
double-stranded circular DNA. Viruses included in the family are: (1) 
human and animal papillomaviruses, (2) mouse polyomavirus, (3) simian 
vacuolating virus, and (4) human viruses BK and JC. 
Human papillomaviruses (HPV) infect cutaneous, genital, oral, and 
respiratory epithelia in a tissue-specific manner. Infection with HPV has 
been associated closely with the development of both benign lesions and 
malignancies (Reichman et al., Papillomaviruses, 1990, pp. 1191-1200; and 
Mandell et al., Principles and Practice of Infectious Diseases, 3rd 
Edition, Churchill Livingstone, New York, N.Y.). For example, HPV type 1 
(HPV-1) is present in plantar warts, HPV types 6 or 11 (HPV-6 or HPV-11) 
in condylomata acuminata (anogenital warts), while HPV types 16 or 18 
(HPV-16 or HPV-18) are common in premalignant and malignant lesions of the 
cervical squamous epithelium (See Crum et al., "Human papillomavirus 
infection and cervical neoplasia: New perspectives," Int. J. Gynecol. 
Pathol. 3:376-388 (1984); zur Hausen, Genital Papillomavirus Infections, 
1985, pp. 83-90; Rigby et al., Viruses and Cancer, Cambridge University 
Press, Cambridge, UK; and Koutsky et al., "Epidemiology of genital human 
papillomavirus infection," Epidemiol. Rev. 10:122-163 (1988)). 
However, difficulties in propagating HPV in vitro has led to the 
development of alternative approaches to antigen production for 
immunologic studies. For example, Bonnez et al., "The Pstl-XhoII 
restriction fragment of the HPV-6b L1 ORF lacks immunological specificity 
as determined by sera from HPV 6 condyloma acuminatum patients and 
controls," UCLA Symp. Mol. Cell. Biol., New Series, 124:77-80 (1990); 
Jenison et al., "Identification of immunoreactive antigens of human 
papillomavirus type 6b by using Escherichia coli-expressed fusion 
proteins," J. Virol. 62:2115-2123 (1988); Li et al., "Identification of 
the human papillomavirus type 6b L1 open reading frame protein in 
condylomas and corresponding antibodies in human sera," J. Virol. 
61:2684-2690 (1987); Steele et al., "Humoral assays of human sera to 
disrupted and nondisrupted epitopes of human papillomavirus type 1," 
Virology 174:388-398 (1990); and Strike et al., "Expression in Escherichia 
coli of seven DNA segments comprising the complete L1 and L2 open reading 
frames of human papillomavirus type 6b and the location of the `common 
antigen`," J. Gen. Virol. 70:543-555 (1989), have expressed recombinant 
capsid protein coding sequences in prokaryotic systems, and used them in 
Western blot analyses of sera obtained from individuals with HPV infection 
of the genital tract. Results from these studies have suggested that 
antibodies to denatured, i.e. linear, epitopes of HPV capsid proteins can 
be detected in the sera of some infected individuals. 
Whole virus particles have also been used to detect antibodies in human 
sera, including antibodies directed against conformational epitopes. These 
studies have been difficult to conduct because most naturally occurring 
HPV-induced lesions produce few particles. Whole virus particles can be 
obtained, however, in amounts sufficient to conduct immunologic assays 
from HPV type 1-induced plantar warts (Kienzler et al., "Humoral and 
cell-mediated immunity to human papillomavirus type 1 (HPV-1) in human 
warts," Br. J. Dermatol. 108:65-672 (1983); "Pfister et al., 
Seroepidemiological studies of human papilloma virus (HPV-1) infections," 
Int. J. Cancer 21:161-165 (1978); and Steele et al., "Humoral assays of 
human sera to disrupted and nondisrupted epitopes of human papillomavirus 
type 1," Virology 174:388-398 (1992)) and experimentally-induced HPV-11 
athymic mouse xenographs (Kreider et al., "Laboratory production in vivo 
of infectious human papillomavirus type 11," J. Virol. 61:590-593 (1991); 
and Kreider et al., "Morphological transformation in vivo of human uterine 
cervix with papillomavirus from condylomata acuminata," Nature 317:639-641 
(1985)). More particularly, U.S. Pat. No. 5,071,757 to Kreider et al., 
discloses a method of propagating infectious HPV-11 virions in the 
laboratory using an athymic mouse xenograph model system. Although this 
system is capable of producing quantities of infectious virus that could 
be used for the development of a serologic test for genital HPV infection, 
this system is very expensive and cumbersome. Furthermore, only one 
genital HPV type has so far been propagated in this system, thus, limiting 
its usefulness. In addition, the infectious virus produced using this 
system represents a biohazard and, therefore, would be difficult to use in 
a vaccine formulation. 
Zhou et al., in "Expression of vaccinia recombinant HPV 16 L1 and L2 ORF 
proteins in epithelial cells is sufficient for assembly of HPV virion-like 
particles", Virology 185:251-257 (1992), have reported the formation of 
HPV-16 virus-like particles in CV-1 cell nuclei following infection with a 
vaccinia virus HPV-16 L1/L2 double recombinant expression vector. However, 
the authors were not able to produce VLPs with a vector expressing L1 
alone. Furthermore, the VLPs produced lacked a well-defined symmetry, and 
were more variable in size and smaller, only about 35-40 nm in diameter, 
than either HPV virions (55 nm) or the VLPs of the present invention 
(baculovirus produced HPV-11 VLPs, about 50 nm in diameter). 
U.S. Pat. No. 5,045,447, to Minson, discloses a method of screening 
hybridoma culture supernatants for monoclonal antibodies with desired 
specificities. Minson's method is exemplified by the production of 
antibodies to the L1 protein of human papillomavirus type 16 (HPV-16) 
using this protein as the target antigen in mice. However, Minson fails to 
disclose the expression of the L1 protein or production of HPV virus-like 
particles (VLPs). 
U.S. Pat. No. 4,777,239, to Schoolnik et al., discloses short peptide 
sequences derived from several of the papillomavirus early region open 
reading frames which elicit type-specific antibodies to papillomavirus. 
However, the inventors fail to disclose any sequences directed to the 
major late open reading frame, L1. 
U.S. Pat. No. 5,057,411 to Lancaster et al., discloses a polynucleotide 
sequence of about 30 nucleotides of the papillomavirus L1 capsid protein 
open reading frame that the inventors contend encode a papillomavirus 
type-specific epitope. However, the inventors do not disclose infected 
animals that produced antibodies which recognize this sequence. Instead, 
they synthesized a bovine papillomavirus type 1 (BPV-1) version of the 
sequence (a 10 amino acid peptide, or decapeptide), then immunized rabbits 
and tested the antiserum's ability to react with either BPV-1 or BPV-2 
induced fibropapilloma tissue. The peptide antiserum only reacted with 
BPV-1 and not BPV-2 tissue. The inventors then concluded that the peptide 
contained an antigenic determinant that was type-specific, and therefore, 
all papillomavirus L1 coding sequences contain a type-specific epitope at 
this locus. This is theoretical speculation on the part of the inventors, 
who give no supporting data for this hypothesis. In addition, the amino 
acid sequences disclosed (10 amino acids) are generally thought not to be 
capable of adopting higher order antigenic structures, i.e., 
conformational epitopes that possess a three-dimensional structure such as 
those produced by the method described herein. 
Another problem associated with papillomavirus infections is the need for 
alternative therapeutic and prophylactic modalities. One such modality 
which has received little recent study, would be papillomavirus vaccines. 
In 1944, Biberstein treated condyloma acuminatum patients with an 
autogenous vaccine derived from the patients' warts (Biberstein, 
"Immunization therapy of warts," Arch. Dermatol Syphilol. 50:12-22 
(1944)). Thereafter, Powell et al., developed the technique typically used 
today for preparing autogenous wart vaccines for the treatment of 
condyloma acuminatum (Powell et al., "Treatment of condylomata acuminata 
by autogenous vaccine," South Med. J. 63:202-205 (1970)). Only one 
double-blind, placebo-controlled study has attempted to evaluate the 
efficacy of the autogenous vaccine (Malison et al., "Autogenous vaccine 
therapy for condyloma acuminatum: A double-blind controlled study," Br. J. 
Vener. Dis. 58:62-65 (1982)). The authors concluded that autogenous 
vaccination was not effective in the treatment of condylomata acuminata, 
although this interpretation may be erroneous. The small number of 
patients studied precluded drawing valid negative conclusions. In any 
event, autogenous vaccines, as presently described, have several 
disadvantages. First, the patient needs to have relatively large warts (2 
g to 5 g) in order to prepare the vaccine. Secondly, the practitioner 
needs access to laboratory equipment and expertise each time a new patient 
is to be treated. Thus, vaccine preparation is very expensive, tedious, 
and in cases involving relatively small lesion mass, not possible. 
Unfortunately, traditional methods of virus propagation have not yet been 
adapted to the study of papillomaviruses, and the alternative methods 
previously described fail to produce infectious virions in any significant 
amounts for immunologic studies. Also, in vivo propagation of HPV-11 in 
the athymic mouse system is not very practical because it is expensive, 
labor intensive and currently limited to HPV-11. Consequently, an 
alternative method of producing epitopes of HPV capsid for use in 
immunologic studies and vaccine production is needed. 
SUMMARY OF THE INVENTION 
The present invention is directed to a method of expressing the capsid 
protein coding sequence of papillomavirus (PV) in a cell, comprising 
transfecting the cell with an expression vector containing the 
papillomavirus capsid protein coding sequence under conditions 
facilitating expression of the protein in the cell. 
In another aspect of the invention, there is provided a virus-like 
particle(s) (VLPs), fragment(s), capsomer(s) or portion(s) thereof, formed 
from papillomavirus capsid protein. It has been discovered that the 
virus-like particle(s) comprises antigenic characteristic(s) similar to 
those of native infectious papillomavirus particles. 
In a preferred embodiment of the invention, there is provided a method of 
expressing the L1 capsid protein coding sequence of human papillomavirus 
type-6 (HPV-6) and type-11 (HPV-11) in Sf-9 insect cells using the 
baculovirus expression system. The HPV-6 and HPV-11 coding sequences were 
cloned using standard techniques in the art into a baculovirus transfer 
vector. The resulting baculovirus transfer vector were used to 
co-transfect Sf-9 insect cells with Autographa californica nuclear 
polyhedrosis virus (AcNPV) forming a recombinant baculovirus (Ac6L1 or 
Ac11L1) which was recovered. Sf-9 insect cells were thereafter infected 
with either Ac6L1 or Ac11L1 under conditions facilitating expression of 
the protein in the cells. It was discovered that the L1 protein formed 
virus-like particles (VLPs). VLPs were identified by electron microscopy 
of negatively-stained sucrose band fractions obtained from Sf-9 cells 
infected with the Ac11L1 recombinant baculovirus. It was further 
discovered that the VLPs possessed immunological and morphological 
characteristics similar to those of native HPV-11 virions, as defined by 
rabbit antisera. 
Virus-like particle(s) produced in accordance with the invention, can be 
used in diagnostic assays, can play a role in the identification and 
characterization of an HPV cell receptor, and can be used for vaccine 
development (both therapeutic and prophylactic). It is understood that the 
method of the invention as described herein for production of HPV-11 and 
HPV-6 can be used to produce similar immunologic reagents from other 
animal and/or human papillomaviruses. In addition, VLPs produced in 
accordance with the invention will provide abundant reagents with which to 
carry out immunologic studies of papillomaviruses and for developing 
vaccines against papillomaviruses. 
The present invention also provides a method of vaccinating a mammal 
against papillomavirus infection by administering papillomavirus 
virus-like particles orally to a mammal in an amount sufficient to induce 
an immune response to the papillomavirus.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention is directed to a method of expressing the 
papillomavirus capsid protein coding sequence in a cell using the 
baculovirus expression system under conditions facilitating expression of 
the protein in the cell. In another aspect of the invention, it has been 
discovered that virus-like particle(s) (VLPs), fragment(s), capsomer(s) or 
portion(s) thereof are formed from the papillomavirus capsid protein. It 
was further discovered that the virus-like particle(s) comprises antigenic 
characteristics similar to those of native infectious papillomavirus 
particles. 
As used herein, "virus-like particle(s) (VLPs)" refer to a virus-like 
particle(s), fragment(s), capsomer(s) or portion(s) thereof produced from 
the capsid protein coding sequence of papillomavirus and comprising 
antigenic characteristic(s) similar to those of infectious papillomavirus 
particles. As used herein, "antigenic characteristic(s)" refers to 
(1) the ability of the virus-like particle(s) to cross-react with wild-type 
particles (native infectious virus particles of the same HPV type) as 
determined by antisera generated in animals and/or humans by immunization 
with either VLPs or infectious virus; and/or 
(2) the ability to recognize or detect antibodies in human sera from 
persons known to be infected with homologous virus. 
As used herein, "L1 protein coding sequence" or "L1 capsid protein coding 
sequence" or "L1 coding sequence" refers to the open reading frame which 
codes for the L1 protein in papillomavirus. When expressed, the L1 protein 
coding sequence produces a protein, or protein complex, or aggregate, 
which possesses immunological and morphological characteristics similar to 
those of native papillomavirus virions. The L1 coding sequence used in the 
invention can be isolated and purified from papillomavirus genomic DNA or 
synthesized using standard genetic engineering techniques. 
As used herein, the term "transfecting" refers to any means for introducing 
a virus, plasmid or vector into a cell. Examples of such means include 
infection, calcium phosphate precipitation and electroporation. 
In a preferred embodiment of the invention, there is provided a method of 
expressing the coding sequence for the L1 capsid protein of human 
papillomavirus type-11 (HPV-11) or human papillomavirus type-6 (HPV-6) in 
Sf-9 insect cells using the baculovirus expression system. It is 
understood that the capsid protein coding sequences of these HPV types are 
used for purposes of illustration only, and that any L1 capsid protein 
coding sequence for any animal or human papillomavirus type can be used 
without deviating from the intended scope of the invention. Such HPV types 
include, without limitation, HPV types 16, 18, 31, 33, 35 (Gissman et al., 
Cancer Cells 5:275 (1987), which is hereby incorporated by reference); and 
those HPV types disclosed in PCT publication no. WO 92/16636 to Boursnell 
et al., which is hereby incorporated by reference. 
The preferred expression system used in the method of the invention is the 
baculovirus expression system, however, it is understood that any other 
expression system(s) can be employed herein provided the system(s) can 
express the L1 protein coding sequence. Examples of such systems include, 
without limitation, any prokaryotic and/or eukaryotic system(s) including 
adenovirus, SV40, E. coli, CHO cells, vaccinia virus, insect viruses, 
yeast, bacteriophage virus or modified viruses, DNA plasmids, vectors and 
the like. 
The host cell for expression of the L1 coding sequence is dependent on the 
expression system used. Examples of suitable host cells include, without 
limitation, bacteria (prokaryotic), microorganisms such as yeast, 
mammalian cells (eukaryotic) and insect cells. When using the baculovirus 
expression system. insect cells, such as Sf-9 or Sf-21 are preferred. 
In another aspect of the invention, it was discovered that the L1 protein 
produces virus-like particles (VLPs), fragment(s), capsomer(s) or 
portion(s) thereof, formed from papillomavirus capsid protein. It has been 
discovered that the virus-like particle(s) comprises antigenic 
characteristic(s) similar to those of native infectious papillomavirus 
particles. More particularly, these VLPs contain an antigenic determinant 
that is specifically recognized by antibodies present in sera obtained 
from genital HPV-infected patients. For example, reaction of 
VLP-containing insect cell extracts with antisera directed against either 
denatured or non-denatured capsid epitopes, as deduced by 
immunoreactivities in Western blot and immunodotblot assays, suggested 
that conformational epitopes present in native HPV-11 infectious virions 
were also present on the baculovirus-produced HPV-11 VLPs of the present 
invention. Immunodotblot assays using human sera obtained from individuals 
with biopsy proven condylomata acuminatum correlated closely with results 
previously obtained in HPV-11 whole virus particle-based ELISA tests as 
described by Bonnez et al., "Use of human papillomavirus type 11 virions 
in an ELISA to detect specific antibodies in humans with condylomata 
acuminata," J. Gen. Virol. 72:1343-1347 (1991), which is hereby 
incorporated by reference. 
These morphologic and immunologic similarities to native HPV-11 virions 
suggest that recombinant VLPs produced in the baculovirus system will be 
useful in sero-epidemiology and pathogenesis studies of not only genital 
HPV infection but for any papillomavirus and for vaccine development. L1 
has an intrinsic capacity for self-assembly. Thus, other papillomavirus 
proteins are not required for VLP formation in the baculovirus system. 
This supports the contention that VLPs to all types of papillomaviruses 
can be produced in accordance with the method described herein. 
The VLPs of the invention can be used to raise antibodies, either in 
subjects for which protection against infection by HPV is desired, i.e., 
vaccines, or to heighten the immune response to an HPV infection already 
present. The VLPs of the invention can be injected into animal species to 
obtain antisera useful in diagnosis. In addition to polyclonal antisera, 
monoclonal antibodies can be obtained using the methods of Kohler and 
Milstein, or by modifications thereof, by immortalizing spleen or other 
antibody-producing cells from injected animals to obtain 
antibody-producing clones, i.e., hybridomas. 
The antibodies obtained can be used for diagnosis of HPV infection in 
cervical biopsies or Papanicolaou smears and in assessing disease levels 
in humans or other subjects. In particular, diagnosis using the antibodies 
of the invention permits monitoring the evolution of the disease. The 
antibodies can be used in analysis of serum to detect the virus, as well 
as to monitor the progress of therapy with antiviral or other therapeutic 
agents directed to control of the infection or carcinoma. The antibodies 
can also be used as passive therapy, taking into account species 
variations. 
The VLPs of the invention can be used in immunoassays to detect the 
presence of antibodies raised against HPV in the serum of patients 
suspected of harboring HPV infections or to titrate the sera of patients 
being treated with an anti-HPV vaccine. 
The VLPs of the invention can be directly administered to a host to induce 
the formation of neutralizing antibodies (Bonnez et al., 
"Antibody-mediated neutralization of human papillomavirus type 11 (HPV-11) 
infection in the nude mouse: Detection of HPV-11 mRNAs by the polymerase 
chain reaction," J. Inf. Dis., 165: 376-380 (1992); Rose, R. C., et al., 
"Human Papillomavirus (HPV) Type 11 Recombinant Virus-Like Particles 
Induce the Formation of Neutralizing Antibodies and Detect HPV-Specific 
Antibodies in Human Sera," J. Gen. Virol. 75:2075-2079 (1994), which are 
hereby incorporated by reference), to confer either protective immunity 
against HPV or, if the patient is already infected, to boost the patient's 
own immune response. For all applications, the VLPs are administered in 
immunogenic form. Optionally, the VLPs can be conjugated to an 
immunogenicity conferring carrier material, the material preferably being 
antigenically neutral. Depending on the use required, the VLPs of the 
invention have the ability to serve as type specific or broad range 
vaccines and diagnostics. 
VLPs which are to be administered as vaccines can be formulated according 
to conventional and/or future methods for such administration to the 
subject to be protected and can be mixed with conventional adjuvants. The 
peptide expressed can be used as an immunogen in subunit vaccine 
formulations, which may be multivalent. The multivalent vaccine 
formulation can comprise VLPs each encoding a different L1 protein from 
different HPVs. The product may be purified for purposes of vaccine 
formulation from any vector/host systems that express the heterologous 
protein. The purified VLPs should be adjusted to an appropriate 
concentration, formulated with any suitable vaccine adjuvant and packaged 
for use. Suitable adjuvants include, but are not limited to: mineral gels, 
e.g., aluminum hydroxide; surface active substances such as lysolecithin, 
pluronic polyols; polyanions; peptides; oil emulsions; and potentially 
useful human adjuvants such as BCG (Bacille Calmette-Guerin) and 
Corynebacterium parvum. The immunogen may also be incorporated into 
liposomes, or conjugated to polysaccharides and/or other polymers for use 
in a vaccine formulation. Many methods may be used to introduce the 
vaccine formulations described above; these include, but are not limited 
to, oral, intradermal, intramuscular, intraperitoneal, intravenous, 
subcutaneous and intranasal routes. If they are to be used directly, as 
diagnostic reagents, they are purified using conventional methods and 
packaged accordingly for such use. If they are to be used to produce 
antibodies for diagnostic purposes, convenient test animals can be used to 
prepare the appropriate antisera. Suitable hosts include mice, rats, 
rabbits, guinea pigs, or even larger mammals such as sheep. The antibodies 
can be used therapeutically so long as they are compatible with the host 
to be treated. Monoclonal antibodies having the proper species 
characteristics are preferred for this application. 
In a preferred embodiment, the invention provides a method of vaccinating a 
mammal for papillomavirus by administering papillomavirus virus-like 
particles orally to a mammal in an amount sufficient to induce an immune 
response to the papillomavirus. In order to obtain a high degree of 
protection, the method may also involve administering one or more vaccine 
booster inoculations of papillomavirus virus-like particles orally to the 
mammal. In a preferred embodiment of the invention, the immune response 
induced by oral immunization will protect the mammal from infection by 
papillomavirus. 
The preferred papillomavirus is a human papillomavirus, in particular Human 
Papillomavirus Type 6 and Type 11. 
The present invention also provides oral vaccines having papillomavirus 
virus-like particles and a pharmaceutically acceptable carrier. Oral 
vaccines may also include flavorings, colorings, and other food additives 
to make the vaccine more palatable. In addition, oral vaccines may also 
contain stabilizers and preservatives to extend the shelf life of the 
vaccine. 
Prophylactic vaccination with recombinant VLPs has emerged as a strategy 
for the prevention of anogenital HPV infection (Kimbauer, R., 
"Papillomavirus-Like Particles For Serology and Vaccine Development," 
Intervirology 39(1-2):54-61 (1996); Rose, R. C., et al., "Human 
Papillomavirus Infections," p. 343-368. In G. J. Galasso, R. J. Whitley, 
and T. C. Merigan (eds.), "Antiviral Agents and Human Viral Diseases," 
4.sup.th ed. Lippincott-Raven Publishers, Philadelphia (1997); Schiller, 
J. T., et al., "Papillomavirus-Like Particles and hpv Vaccine 
Development," Seminars in Cancer Biology 7(6):373-382 (1996), which are 
hereby incorporated by reference). VLPs are highly immunogenic when 
administered parenterally (Kimbauer, R. F., et al., "Papillomavirus L1 
Major Capsid Protein Self-Assembles into Virus-Like Particles That Are 
Highly Immunogenic," Proceedings of the National Academy of Sciences of 
the United States of America 89(24):12180-12184 (1992); Rose, R. C., et 
al., "Human Papillomavirus (HPV) Type 11 Recombinant Virus-Like Particles 
Induce the Formation of Neutralizing Antibodies and Detect HPV-Specific 
Antibodies in Human Sera," J. Gen. Virol. 75:2075-2079 (1994), which are 
hereby incorporated by reference), and have been shown to elicit 
protective immune responses (Breitburd, F., et al., "Immunization With 
Virus-Like Particles From Cottontail Rabbit Papillomavirus (CRPV) Can 
Protect Against Experimental CRPV Infection," J. Virology 69(6):3959-3963 
(1995); Christensen, N. D., et al., "Assembled Baculovirus-Expressed Human 
Papillomavirus Type 11 L1 Capsid Protein Virus-Like Particles Are 
Recognized By Neutralizing Monoclonal Antibodies and Induce High Titres of 
Neutralizing Antibodies," J. Gen. Virol. 75:2271-2276 (1994); Kimbauer, 
R., et al., "Virus-Like Particles of Bovine Papillomavirus Type 4 in 
Prophylactic and Therapeutic Immunization," Virology 219(1):37-44 (1996); 
Rose, R. C., et al., "Human Papillomavirus (HPV) Type 11 Recombinant 
Virus-Like Particles Induce the Formation of Neutralizing Antibodies and 
Detect HPV-Specific Antibodies in Human Sera," J. Gen. Virol. 75:2075-2079 
(1994); Suzich, J. A., et al., "Systemic Immunization With Papillomavirus 
L1 Protein Completely Prevents the Development of Viral Mucosal 
Papillomas," Proc. Natl. Acad. Sci., USA 92:11553-11557 (1995); White, W. 
I., et al., "In Vitro Infection and Type-Restricted Antibody-Mediated 
Neutralization of Authentic Human Papillomavirus Type 16," J. Virology 
72:959-964 (1998), which are hereby incorporated by reference). The 
present results demonstrate that similar responses can be induced by oral 
VLP immunization. Antigenic specificities of orally induced antibodies 
were found to be dependent on native VLP structure, and restricted 
according to HPV genotype. Results from the epitope-blocking ELISA 
indicated that post-immune serum antibodies efficiently inhibited VLP 
binding by HPV-11 virus-neutralizing antibodies. The detection of antigen 
structure-dependent antibody specificities in the murine post-immune sera 
indicated that HPV-11 VLPs maintained their native structure and 
antigenicity despite acid pH in the stomach and abundant proteases in the 
small intestine. This demonstrates the usefulness of VLPs as oral 
immunogens for the prevention of anogenital HPV disease. 
Oral immunization offers certain advantages over other routes of 
vaccination. For example, oral vaccines are more easily administered and 
thus may be more acceptable to vaccine recipients. Also, oral vaccines can 
be less pure than vaccines formulated for injection, making production 
costs lower. Interestingly, in some instances orally administered antigens 
have been shown to elicit mucosal immune responses, which may be important 
for protection against infection with certain pathogens (Ball, J. M., et 
al., "Oral Immunization With Recombinant Norwalk Virus-Like Particles 
Induces a Systemic and Mucosal Immune Response in Mice," J. Virology 
72(2):1345-1353 (1998); Oneal, C. M., et al., "Rotavirus Virus-Like 
Particles Administered Mucosally Induce Protective Immunity," J. Virology 
71(11):8707-8717 (1997), which are hereby incorporated by reference). 
Based on previous work reported by others (Kimbauer, R., et al., 
"Virus-Like Particles of Bovine Papillomavirus Type 4 in Prophylactic and 
Therapeutic Immunization," Virology 219(1):37-44 (1996); Suzich, J. A., et 
al., "Systemic Immunization With Papillomavirus L1 Protein Completely 
Prevents the Development of Viral Mucosal Papillomas," Proc. Natl. Acad. 
Sci., USA 92:11553-11557 (1995), which are hereby incorporated by 
reference), it was not the intent of the present study to investigate 
mucosal responses after oral VLP administration. However, such responses 
may enhance vaccine efficacy, and this possibility is now being 
investigated. 
Intestinal antigens are believed to gain access to gut-associated lymphoid 
tissue (GALT) via M cells in the Peyer's Patch (PP) epithelium (Neutra, M. 
R., "Antigen Sampling Across Epithelial Barriers and Induction of Mucosal 
Immune Responses," Annual Review of Immunology 14:275-300 (1996), which is 
hereby incorporated by reference), and M-cell-mediated uptake into PP has 
been demonstrated for a number of microorganisms (Amerongen, H. M., et 
al., "Transepithelial Transport of HIV-1 By Intestinal M Cells: a 
Mechanism For Transmission of AIDS," Journal of Acquired Immune Deficiency 
Syndromes 4(8):760-765 (1991); Buller, C. R., et al., "Natural Infection 
of Porcine Ileal Dome M Cells With Rotavirus and Enteric Adenovirus," Vet. 
Pathol. 25(6):516-517 (1988); Inman, L. R., et al., "Specific Adherence of 
Escherichia Coli (Strain RDEC-1) to Membranous (M) Cells of the Peyer's 
Patch in Escherichia Coli Diarrhea in the Rabbit," Journal of Clinical 
Investigation 71(1):1-8 (1983); Keren, D. F., et al., "The Enteric Immune 
Response to Shigella Antigens," Current Topics in Microbiology & 
Immunology 146:213-223 (1989); Sicinski, P., et al., Poliovirus Type 1 
Enters the Human Host Through Intestinal M Cells," Gastroenterology 
98(1):56-58 (1990); Wolf, J. L., et al., Intestinal M Cells: a Pathway for 
Entry of Reovirus Into the Host," Science 212(4493):471-472 (1981), which 
are hereby incorporated by reference). M cells may be able to deliver 
intact VLPs directly to professional antigen presenting cells (Ball, J. 
M., et al., "Oral Immunization With Recombinant Norwalk Virus-Like 
Particles Induces a Systemic and Mucosal Immune Response in Mice," J. 
Virology 72(2):1345-1353 (1998), which is hereby incorporated by 
reference), which are abundant in the underlying areas of the PP (Mowat, 
A. M., et al., "The Anatomical Basis of Intestinal Immunity," Immonol. 
Rev. 156:145-166 (1997), which is hereby incorporated by reference). 
Previous work has shown that several types of antigen can elicit systemic 
responses after oral delivery, and that cellular binding activity may be 
involved in this phenomenon (de Aizpurua, H. J., et al., "Oral 
Vaccination. Identification of Classes of Proteins That Provoke an Immune 
Response Upon Oral Feeding," J. Exp. Med. 167(2):440-451 (1988), which is 
hereby incorporated by reference). It may be that the ability to bind 
glycolipids or glycoproteins on the intestinal mucosa may stimulate 
mucosal cells to transport such antigens into the circulation, thereby 
eliciting a systemic response (de Aizpurua, H. J., et al., "Oral 
Vaccination. Identification of Classes of Proteins That Provoke an Immune 
Response Upon Oral Feeding," J. Exp. Med. 167(2):440-451 (1988), which is 
hereby incorporated by reference). Papillomavirus VLPs bind to a variety 
of eukaryotic cell types (Muiller, M., et al., "Papillomavirus Capsid 
Binding and Uptake by Cells From Different Tissues and Species," J. 
Virology 69(2):948-954 (1995); Volpers, C., et al., "Binding and 
Internalization of Human Papillomavirus Type 33 Virus-Like Particles by 
Eukaryotic Cells," J. Virology 69(6):3258-3264 (1995), which are hereby 
incorporated by reference), and this ability may be involved in the 
induction of responses such as those described in the present study. 
Although natural infection by papillomaviruses is thought to be 
receptor-mediated, a role for a specific receptor in the induction of 
immune responses after oral vaccination is unlikely, as papillomaviruses 
are not known to infect intestinal mucosal epithelial tissues, and mice 
are not naturally susceptible to HPV infection. 
Results obtained in the epitope-blocking ELISA indicate that orally induced 
antibodies efficiently block VLP binding by HPV-11 virion neutralizing 
antibodies. Native virions are costly to produce (Bonnez, W., et al., 
"Propagation of Human Papillomavirus Type 11 in Human Xenografts Using the 
Severe Combined Immunodeficiency (SCID) Mouse and Comparison to the Nude 
Mouse Model," Virology 197(1):455-458 (1993); Kreider, J. W., et al., 
"Laboratory Production in vivo of Infectious Human Papillomavirus Type 
11," J. Virology 61:590-593 (1987), which are hereby incorporated by 
reference), and are not available for most clinically relevant virus 
genotypes. Therefore, the epitope-blocking ELISA is a useful alternative 
to virion infectivity assays for predicting vaccine efficacy. It may also 
be more reliable than other recently described surrogate assays. For 
example, a hemagglutination inhibition assay (HAI) (Roden, R. B., et al., 
"Assessment of the Serological Relatedness of Genital Human 
Papillomaviruses by Hemagglutination Inhibition," J. Virology 
70(5):3298-3301 (1996), which is hereby incorporated by reference), which 
suggests the possibility that the mechanism(s) by which antibodies 
neutralize infectious HPV virions may differ from the mechanism(s) 
involved in VLP-mediated hemagglutination. By contrast, results from the 
epitope-blocking ELISA indicated the presence of H11.H3 neutralizing 
antigenic specificity in rabbit HPV-11 N-pAb, and thus indicated 
indirectly the presence of the same specificity in antibodies induced 
after oral immunization. 
Roughly 450,000 new cases of invasive uterine cervical carcinoma are 
diagnosed annually worldwide (Munos, N., "Disease-Burden Related to Cancer 
Induced by Viruses and H.pylori," World Health Organization (WHO) Vaccine 
Research and Development: Report of the Technical Review Group Meeting 
Jun. 9-10, 1997 (1997), which is hereby incorporated by reference). 
Therefore, efficient methods of vaccine delivery will be needed for the 
immunization of large numbers of susceptible individuals. Thus, oral 
immunization strategies will certainly facilitate implementation of mass 
immunization programs designed to reduce the incidence of cervical cancer 
and other HPV-associated diseases. 
The following Examples are provided to further illustrate the present 
invention. 
EXAMPLE I 
Methods 
1. HPV-11 Viral DNA And pVL11L1 Baculovirus Transfer Vector Construction. 
HPV-11 genomic DNA was obtained from virus particles which were purified 
from experimentally induced athymic mouse xenografts as described by Rose 
et al., "Expression of the full-length products of the HPV-6b and HPV-11 
L2 open reading frames by recombinant baculovirus, and antigenic 
comparisons with HPV-11 whole virus particles," J. Gen. Virol. 
71:2725-2729 (1990), which is hereby incorporated by reference. The L1 
coding sequence was cloned by PCR amplification of purified genomic DNA, 
using primers designed to introduce BglII and EcoRl restriction enzyme 
sites at the 5' and 3' ends, respectively. The forward and reverse primer 
sequences, respectively, were, 5'-CGC AGA TCT ATG TGG CGG CCT AGC-3'(SEQ 
ID NO. 1) and 5'-CAT ATG AAT TCC CAC AAC ACA CTG ACA CAC-3' (SEQ ID NO. 
2). Restriction sites (underlined) were introduced proximal to the 
putative L1 start codon (bold text), and approximately 30 nucleotides 
downstream from the putative L1 stop codon, by primer-directed 
mutagenesis. Amplification was performed essentially as described by 
Bonnez et al., "Antibody-mediated neutralization of human papillomavirus 
type 11 (HPV-11) infection in the nude mouse: Detection of HPV-11 mRNAs by 
the polymerase chain reaction," J. Inf. Dis., 165: 376-380 (1992), which 
is hereby incorporated by reference, using 500 ng of each primer and 2 
units of Taq DNA polymerase (Amplitaq, Perkin-Elmer Cetus Corp., Norwalk, 
Conn.). After amplification, the PCR product was digested with BglII and 
EcoRI. The 1539 base pair (bp) digestion product, which contained the 
entire HPV-11 L1 open reading frame (ORF), was purified by agarose gel 
electrophoresis as described by Rose et al., "Expression of the 
full-length products of the HPV-6b and HPV-11 L2 open reading frames by 
recombinant baculovirus, and antigenic comparisons with HPV-11 whole virus 
particles," J. Gen. Virol. 71:2725-2729 (1990), which is hereby 
incorporated by reference, and cloned into the corresponding sites of a 
baculovirus transfer vector, pVL-1392 (M. D. Summers, Texas A&M 
University, College Station, Tex.). The resulting construct, pVL11L1, was 
used to co-transfect Sf-9 cells with Autographa californica nuclear 
polyhedrosis virus (AcNPV) genomic DNA according to the methods of Summers 
et al., A Manual of Methods for Baculovirus Vectors and Insect Cell 
Culture Procedures, 1987, Texas A&M University, College Station, Tex., 
which is hereby incorporated by reference. Recombinant baculoviruses were 
recovered by visual examination and selection of occlusion-negative (occ-) 
plaques, and were subjected to two further rounds of plaque-purification 
according to the methods of Summers et al., A Manual of Methods for 
Baculovirus Vectors and Insect Cell Culture Procedures, 1987, Texas A&M 
University, College Station, Tex., which is hereby incorporated by 
reference. Protein expression from isolated virus stocks was determined by 
Western blot. 
2. SDS-PAGE And Western Blot Detection Of Recombinant L1 Expression In Sf-9 
Cells. 
Infected Sf-9 cell cultures were grown in 150 cm.sup.2 tissue culture 
flasks and prepared for analytical SDS-PAGE and Western Blot assay. 
Non-recombinant or recombinant L1-infected cells were collected from 
flasks by resuspending with a pasteur pipet, and equal numbers of 
wild-type or recombinant L1-infected cells were centrifuged at 500.times.g 
for 10 minutes at 4.degree. C. Supernatants were removed and cell pellets 
were transferred to ice, immediately resuspended in 1 ml lysis buffer (30 
mM Tris, pH 7.6; 10 mM MgCl.sub.2 ; 1 mM CaCl.sub.2 ; 1 MM 
phenylmethylsulfonyl fluoride (PMSF); leupeptin (10 .mu.g/ml); 1% NP-40) 
and allowed to stand at room temperature for 15 minutes with periodic 
vortexing. After centrifugation at 500.times.g for 2 minutes at 4.degree. 
C., the NP40-soluble fraction contained in the supernatant was removed and 
diluted 1:1 with 2.times. Laemmli sample buffer as described by Laemmli, 
"Cleavage of structural proteins during the assembly of the head of the 
bacteriophage T4," Nature 277:680-685 (1970), which is hereby incorporated 
by reference, and heated to 95.degree. C. for 3 minutes. The 
NP40-insoluble pellet (containing nuclear material) was washed once with 
cold PBS (1 mM PMSF: 10 .mu.g/ml leupeptin) and solubilized by boiling and 
vortexing in 1.times. Laemmli buffer. Samples were electrophoresed in 10% 
SDS polyacrylamide gels, followed by Coomassie-blue staining (FIG. 1, 
panel A) or blotting (FIG. 1, panel B) to an Immobilon-P membrane 
(Millipore Corp., New Bedford, Mass.) as described by Rose et al., 
"Expression of the full-length products of the HPV-6b and HPV-11 L2 open 
reading frames by recombinant baculovirus, and antigenic comparisons with 
HPV-11 whole virus particles," J. Gen. Virol. 71:2725-2729 (1990), which 
is hereby incorporated by reference. 
3. Preparation Of Non-Recombinant And Recombinant L1 Stock Solutions. 
These assays were performed using dilutions of clarified (high-speed) 
supernatant stock solutions prepared from extracts of either AcNPV or 
Ac11L1-infected insect cells. Suspension cultures (100 ml) of Sf-9 cells 
infected either with AcNPV or Ac11L1 at an approximate multiplicity of 
infection of 10 plaque forming units per cell were incubated at 27.degree. 
C. for 72 hours. Cultures were then centrifuged at 1,000.times.g for 10 
minutes at 4.degree. C. and cell pellets were resuspended in 20 ml 
homogenization buffer (lysis buffer with 1 M NaCl) and homogenized with 50 
strokes in a Dounce homogenizer on ice. Homogenates were transferred to 
cold 30 ml screw-cap Corex tubes and centrifuged at 3,000.times.g for 10 
minutes at 4.degree. C. Low-speed supernatant fractions were then 
transferred to a clean tube and centrifuged at 100,000.times.g for 30 
minutes at 4.degree. C. Total protein concentrations of high speed 
supernatant fractions were measured by spectrophotometric absorption at 
280 nm according to the procedure of Stoscheck, "Quantitation of 
proteins," 1990, in Methods in Enzvmology, vol. 182, p.54, Academic Press, 
Inc., New York, which is hereby incorporated by reference, and adjusted to 
equivalence with fresh homogenization buffer (protein concentrations 
approximately equal to 30 mg/ml). Glycerol was added to 10% (v/v) and 
stock solutions were aliquoted and stored at -20.degree. C. 
4. Western Blot And Immunodotblot Assays. 
Western blot and immunodotblot assays were used to determine linear and 
conformational epitope antibody specificities in rabbit antisera and human 
sera. The Western blot assays (FIG. 3, panel A, and FIG. 4) were performed 
using 2 .mu.l (about 60 .mu.g total protein) of recombinant L1 stock 
solution diluted 1:100 with 1.times. Laemmli sample buffer, which contains 
protein denaturation reagents as described by Laemmli, "Cleavage of 
structural proteins during the assembly of the head of the bacteriophage 
T4," Nature 277:680-685 (1990), which is hereby incorporated by reference, 
and heated to 95.degree. C. for 3 minutes. The denatured sample was loaded 
in a single 100 mm wide sample well, electrophoresed in a 10% SDS 
polyacrylamide gel, and blotted to an Immobilon-P membrane. After blocking 
with a 2% BSA solution (Kirkegaard and Perry Labs, Inc., Gaithersburg, 
Md.) for 2 hours at 37.degree. C., the membrane was sliced into 24, 4 mm 
wide strips, each containing about 2.5 .mu.g total protein. Thereafter, 
the strips were probed with antisera (FIG. 3, panel A, and FIG. 4). 
For immunodotblot analysis, non-recombinant or recombinant L1 stock 
solutions were diluted 1:1,000 with cold PBS (1 mM CaCl.sub.2 and 100 
.mu.l aliquots (containing about 3.0 .mu.g total protein) were dotted onto 
an Immobilon-P membrane. Protein denaturation reagents were omitted from 
the immunodotblot sample preparation to preserve the native conformation 
of recombinant L1. Blocking, primary and secondary antibody diluent 
solutions, washes, and substrate used are as described by Strike et al., 
"Expression in Escherichia coli of seven DNA segments comprising the 
complete L1 and L2 open reading frames of human papillomavirus type 6b and 
the location of the (common antigen)," J. Gen. Virol. 70:543-555 (1989), 
which is hereby incorporated by reference. Primary antibody incubations 
were performed overnight at 4.degree. C., second antibody incubations were 
done at room temperature for 90 minutes. For immunodotblots, all solutions 
except the substrate solution contained CaCl.sub.2 at 1 mM. Primary 
antibody dilutions were 1:2,000 for rabbit antisera and 1:1,000 for human 
sera. Specifically-bound antibodies were detected with affinity-purified 
anti-rabbit (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, Md.), 
or anti-human (TAGO Immunodiagnostics, Burlingame, Calif.) IgG-alkaline 
phosphatase conjugates used at dilutions of 1:2,000 and 1:5,000, 
respectively, using BCIP/NBT (Kirkegaard and Perry Laboratories, Inc.) as 
substrate. Immunodotblot reactions were assessed by visual comparison of 
non-recombinant and recombinant L1 dot intensities. A reaction was 
considered positive if the color intensity of the recombinant L1 dot was 
greater than the color intensity of the non-recombinant control dot 
present on the same strip. 
5. Antisera. 
The denatured L1 antiserum used was described previously as anti-pEX480 by 
Strike et al., "Expression in Escherichia coli of seven DNA segments 
comprising the complete L1 and L2 open reading frames of human 
papillomavirus type 6b and the location of the (common antigen)," J. Gen. 
Virol. 70:543-555 (1989), which is hereby incorporated by reference. This 
antiserum was obtained by rabbit immunization with a gel-purified 
bacterially-expressed fusion protein that contained a 160 amino acid 
sequence derived from the mid-region of the HPV-6b L1 open reading frame 
fused to the carboxy terminus of betagalactosidase, as described by 
Stanley et al., "Construction of a new family of high efficiency bacterial 
expression vectors: Identification of cDNA clones coding for human liver 
proteins," EMBO. J. 3:1429-1434 (1984); and Strike et al., "Expression in 
Escherichia coli of seven DNA segments comprising the complete L1 and L2 
open reading frames of human papillomavirus type 6b and the location of 
the (common antigen)," J. Gen. Virol. 70:543-555 (1989), which are hereby 
incorporated by reference. This sequence contains the papillomavirus L1 
common antigen as described by Strike et al., "Expression in Escherichia 
coli of seven DNA segments comprising the complete L1 and L2 open reading 
frames of human papillomavirus type 6b and the location of the (common 
antigen)," J. Gen. Virol., 70:543-555 (1989), which is hereby incorporated 
by reference. The rabbit whole virus particle antiserum used was as 
described by Bonnez et al., "Antibody-mediated neutralization of human 
papillomavirus type 11 (HPV-11) infection in the nude mouse: Detection of 
HPV-11 mRNAs by the polymerase chain reaction," J. Inf. Dis., 165:376-380 
(1992), which is hereby incorporated by reference, and produced by 
immunization of rabbits with purified non-denatured HPV-11 virions, which 
were obtained from athymic mouse foreskin xenografts according to Bonnez 
et al., "Antibody-mediated neutralization of human papillomavirus type 11 
(HPV-11) infection in the nude mouse: Detection of HPV-11 mRNAs by the 
polymerase chain reaction," J. Inf. Dis., 165:376-380 (1992); and Kreider 
et al., "Laboratory production in vivo of infectious human papillomavirus 
type 11," J. Virol., 61:590-593 (1989), which are hereby incorporated by 
reference. Patients' sera were obtained from individuals with 
biopsy-proven condyloma acuminatum. Serum specimens previously found 
positive by HPV-11 whole virus particle-based ELISA as described by Bonnez 
et al., "Use of human papillomavirus type 11 virions in an ELISA to detect 
specific antibodies in humans with condylomata acuminata," J. Gen. Virol., 
72:1343-1347 (1991), which is hereby incorporated by reference, were used 
to maximize the ability to detect antibodies directed against VLPs. 
Control sera were obtained from nuns who professed no lifetime sexual 
contact. These sera were negative for HPV-11 antibodies as determined by 
the HPV-11 particle-based ELISA as described by Bonnez et al., "Use of 
human papillomavirus type 11 virions in an ELISA to detect specific 
antibodies in humans with condylomata acuminata," J. Gen. Virol., 
72:1343-1347 (1991), which is hereby incorporated by reference. 
6. Production and Purification of HPV-11 L1 Virus-like Particles. 
Recombinant VLPs were purified directly from the cell-free culture 
supernatant of Ac11L1-infected Sf-9 cell suspension cultures by a series 
of low and high speed centrifugation steps. Infected Sf-9 cells were 
pelleted from a 200 ml suspension culture a low speed (1,000.times.g) and 
the cell-free supernatant was centrifuged again at high speed 
(100,000.times.g) for 90 minutes at 4.degree. C. The high-speed pellet was 
resuspended in buffer A (50 mM Tris, pH 8.0; 1 M NaCl; 10 mM MgCl.sub.2 ; 
10 mM CaCl.sub.2 ; 2 mM phenylmethylsulfonyl fluoride (PMSF); 10 .mu.g/ml 
Leupeptin), 5.2 g solid CsCl were added, and the final volume was adjusted 
to a total of 13 ml with fresh buffer A (0.4 g/ml final concentration). 
After centrifugation (100,000.times.g, 22 hours, 10.degree. C.), the 
single band obtained was removed and diluted with 12 ml of fresh buffer A 
(without CsCl) and centrifuged again (100,000.times.g, 90 minutes, 
4.degree. C.) to pellet purified VLPs. VLPs purified by sucrose density 
gradient centrifugation were identified by electron microscopy after 
staining with 2% neutral buffered phosphotungstic acid (FIGS. 2, 6, and 
7). 
EXAMPLE II 
Expression And Immunologic Detection Of Recombinant HPV-11 L1 Protein In 
Sf-9 cells 
SDS-PAGE analysis of total Sf-9 cell proteins from insect cells infected 
with the recombinant virus Ac11L1 demonstrated a novel 55 kD protein seen 
by Coomassie-blue staining in Ac11L1-infected cells (FIG. 1A, lane 3). 
With reference to FIGS. 1 (A and B), FIG. 1A shows Coomassie-stained SDS 
polyacrylamide gel of wild-type AcNPV and recombinant Ac11L1-infected Sf-9 
cell lysates and FIG. 1B shows Western blot of wild-type AcNPV and 
recombinant Ac11L1-infected Sf-9 cell lysates probed with a rabbit 
polyclonal antiserum specific for the HPV L1 common epitope. 
Non-recombinant (lanes 1,2) and recombinant L1-infected (lanes 3,4) Sf-9 
cell lysates were fractionated into insoluble (lanes 1,3) and soluble 
(lanes 2,4) fractions, and electrophoresed on 10% polyacrylamide gels. 
Molecular reference (M.sub.r) markers are displayed at the left, and the 
arrow at the right indicates the approximate position of recombinant L1 
(about 55 kD M.sub.r). This protein is not present in wild-type AcNPV 
lysates, and co-migrates with a protein that is immunoreactive (FIG. 1B, 
lanes 3 and 4) with a rabbit antiserum prepared against the linear HPV L1 
common antigen as described by Strike et al., "Expression in Escherichia 
coli of seven DNA segments comprising the complete L1 and L2 open reading 
frames of human papillomavirus type 6b and the location of the (common 
antigen)," J. Gen. Virol. 70:543-555 (1989), which is hereby incorporated 
by reference. Lower M.sub.r L1-immunoreactive bands were also detected and 
may be derived from degradation of the full-length L1 product (FIG. 1B, 
lanes 3 and 4). Although the predominant portion of L1 produced in this 
system appeared in the NP40-insoluble fraction, approximately 25-30% was 
present in the NP40-soluble fraction (FIG. 1B, lane 4). Maximal L1 
accumulation occurred at 72 hours post-infection. 
EXAMPLE III 
Electron microscopic visualization of VLPs 
Electron micrographs of negatively stained preparations of sucrose banded 
VLPs (FIGS. 2, 6, and 7) showed distinct VLPs. FIG. 2 shows HPV-11 
capsid-like particles which were present at the 50-60% interface of the 
sucrose density gradient. FIG. 6 shows HPV type 6b (HPV-6b) capsid-like 
particles which resulted from the expression of the HPV-6b L1 coding 
sequence in the baculovirus system, and which were purified in exactly the 
same manner. FIG. 7 demonstrates that this method is also suitable for the 
production of HPV type 16 (HPV-16) VLPs, upon expression of the HPV-16 L1 
coding sequence. FIGS. 12 & 16 demonstrate that VLPs can be purified by 
cesium chloride density gradient centrifugation as well. Particle 
diameters determined by direct measurement of the VLPs in FIG. 2, were 
approximately 52 nm. This measurement is consistent with the diameter of 
isolated papillomavirus virions as described by Klug et al., "Structure of 
viruses of the papilloma-polyoma type I: Human wart virus," J. Mol. Biol. 
11:403-423 (1965), which is hereby incorporated by reference. 
EXAMPLE IV 
Immunoreactivity Of HPV-11 VLP-Containing Insect Cell Extracts With Rabbit 
Antisera 
The immunologic properties of the recombinant L1 protein were studied using 
rabbit antisera that reacted with native or denatured L1 protein epitopes. 
Rabbit antiserum pEX480, directed against the common papillomavirus 
antigen, reacted well with denatured recombinant L1 in Western blot 
assays, but did not react with the same antigen preparation by 
immunodotblot, a type of immunoassay in which the antigen is placed on the 
blotting membrane under non-denaturing conditions (FIG. 3, compare strips 
A). In contrast to the pattern of reactivity exhibited by anti-pEX480, the 
rabbit polyclonal antiserum raised against HPV-11 whole virus particles 
did not react with recombinant L1 by Western blot, but reacted strongly 
with recombinant L1 in the immunodotblot assay (FIG. 3, compare strips C). 
This reactivity was specific as demonstrated by lack of reactivity in the 
post-immune serum against the native non-recombinant control preparation 
(FIG. 3, panel B, strip C). Rabbit antiserum pEX215 was included in these 
immunoassays to allow comparison of the relative amounts of L1 present in 
the two types of immunoassays. The level of immunoreactivity of the pEX215 
antiserum with recombinant L1 in both formats is roughly equivalent (FIG. 
3, strips B), indicating that the amounts of L1 present are approximately 
equal. Furthermore, the observation that this antiserum is able to react 
with L1 in both formats suggests that the linear immunoreactive L1 
amino-terminal epitope(s) recognized by the pEX215 antiserum is not 
obscured by the adoption of higher-order L1 conformation. 
EXAMPLE V 
Immunoreactivity Of VLP-Containing Insect Cell Extracts With Human Sera 
To determine the prevalence of antibodies in human sera directed against 
linear versus conformational epitopes, sera obtained from individuals with 
biopsy-proven condyloma acuminatum were evaluated in Western blot and 
immunodotblot assays using VLPs as antigen. None of the patients' or 
control sera were immunoreactive with denatured recombinant L1 by Western 
blot (FIG. 4, strips D-O (patients) and P-X (controls)). Conversely, 11 of 
12 patients' sera (FIG. 5, strips D-O were read as positive, with the 
exception of strip H) and 0 of 9 control sera (FIG. 5, strips P-X) were 
immunoreactive with recombinant L1 by immunodotblot, a highly 
statistically significant difference (p=7.times.10-.sup.5 ; Fisher's exact 
test). This result correlates well with results previously obtained using 
the same sera in an HPV-11 particle-based ELISA as described by Bonnez et 
al., "Use of human papillomavirus type 11 virions in an ELISA to detect 
specific antibodies in humans with condylomata acuminata," J. Gen. Virol. 
72:1343-1347 (1991), which is hereby incorporated by reference. 
EXAMPLE VI 
ELISA Assay 
CsCl-purified VLPs were quantitated by spectrophotometer (A.sub.280) and 
diluted to a concentration of 8 ng/ul in cold PBS. Aliquots (100 .mu.l) of 
either PBS or diluted VLP solution (800 ng total protein) were loaded into 
wells and plates were allowed to stand at 4.degree. C. overnight. Plates 
were blocked for 2 hours at room temperature with a 1% BSA solution, 
followed by the addition of antisera, in duplicate, at a dilution of 
1:100. Primary antisera were reacted at room temperature for 90 minutes. 
Plates were washed four times and secondary antibody (goat anti-Human 
IgG-alkaline phosphatase conjugate) was added (TAGO, 1:5000) and plates 
were allowed to stand at room temperature for 90 minutes. Substrate was 
added to each well and absorbance at 405 nm was read. Specific absorbance 
was calculated by subtracting the PBS absorbance from the VLP absorbance 
for each replicate, and taking the average absorbance value. 
The result obtained using VLPs (FIG. 8) were equivalent to results 
previously reported in an ELISA test of the same sera (from RRP patients), 
which used HPV-11 whole virus particles as antigen (50%). Good correlation 
with results from a previous whole virus particle-based ELISA is given in 
FIG. 9 (r.sup.2 =0.75). 
EXAMPLE VII 
Western Blot and Immunodotblot 
Sf-9 suspension cultures (100 ml) were infected with either AcNPV 
(non-recombinant control), Ac11L1, or Ac16L1 recombinant baculoviruses as 
previously described by Rose et al., J. Virol., 67:1936-1944 (1993), which 
is hereby incorporated by reference, and incubated 72 hours at 27.degree. 
C. With reference to FIG. 11, samples were prepared, electrophoresed, and 
immunoblotted as previously described by Rose et al., J. Virol., 
67:1936-1944 (1993); and Rose et al., J. Gen. Virol., 71:2725-2729 (1990), 
which are hereby incorporated by reference. VLPs were present in both 
sample preparations, as verified by electron microscopy (data not shown). 
Total sample protein concentrations were equilibrated prior to use by 
spectrophotometer (A.sub.280). 
With reference to FIG. 10(a), samples (20 .mu.g total protein/lane) were 
electrophoresed in a 10% SDS-polyacrylamide gel and Western blotted 
overnight as previously described by Bonnez et al., J. Inf. Dis. 
165:376-380 (1992), which is hereby incorporated by reference. The 
nitrocellulose blot was probed with rabbit antiserum R5-409, used at a 
dilution of 1:1000 as described by Christensen et al., Virus Research 
21:169-179 (1991), which is hereby incorporated by reference. As shown in 
FIG. 10(a) (left panel), recombinant HPV-11 L1 (lane 2) and recombinant 
HPV-16 L1 (lane 3) proteins were detected in approximately equal amounts 
by anti-papillomavirus L1 common epitope antiserum R5-409. The predicted 
amino acid sequence of the HPV-16 L1 protein is five amino acids longer 
than the predicted sequence of the HPV-11 L1 protein, which is consistent 
with the slightly slower rate of migration exhibited by the recombinant 
HPV-16 L1 protein. 
With reference to FIG. 10(b), samples were diluted (two-fold serial 
dilutions made with PBS) and applied to nitrocellulose under 
non-denaturing conditions, beginning with a total protein concentration of 
25 .mu.g (bottom), and ending with a total protein concentration of 25 ng 
(top)). Rabbit antiserum R-366 was used at a dilution of 1:1000. In the 
right panel (i.e., the immunodotblot), the whole virus particle antiserum 
detected the native recombinant HPV-11 L1 VLP preparation over a 1000-fold 
dilution range. However, this same hyperimmune rabbit antiserum was not 
immunoreactive with the native recombinant HPV-16 L1 VLP preparation, even 
at a higher concentration of antigen (25 .mu.g) than that used for 
analysis by Western blot (20 .mu.g). 
The hyperimmune rabbit native HPV-11 virion neutralizing antiserum did not 
cross-react with native HPV-16 L1 protein, suggesting that the 
conformational epitope(s) of the HPV-11 capsid that is recognized by this 
antiserum is immunologically distinct from conformational epitopes present 
in the HPV-16 VLP preparation. 
EXAMPLE VIII 
Western Immunoblot Assay 
VLPs were detected in, and purified directly from, the supernatant medium 
of an Ac11L1-infected Sf-9 cell suspension culture (200 ml). Cells were 
pelleted at low speed (1000.times.g) and the cell-free supernatant was 
then centrifuged at high speed (100,000.times.g). Cells were removed by 
low speed centrifugation (1000.times.g), and VLPs were prepared from 
culture supernatants as previously described in Rose, R. C., et al., 
"Human Papillomavirus (HPV) Type 11 Recombinant Virus-Like Particles 
Induce the Formation of Neutralizing Antibodies and Detect HPV-Specific 
Antibodies in Human Sera," J. Gen. Virol. 75:2075-2079 (1994), which is 
hereby incorporated by reference. FIG. 11A is a 10% SDS-polyacrylamide gel 
stained with coomassie blue. FIG. 11B is a western immunoblot of an 
identically loaded gel, probed with a rabbit antiserum specific for the 
HPV common antigen as described by Strike et al., J. Gen. Virol 70:543-555 
(1989), which is hereby incorporated by reference, used at a dilution of 
1:1000. Examination of the high-speed pellets obtained from 
non-recombinant or recombinant L1-infected Sf-9 cell culture supernatants 
indicated the presence of VLPs in the recombinant L1-infected supernatant 
fraction. The resuspended recombinant L1 high-speed pellet was purified by 
equilibrium density gradient centrifugation as previously described in 
Bonnez et al., J. Inf. Dis. 165:376-380 (1992), which is hereby 
incorporated by reference. The single band obtained by this method was 
removed with a sterile 18 gauge needle, diluted with fresh buffer A (50 mM 
Tris, pH 8.0; 1 M NaCl; 10 mM MgCl.sub.2 ; 10 m M CaCl.sub.2 ; 2 mM 
phenylmethylsulfonyl fluoride (PMSF); 10 .mu.g/ml Leupeptin) to a volume 
of 12 ml, and again centrifuged at 100,000.times.g for 90 minutes at 
4.degree. C. After resuspension of the pellet in 0.5 ml of fresh buffer A 
(50% glycerol), electron microscopic analysis of a portion of the sample, 
negatively stained with 2% phosphotungstic acid, confirmed the presence of 
intact HPV VLPs (FIG. 12). 
As previously described, recombinant VLPs were immunoreactive with 
antibodies directed against HPV-11 whole virus particles. (See Rose et 
al., J. Virol. 67:1936-1944 (1993), which is hereby incorporated by 
reference.) In this study, applicants immunized rabbits with purified VLPs 
and tested the post-immune sera for immunoreactivity with whole virions. 
New Zealand white rabbits were immunized intramuscularly at two sites with 
a 1:1 emulsion of purified VLPs (.about.20 .mu.g protein) in complete 
Freund's adjuvant (0.25 ml per site). Boosts were given after 30 days with 
a VLP emulsion prepared in incomplete Freund's adjuvant, and immune sera 
were collected 14 days later. Sera were reacted with either native HPV-11 
virions or recombinant VLPs in a dotblot immunoassay, as previously 
described in Rose et al., J. Virol. 67:1936-1944 (1993), which is hereby 
incorporated by reference. The immunologic cross-reactivity of anti-VLP 
antibodies with whole virions, as shown in FIG. 13, demonstrates that VLPs 
are immunogenic, and appear to faithfully replicate the antigenic profile 
of infectious HPV-11 virions. With reference to FIG. 13, non-denatured 
purified sample preparations were applied to nitrocellulose as described 
by Rose et al., J. Virol. 67:1936-1944 (1993), which is hereby 
incorporated by reference. 
EXAMPLE IX 
Neutralization Activity 
The preparation of the infection HPV-11 .sub.Hershey viral suspension 
(originally provided by John Kreider, Department of Pathology and 
Microbiology and Immunology, The Milton S. Hershey Medical Center, 
Hershey, Pa.) has been described by Bonnez et al., J. Inf. Dis. 
165:376-380 (1992), which is hereby incorporated by reference. In four 
parallel experiments, 450 .mu.l of the infecting viral suspension (batch 
4/90) were incubated at 37.degree. C. for 1 hour with 50 .mu.l (1:10 final 
dilution) of either preimmune anti-HPV-11 serum (group 1), post-immune 
anti-HPV-11 serum (group 2), preimmune anti-VLP serum (group 3), or 
post-immune anti-VLP serum (group 4). Groups 1 and 2 were neutralization 
controls that have described previously by Bonnez et al., J. Inf. Dis. 
165:376-380 (1992), which is hereby incorporated by reference., and groups 
3 and 4 were the test groups. The preparation of human foreskins excised 
for routine circumcision has also been described by Bonnez et al., J. Gen. 
Virol. 72:1343-1347 (1991), which is hereby incorporated by reference. 
Foreskins were cut into 1.times.1 mm squares and small number of fragments 
from each foreskin used were snap frozen and saved. The remaining 
fragments were divided equally into four groups, and each group was added 
to one of the four viral suspension-serum mixtures at the end of the 
incubation period. Mixtures were incubated for 1 hour at 37.degree. C. For 
each experimental group, one foreskin fragment was placed under the renal 
capsule of each kidney of 3 female, litter-matched, 4-6 week old athymic 
nu/nu mice on a BALB/c background (Taconic Farms, Germantown, N.Y.). The 
experiment was replicated on a different day, with a different foreskin. 
Thus, for each experimental group a total of 12 grafts were implanted. The 
animals were sacrificed 12 weeks after grafting, at which time the grafts 
were removed and processed. (See Bonnez et al., J. Inf. Dis. 165:376-380 
(1992), which is hereby incorporated by reference). With reference to FIG. 
14, grafts were prepared for analysis as described herein and infected 
with viral lysate that was pre-treated with either (1) pre-, and (2) 
post-immune rabbit HPV-11 whole virus particle sera, or (3) pre-, and (4) 
post-immune rabbit HPV-11L1 virus-like particle sera. The filled circles 
correspond to the first replicate experiment, the open circles to the 
second replicate experiment. The horizontal bars indicate the median GMD. 
For graft size comparison, the geometric mean diameter (GMD) was 
calculated by taking the cubic root of the product of the length, width, 
and height of the recovered grafts. 
At the time of euthanasia, one graft was missing from each of the 
neutralization control pre- and post-immune anti-HPV-11 treated groups. 
Thus, the number of grafts available for analysis in each of these groups 
was 11 (FIG. 14). The median [range] GMDs (mm) of the grafts in the pre- 
and post-immune control groups were respectively 2.9 [1.0, 4.9] and 1.3 
[1.0, 2.6]. The difference, 1.6 mm, was statistically significant 
(P=0.004, Mann-Whitney U test). All 12 implanted grafts were available for 
analysis in both the pre- and post-immune anti-VLP antibody-treated groups 
(FIG. 4). The median [range] GMDs (in mm) of the grafts were respectively 
2.3 [1.3, 4.2] and 1.0 [1.0, 1.8]. The difference in size, 1.3 mm, was 
statistically significant (p&lt;10.sup.-4). Although the difference in graft 
sizes between the first and second experiment was not statistically 
significant (P=0.62) in the preimmune group, it was significant (P=0.007) 
in the post-immune group. Therefore, applicants compared the differences 
in graft sizes between the pre- and post-immune anti-VLP antibody-treated 
groups within each replicate experiment. Both were statistically 
significant (P=0.002 and P=0.04, respectively for the first and second 
replicate). cl EXAMPLE X 
Source of Viral DNAs 
The source of HPV-11 genomic DNA (Bonnez et al., J. Gen. Virol. 72:343-1347 
(1991), which is hereby incorporated by reference) and construction of the 
Ac11L1 recombinant baculovirus (Rose et al., J. Virol., 67:1936-1944 
(1993), which is hereby incorporated by reference) have been described. 
The HPV-16 genomic DNA was recovered from a CIN III lesion and standard 
cloning methods were used to construct the Ac16L1 baculovirus (Chesters 
and McCance, unpublished data). The HPV-18 L1 sequence was amplified by 
polymerase chain reaction from the HPV-18 prototype (provided by H. zur 
Hausen) and used to construct the Ac 18L1 baculovirus by the same 
procedure used for the construction of Ac11L1 (Rose et al., J. Virol., 
67:1936-1944 (1993), which is hereby incorporated by reference). 
EXAMPLE XI 
Purification of Recombinant VLPs 
Recombinant VLPs were purified as described by Rose, R. C., et al., "Human 
Papillomavirus (HPV) Type 11 Recombinant Virus-Like Particles Induce the 
Formation of Neutralizing Antibodies and Detect HPV-Specific Antibodies in 
Human Sera," J. Gen. Virol. 75:2075-2079 (1994), which is hereby 
incorporated by reference. Single bands containing purified HPV-11, 
HPV-16, or HPV-18 VLPs were removed from CsCl density gradients by 
syringe, diluted with buffer A (phosphate-buffered saline (PBS); 1 mM 
Mgcl.sub.2 ; 1 mM CaCl.sub.2 ; 1 mM phenylmethylsulfonylfluoride (PMSF)) 
to 12 ml, and sedimented at 100,000.times.g for 90 minutes at 4.degree. C. 
Pellets were resuspended in 200 .mu.l of buffer A containing 50% glycerol, 
quantitated by spectrophotometry (280 nm), and stored at -20.degree. C. 
Recombinant L1 proteins were analyzed by SDS-PAGE and Western blot 
immunoassay as previously described (Rose et al., J. Virol. 67:1936-1944 
(1993), which is hereby incorporated by reference). Samples containing 5 
.mu.g of purified HPV-11, -16, or -18 VLPs were electrophoresed, blotted, 
and probed with anti-papillomavirus L1 (anti-PVL1) common antigen rabbit 
antiserum as previously described (Strike et al., J. Gen. Virol. 
70:543-555 (1989); and Rose et al., J. Virol. 67:1936-1944 (1993), which 
are hereby incorporated by reference). Predicted coding capacities of the 
HPV-11, -16, and -18 L1 open reading frames (ORFs) are 501 amino acids 
(Dartmann et al., Virology 151:124-130 (1986), which is hereby 
incorporated by reference), 505 amino acids (Seedorf et al., Virology 
145:181-185 (1985), which is hereby incorporated by reference), and 507 
amino acids (Cole et al., J. Mol Biol. 193:599-608 (1987), which is hereby 
incorporated by reference), respectively, and an L1-immunoreactive band of 
the expected size (.about.55 kD M.sub.r) appeared in each of the three 
sample preparations tested by Western blot immunoassay (FIG. 15). Lower 
molecular weight L1-immunoreactive proteins were also detected by Western 
blot immunoassay of the CsCl-purified VLP preparations (FIG. 15), and are 
likely to be degradation products of full-length L1 proteins, as relative 
amounts of these proteins varied in subsequent analyses (data not shown). 
However, the major 55 kD M.sub.r L1-immunoreactive bands in each of the 
samples did not vary, either in their mobilities or their relative amounts 
(data not shown). Electron microscopy of purified samples 
(negatively-stained with 2% phosphotungstic acid) confirmed VLP formation 
in HPV-11 (FIG. 16A), HPV-16 (FIG. 16B) and HPV-18 (FIG. 16C) VLP 
preparations. 
EXAMPLE XII 
Preparation of Rabbit VLP Immune Sera and Conditions of the ELISA 
HPV-11, HPV-16, and HPV-18L1 VLP rabbit immune sera were prepared by 
immunizing two New Zealand white rabbits intramuscularly at two sites with 
each of the VLP preparations (i.e., six rabbits were immunized), using 
previously described methods (Bonnez et al., J. Inf. Dis. 165:376-380 
(1992); Rose et al., J. Virol. 67:1936-1944 (1993), which are hereby 
incorporated by reference). Rabbit anti-PVL1 common antigen (Strike et 
al., J. Gen. Virol. 70:543-555 (1989), which is hereby incorporated by 
reference), HPV-11 whole virion (Bonnez et al., J. Inf. Dis. 165:376-380 
(1992), which is hereby incorporated by reference), and HPV-11, -16, and 
-18 VLP antisera were tested with ELISA against the three recombinant VLP 
preparations (FIG. 17). For this ELISA, purified VLPs were diluted to a 
concentration of 10 ng/.mu.l in PBS, and aliquots containing approximately 
1 .mu.g of antigen or PBS alone were dispensed into alternate rows of 
96-well ELISA plates. The conditions of the assay were exactly as 
previously described (Rose, R. C., et al., "Human Papillomavirus (HPV) 
Type 11 Recombinant Virus-Like Particles Induce the Formation of 
Neutralizing Antibodies and Detect HPV-Specific Antibodies in Human Sera," 
J. Gen. Virol. 75:2075-2079 (1994), which is hereby incorporated by 
reference), except primary antisera were pre-absorbed with non-recombinant 
(AcNPV) baculovirus-infected Sf-9 cell lysate diluted in blocking solution 
(2% v/v) prior to testing. All antisera were tested in duplicate, on 
numerous occasions, at dilutions ranging from 1:1000 to 1:128,000. 
Absorbance values for all of the rabbit anti-VLP antisera shown in FIG. 17 
were obtained at the optimal dilution for these antisera of 1:16,000. 
Absorbance values for the anti-PVL1 common antigen and HPV-11 whole virion 
rabbit antisera were obtained at lower dilution (1:1,000). Specific 
absorbance values were determined by subtracting control values (PVS 
wells) from experimental values (antigen-containing wells) for each 
replicate, and mean (405 nm) absorbance values were determined. 
EXAMPLE XIII 
VLP ELISA 
VLPs were tested in ELISA immunoassay to assess their ability to detect 
specific antibodies in patients' sera, and the results were compared with 
results previously obtained using the same sera in an HPV-11 whole virion 
ELISA immunoassay (Bonnez et al., J. Med. Virol. 39:340-344 (1993), which 
is hereby incorporated by reference). The antigen was diluted in 
phosphate-buffered saline (PBS) to give an amount equivalent to that of 
the amount used in the previous whole virion ELISA (Bonnez et al., J. Med. 
Virol. 39:340-344 (1993), which is hereby incorporated by reference), and 
either the antigen solution or PBS without any antigen was aliquoted into 
alternate rows of 96-well plates. After coating for 16 hours at 4.degree. 
C., these solutions were aspirated and wells were blocked with 
diluent/blocking solution (Kirkegaard and Perry Laboratories, Inc., 
Gaithersburg, Md.) at room temperature for 2 hours. A total of 59 human 
sera (43 patients, 16 controls) previously tested by HPV-11 whole virus 
particle ELISA (Bonnez et al., J. Med. Virol. 39:340-344 (1993), which is 
hereby incorporated by reference) were diluted 1:100 in diluent/blocking 
solution and 100 .mu.l aliquots were added to wells treated either with 
PBS alone or with antigen solution (two replicates per serum sample). 
Plates were incubated at room temperature for 90 minutes, and then washed 
four times (wash solution, Kirkegaard and Perry Laboratories, Inc., 
Gaithersburg, Md.). Anti-human IgG-alkaline phosphatase conjugate (100 
.mu.l aliquots, diluted 1:5000, TAGO, Burlingame, Calif.), was added to 
each well and plates were incubated at room temperature for 90 minutes. 
Plates were washed four times and developed with alkaline phosphatase 
substrate (p-nitrophenyl phosphate in diethanolamine buffer). Specific 
absorbance at 405 nm for each serum sample was calculated by subtracting 
the value obtained from the PBS-treated well from the value obtained from 
the antigen-containing well for each replicate and mean replicate 
differences were calculated. In the whole virion ELISA discussed elsewhere 
herein, 42 patients' sera was analyzed (and 20 control sera) for changes 
in capsid antibody levels during the course of treatment (Bonnez et al., 
J. Med. Virol. 39:340-344 (1993), which is hereby incorporated by 
reference). All sera tested in the present ELISA study were collected at 
entry into the previous study. One of the patients' sera analyzed in the 
previous study were subsequently excluded for reasons related to treatment 
outcome and not to the results of the immunoassay. However, because the 
absorbance value of this serum was available, the serum was included in 
the present assay, which increased the number of patients' sera analyzed 
in the present ELISA study to 43. The number of control sera analyzed was 
reduced from 20 to 16 for logistical considerations pertaining to the 
assay. 
The median [range] seroreactivity of the 16 control sera, expressed as an 
OD value, was 0.005 [-0.029, 0.025], compared to 0.024 [-0.063, 0.512] for 
the 43 patients' sera, a statistically significant difference (P=0.01; 
Mann-Whitney U test). Using the highest OD value in the control group as a 
cut-off, the sensitivity of the assay was 49% (P=2.times.10.sup.-4 ; 
Fisher's exact test). Therefore, the HPV-11 VLP ELISA was able to 
discriminate between patients with condyloma acuminatum and controls. In 
addition, there was excellent correlation (Pearson's product-moment 
r=0.87; P &lt;10.sup.-6) between sample seroreactivities with the HPV-11 VLP 
ELISA and the HPV-11 virion ELISA when all sera were included, or when 
only the 21 sera positive by HPV-11 VLP ELISA were considered (r=0.87; 
P&lt;10.sup.-6). 
EXAMPLE XIV 
Orally administered VLPs induce systemic immunoglobulin G (IgG) and IgA 
antibody responses. 
HPV-11 VLPs were produced by co-infecting insect cells with recombinant 
baculoviruses Ac11L1 and Ac11L2, which were constructed as previously 
described (Rose, R. C., et al., "Expression Of Human Papillomavirus Type 
11 L1 Protein In Insect Cells: In Vivo And In Vitro Assembly of Virus-like 
Particles," J. Virology 67(4):1936-1944 (1993); Rose, R. C., et al., 
"Expression Of the Full-Length Products Of the Human Papillomavirus Type 
6b (HPV-6b) and HPV-11 L2 Open Reading Frames By Recombinant Baculovirus, 
And Antigenic Comparisons With HPV-11 Whole Virus Particles," J. Gen. 
Virol. 71:2725-2729 (1990), which are hereby incorporated reference.) 
Purified VLPs were formulated in three dosage levels (100 .mu.g, 50 .mu.g, 
or 10 .mu.g, in phosphate-buffered saline) and were administered 
intragastrically on three occasions over a six-week period to three groups 
of five female BALB/c mice (ages 8-10 weeks). Booster inoculations were 
also administered orally 14 and 41 days after primary immunizations. A 
fourth group of mice received no inoculations. Sera were collected every 
two weeks by retro-orbital puncture. Pre- and post-immune sera were pooled 
within each group of mice after collection, and were tested in an 
enzyme-linked immunosorbent assay (ELISA), as previously described (Li, 
M., et al., "Expression of The Human Papillomavirus Type 11 L1 Capsid 
Protein In Escherichia Coli: Characterization of Protein Domains Involved 
in DNA-Binding And Capsid Assembly," J. Virologv, 71:2988-2995 (1997); 
White, W. I., et al., "In Vitro Infection And Type-Restricted 
Antibody-Mediated Neutralization Of Authentic Human Papillomavirus Type 
16," J. Virology, 72:959-964 (1998), which are hereby incorporated by 
reference.) Specific absorbance Values (405 nm) were determined as 
previously described (Li, M., et al., "Expression of The Human 
Papillomavirus Type 11 L1 Capsid Protein In Escherichia Coli: 
Characterization of Protein Domains Involved in DNA-Binding And Capsid 
Assembly," J. Virology, 71:2988-2995 (1997); White, W. I., et al., "In 
Vitro Infection And Type-Restricted Antibody-Mediated Neutralization Of 
Authentic Human Papillomavirus Type 16," J. Virology, 72:959-964 (1998), 
which are hereby incorporated by reference.) Responses in the intermediate 
and high antigen dose groups became comparable within two weeks after the 
second booster inoculation (FIG. 18A). 
Serum IgA antibody responses were also detected in post-immune sera from 
immunized animals (FIG. 18B) after removing serum IgG antibodies by 
pre-treatment with goat anti-mouse IgG (Kirkegaard and Perry Laboratories, 
Inc., Gaithersburg, Md.) as described (Gray, J. J., et al., "Detection Of 
Immunoglobulin M (IgM), IgA, and IgG Norwalk Virus-Specific Antibodies By 
Indirect Enzyme-Linked Immunosorbent Assay With Baculovirus-Expressed 
Norwalk Virus Capsid Antigen In Adult Volunteers Challenged With Norwalk 
Virus," Journal of Clinical Microbiology 32(12):3059-3063 (1994), which is 
hereby incorporated by reference.) Although the kinetics of IgG and IgA 
antibody responses were comparable (FIGS. 18A,B), the relative magnitude 
of the IgG response was much greater than that of the IgA response (FIG. 
18). 
IgG subclass analysis (Southern Biotechnology Associates, Inc., Birmingham, 
Ala.) indicated there was no clear predominance of any specific subclass, 
although the appearance of IgG2a and IgG2b preceded that of IgG.beta. 
(Table 1). 
TABLE 1 
______________________________________ 
VLP Serum IgG Subclass Analysis 
Weeks Post-Immunization (A.sub.405) 
Subclass 
0 2 4 6 8 10 
______________________________________ 
IgG1 0.010 0.139 0.864 
0.851 2.354 
1.022 
IgG2a 0.021 0.757 1.293 
1.319 2.550 
1.083 
IgG2b 0.021 0.506 1.511 
1.274 1.917 
0.565 
IgG3 0.012 0.213 0.292 
0.164 0.286 
0.067 
______________________________________ 
IgG2a has been shown to be a prominent component of murine immune responses 
to viral infections (Coutelier, J. P., et al., "Virally Induced Modulation 
of Murine IgG Antibody Subclasses," J. Exp. Med. 168(6):2373-2378 (1988), 
which is hereby incorporated by reference). 
EXAMPLE XV 
Antigenic specificities of orally induced serum VLP antibodies. 
Orally induced antibodies were evaluated in an ELISA against native and 
denatured HPV-11 VLPs (Rose, R. C., et al., "Expression of Human 
Papillomavirus Type 11 L1 Protein in Insect Cells: in vivo and in vitro 
Assembly of Virus-like Particles," J. Virology 67(4):1936-1944 (1993), 
which is hereby incorporated by reference), and against heterologous 
native VLPs, as previously described (Rose, R. C., et al., "Serological 
Differentiation of Human Papillomavirus Types 11, 16 and 18 Using 
Recombinant Virus-Like Particles," J. Gen. Virol. 75:2445-2449 (1994), 
which is hereby incorporated by reference). Antigen denaturation was 
accomplished by diluting VLPs in carbonate buffer (pH 9.5; 0.01 mg/ml 
final concentration) followed by incubation in a boiling water bath (10 
minutes) (Dillner, L, et al., "Antigenic and Immunogenic Epitopes Shared 
By Human Papillomavirus Type 16 and Bovine, Canine, and Avian 
Papillomaviruses," J. Virology 65(12):6862-6871 (1991), which is hereby 
incorporated by reference). Native VLP antigens were diluted in 
phosphate-buffered saline (PBS, pH 7.1) to the same final concentration 
and kept on ice prior to evaluation. As shown in FIG. 19, the 
specificities of orally induced serum IgG and IgA VLP antibodies were 
entirely dependent on native antigen conformation. Consistent with 
previously reported results (Rose, R. C., et al., "Serological 
Differentiation of Human Papillomavirus Types 11, 16 and 18 Using 
Recombinant Virus-Like Particles," J. Gen. Virol. 75:2445-2449 (1994), 
which is hereby incorporated by reference), orally induced HPV-11 VLP 
antibodies were highly immunoreactive with VLPs of the same type used for 
immunization, but were not immunoreactive by ELISA with VLPs of a 
heterologous HPV genotype (FIG. 19). 
EXAMPLE XVI 
Epitope-Blocking ELISA. 
A VLP epitope-blocking ELISA was developed as a surrogate assay for 
detecting antibody-mediated virus-neutralizing activity. In this assay, an 
unknown antibody preparation is evaluated for the ability to prevent 
homologous VLP binding by antibodies that are known to neutralize 
homologous infectious virions; blockade of the neutralizing domain is 
interpreted as evidence of neutralizing activity in the unknown. The 
epitope-blocking assay requires that the unknown and virus-neutralizing 
antibodies be raised in alternate host species. The ability of rabbit 
HPV-11 virion-neutralizing polyclonal antibodies (N-pAb) (Rose, R. C., et 
al., "Human Papillomavirus (HPV) Type 11 Recombinant Virus-Like Particles 
Induce the Formation of Neutralizing Antibodies and Detect HPV-Specific 
Antibodies in Human Sera," J. Gen. Virol. 75:2075-2079 (1994), which is 
hereby incorporated by reference) to block the epitope recognized by a 
previously characterized HPV-11 virion neutralizing monoclonal antibody 
(N-mAb), H11.H3 (Christensen, N. D., et al., "Monoclonal Antibody-Mediated 
Neutralization of Infectious Human Papillomavirus Type 11," J. Virology 
64(11):5678-5681 (1990), which is hereby incorporated by reference) was 
tested. Serial three-fold dilutions of Rabbit HPV-11 N-pAb were tested in 
duplicate wells containing 250 ng of HPV-11 VLPs. Following this, 
pre-titered H11.H3 was diluted below the level of antigen saturation 
(i.e., 1:180,000) and added to wells containing rabbit N-pAb/VLP 
complexes. Lastly, relative amounts of bound N-pAb versus bound N-mAb were 
determined by adding anti-rabbit or anti-mouse IgG-alkaline phosphatase 
enzyme conjugates (Kirkegaard and Perry Laboratories, Gaithersburg, Md.) 
to parallel rows. As expected, low dilutions of rabbit N-pAb efficiently 
blocked H11.H3 VLP binding (FIG. 20A), suggesting that the rabbit HPV-11 
N-pAb contained the neutralizing antigenic specificity defined by H11.H3. 
To further validate the assay, HPV-16 virion neutralizing monoclonal and 
polyclonal antibodies (Christensen, N. D., et al., "Surface Conformational 
and Linear Epitopes on HPV-16 and HPV-18 L1 Virus-Like Particles as 
Defined By Monoclonal Antibodies," Virology 223(1):174-184 (1996); White, 
W. I., et al., "In Vitro Infection and Type-Restricted Antibody-Mediated 
Neutralization of Authentic Human Papillomavirus Type 16," J. Virology 
72:959-964 (1998), which are hereby incorporated by reference) were 
evaluated in a similar manner, and comparable results were obtained. 
Serial dilutions of the murine antibodies produced in the present study 
were evaluated against a sub-saturating dilution of pre-titered rabbit 
HPV-11 N-pAb (1:9,000). Results indicated that, at low dilutions, orally 
induced HPV-11 VLP antibodies efficiently blocked rabbit N-pAb VLP binding 
(FIG. 20B), which suggested that a potentially protective humoral response 
may be induced after oral VLP immunization. 
RESULTS 
Immunologic observations suggest that recombinant L1 adopts a native 
conformation. The rabbit antiserum raised against the denatured L1 common 
antigen was immunoreactive only with denatured recombinant L1 (i.e., by 
Western blot), whereas the rabbit antiserum raised against non-denatured 
whole virus particles reacted only with non-denatured recombinant L1 
(i.e., by immunodotblot). Furthermore, human sera from condyloma 
acuminatum patients which were reactive with HPV-11 virions in an ELISA 
according to Bonnez et al., "Use of human papillomavirus type 11 virions 
in an ELISA to detect specific antibodies in humans with condylomata 
acuminata," J. Gen. Virol. 72:1343-1347 (1991), which is hereby 
incorporated by reference, also reacted with non-denatured HPV-11 
recombinant L1 (FIGS. 4, 8 & 9). Therefore, it appears that the 
conformational epitopes of the VLPs of the invention are similar to those 
present in native HPV-11 virions, which are recognized by the human immune 
system during natural infection. Several studies of papillomavirus 
serology demonstrate that conformational epitope antibody specificities 
are good indicators of papillomavirus infection (Bonnez et al., "Use of 
human papillomavirus type 11 virions in an ELISA to detect specific 
antibodies in humans with condylomata acuminata," J. Gen. Virol. 
72:1343-1347 (1991); Bonnez et al., "Evolution of the antibody response to 
human papillomavirus type 11 (HPV-11) in patients with condyloma 
acuminatum according to treatment response," J. Med. Virol. 39:340-44 
(1993); Bonnez et al., "Antibody-mediated neutralization of human 
papillomavirus type 11 (HPV-11) infection in the nude mouse: Detection of 
HPV-11 mRNAs by the polymerase chain reaction," 1992, J. Inf. Dis. 
165:376-380 (1992); Christensen et al., "Detection of human serum 
antibodies that neutralize infectious human papillomavirus type 11 
virions," J. Gen. Virol. 73:1261-1267 (1992); Kienzler et al., "Humoral 
and cell-mediated immunity to human papillomavirus type 1 (HPV-1) in human 
warts," Br. J. Dermatol. 108:665-672 (1983); and Steele et al., "Humoral 
assays of human sera to disrupted and nondisrupted epitopes of human 
papillomavirus type 1," Virology 174:388-398 (1990), which are hereby 
incorporated by reference). These specificities can also play a 
significant role in viral pathogenesis. For instance, a rabbit antiserum 
directed against whole HPV-11 particles neutralizes HPV-11 infectivity 
(Bonnez et al., "Antibody-mediated neutralization of human papillomavirus 
type 11 (HPV-11) infection in the nude mouse: Detection of HPV-11 mRNAs by 
the polymerase chain reaction," J. Inf. Dis. 165:376-380 (1992); and 
Christensen et al., "Antibody-mediated neutralization in vivo of 
infectious papillomavirus," J. Virol., 64:3151-3156 (1990), which are 
hereby incorporated by reference). Furthermore, Christensen et al., 
"Detection of human serum antibodies that neutralize infectious human 
papillomavirus type 11 virions," J. Gen. Virol. 73:1261-1267 (1992), which 
is hereby incorporated by reference, using human sera reported a 
correlation between anti-whole HPV-11 virion antibody and serum 
neutralizing activity. Detection of such antibodies with the recombinant 
L1 VLPs of the present invention can have diagnostic and functional 
significance. 
When taking into account construction of the recombinant baculovirus, some 
of the early recombinant baculoviruses applicants constructed had the 
correct L1 coding sequence, but were not producing detectable levels of L1 
proteins. This caused applicants to look at the 3' untranslated regions of 
the HPV-11 and several other HPV L1 coding sequences. It was determined 
that a pentanucleotide mRNA degradation signal sequence, AUUUA, (Shaw G. 
and Kamen R., "A conserved AU sequence from the 3' untranslated region of 
GM-CSF mRNA mediates selective mRNA degradation," Cell 46:659-67 (1986); 
Cole M D. and Mango S E., "cis-acting determinants of c-myc mRNA 
stability," Enzyme 44:167-80 (1990); Shyu A B et al., "Two distinct 
destabilizing elements in the c-fos message trigger deadenylation as a 
first step in rapid mRNA decay," Genes & Development 5:221-31 (1991); 
Savant-Bhonsale S. and Cleveland D W., "Evidence for instability of mRNAs 
containing AUUUA motifs mediated through translation-dependent assembly of 
a &gt;20S degradation complex," Genes & Development 6:1927-37 (1992), which 
are hereby incorporated by reference) was within 30 nucleotides of the 
stop codon of the HPV-11 L1 coding sequence, and in addition, the other 
HPV types looked at had the AUUUA sequence in the vicinity of the L1 stop 
codon as well. If this sequence were removed, or a mutation introduced, 
the expression level of the L1 protein could be increased. Therefore, PCR 
primers to amplify the L1 coding sequence from HPV-11 genomic DNA which 
not only incorporated restriction enzyme sites for cloning, but also 
mutated the AUUUA pentanucleotide sequence 30 nucleotides downstream from 
the L1 stop codon as well were designed. Scaleup of this clone produced 
extremely high levels of L1 protein. Reports using the BEVS system have 
given levels of recombinant protein production in the range of 300-500 
mg/liter of cell culture. In the present invention, levels for recombinant 
L1 protein production were much greater, about 600-800 mg/liter, possibly 
due to the removal of the L1 degradation signal sequence in the 3' 
untranslated region. 
These results show that, under similar experimental conditions, post-immune 
sera from rabbits immunized with HPV-11 VLPs can block HPV-11 infection of 
human tissue as effectively as sera obtained from rabbits immunized with 
HPV-11 whole virions. The blockage, which was not observed with the 
respective preimmune sera, was associated with the absence of early viral 
gene expression. Therefore, the effect was consistent with classic viral 
neutralization, i.e., the prevention of virus penetration or decapsidation 
(Dimmock, 1993, Neutralization of Animal Viruses, Berlin: Springer-Verlag, 
which is hereby incorporated by reference). 
To provide confirmation of HPV-11 neutralization by analysis of viral gene 
expression, all grafts were analyzed for the presence of the HPV-11 E1 E4 
spliced mRNA transcript (data not shown), as previously described in 
Bonnez et al., J. Inf. Dis. 165:376-380 (1992), which is hereby 
incorporated by reference. The E1 E4 mRNA was detected in 10/12 (83%) and 
0/12 (0%) of the grafts from groups pre-treated with pre- or post-immune 
VLP sera, respectively (p&lt;10.sup.-4). Similarly, for the control groups 
pre-treated with pre- or post-immune anti-whole virion sera, E1 E4 mRNA 
was detected in 8/11 (73%) and 0/11 (0%) grafts, respectively 
(p=10.sup.-3). These results indicate that treatment of the viral inoculum 
with the post-immune VLP serum is associated with marked inhibition of 
graft growth and viral gene expression, effects which are consistent with 
immune neutralization. Thus, recombinant VLPs can induce a neutralization 
response similar in magnitude to the response obtained by immunization 
with infectious virus. 
The HPV 16 L1-L2 VLPs described by Zhou et al., Virology 185:251-257 
(1991), which is hereby incorporated by reference, were variable in size 
and smaller (35-40 nm in diameter) than either HPV virions (50-55) or 
baculovirus produced HPV 11 VLPs (50-55 nm; Rose, R. C., et al., "Human 
Papillomavirus (HPV) Type 11 Recombinant Virus-Like Particles Induce the 
Formation of Neutralizing Antibodies and Detect HPV-Specific Antibodies in 
Human Sera," J. Gen. Virol. 75:2075-2079 (1994), which is hereby 
incorporated by reference). These morphologic characteristics are quite 
different from those of the VLPs described in the present invention. 
Furthermore, using the method of the invention, HPV L1 protein alone is 
sufficient for the formation of particles whose biophysical 
characteristics and antigenic properties closely reflect those of native 
HPV virions. 
Using a similar approach, Kimbauer, et al. reported inhibition of 
BPV-1-mediated transformation of mouse C127 cells in vitro by anti-BPV-1 
VLP antibodies (Kimbauer et al., Proc. Natl. Acad. Sci. USA 89:12180-12184 
(1992), which is hereby incorporated by reference.) The results obtained 
in that system support the results reported in the present invention, in 
which applicants have demonstrated neutralization using a genital HPV and 
it's normal target tissue. Although concordance of results from the 
BPV-1/C127 cell assay and the athymic mouse bovine fetal skin xenograft 
system has been reported as previously described by Ghim et al., Int. J. 
Cancer 49:285-289 (1993), which is hereby incorporated by reference, the 
BPV-1/C127 mouse fibroblast system is non-productive, and therefore 
neutralization can only be inferred from the absence of transformed foci 
in vitro. In addition, BPV-1 does not naturally infect mice, and the 
mechanism by which it gains entry into C127 cells may differ from the 
mechanism involved in the natural infection process. In contrast, the 
athymic mouse model used in the present study relies on infection by a 
genital HPV of its natural target tissue as previously described by 
Kreider et al., Nature 317:639-641 (1985), which is hereby incorporated by 
reference; the infected graft is maintained in vivo and morphologic and 
histologic transformation of the infected graft is accompanied by the 
production of infectious virions. (See Kreider et al., J. Virol. 
61:590-593 (1987), which is hereby incorporated by reference.) 
Antibody-mediated graft growth inhibition as previously described by 
Bonnez et al., J. Inf. Dis. 165:376-380 (1992); Christensen et al., J. 
Virol. 64:3151-3156 (1990); Christensen et al., Virus Research 21:169-179; 
Christensen et al., J. Virol. 64:5678-5681 (1990); and Christensen et al., 
J. Gen. Virol. 73:1261-1267 (1992), which are hereby incorporated by 
reference, and immunocytochemical and molecular biologic evidence of 
inhibition of viral gene expression has been well documented, as 
previously described by Bonnez et al., J. Gen. Virol. 72:1343-1347 (1991); 
and Bonnez et al., J. Inf. Dis. 165:376-380 (1992), which are hereby 
incorporated by reference. Therefore, observations made in the athymic 
mouse system may more accurately reflect the events that occur in the 
natural infection. 
Neutralizing antibodies to HPV-11 have been identified in humans with 
condyloma acuminatum as previously described by Christensen et al., J. 
Gen. Virol. 73:1261-1267 (1992), which is hereby incorporated by 
reference, but their biological significance is unknown. If neutralization 
proves to be a protective immunologic effector mechanism against 
papillomavirus infections in vivo, then immunization with recombinant VLPs 
may provide protective immunity to individuals at risk for infection. 
Applicant's results suggest that the magnitude of neutralization activity 
of HPV-11 VLP antibodies is similar to that of antibodies specific for 
HPV-11 infectious virions. Therefore, VLPs appear to be good vaccine 
candidates. However, the degree of cross-reactivity of capsid 
conformational determinants among different HPV types is not yet known and 
may be low as previously described by Gissmann et al., Virology 76:569-580 
(1977); Gross et al., Oncoienic Viruses, Pergamon Press, New York (1983); 
Hagensee et al., J. Virol. 67:315-322 (1993); Howley et al., 
"Papillomavirinae and their replication," Chap. 58. p. 1625-1650, in B. N. 
Fields and D. M. Knipe (ed.), Virology, 2nd ed., Vol. 2. Raven Press, New 
York (1990); Kirnbauer et al., Proc. Natl. Acad. Sci. USA, 89:12180-12184 
(1992); Kreider et al., J. Virol. 61:590-593 (1987); Kreider et al., 
Nature 317:639-641 (1985); and Orth et al., J. Virol., 24:108-120 (1977), 
which are hereby incorporated by reference. Full characterization of the 
potential of recombinant VLPs for use as immunogens for the prevention of 
genital HPV disease will require further studies involving VLPs derived 
from other genital HPV types. It will be of particular important to 
determine if antibodies to heterologous genital HPV VLPs will be capable 
of neutralizing HPV infection. 
With reference to FIG. 17, HPV-11 whole virus particle (B) and HPV-11 VLP 
antisera (C,D) reacted strongly with HPV-11 VLPs, but none of these 
antisera reacted with the HPV-16 or HPV-18 VLP preparations. Similarly, 
HPV-16 (E,F) and HPV-18 (G,H) L1 VLP rabbit antisera reacted only with 
homotypic VLPs. The specificities of these reactions were verified in 
preabsorption experiments, in which the immunoreactivity of each rabbit 
VLP antiserum was abrogated by preabsorption with homotypic, but not 
heterotypic, VLPs. None of the rabbit preimmune sera reacted with any of 
the VLP preparations. The antiPVL1 common antigen antiserum, which reacted 
well with recombinant L1 proteins by Western immunoblot (FIG. 15), reacted 
only slightly with native VLP preparations in the ELISA (FIG. 17A). This 
observation suggests that epitopes normally recognized by this antiserum 
are masked under the conditions of the ELISA assay, and that the L1 
proteins tested in this assay are predominantly non-denatured. 
The present invention has shown that L1 VLP epitopes of HPV-11, -16, and 
-18 are antigenically distinct. Although L2 capsid proteins were not 
present in these VLP preparations, it is likely that the observed 
antigenic difference between HPV types also applies to virions. L2 
represents approximately 10% of the total protein content of HPV particles 
(Doorbar et al., J. Virol. 61:2793-2799 (1987), which is hereby 
incorporated by reference) and, although its exact location in the 
particle has not been determined (Baker et al., Biophysical J. 
60:1445-1456 (1991), which is hereby incorporated by reference), recent 
studies have suggested that it may be required for DNA encapsidation (Zhou 
et al., J. Gen. Virol. 74:763-768 (1993), which is hereby incorporated by 
reference) and that a domain present in the relatively conserved amino 
terminal portion of the HPV-16 L2 amino acid sequence mediates 
non-specific DNA binding (Zhou et al., J. Virol 68:619-625 (1994), which 
is hereby incorporated by reference). Although the remainder of the L2 
amino acid sequence is very heterogeneous among papillomaviruses (Danos et 
al., J. Invest. Dermatology 83:7-11 (1990), which is hereby incorporated 
by reference), it is unclear if L2-specific antibodies react with intact 
virions (Komly et al., J. Virol. 60:813-816 (1986); and Hagensee et al., 
J. Virol. 67:315-322 (1993), which are hereby incorporated by reference). 
Thus, the L2 protein is not expected to alter substantially the results of 
the present study. 
Previous studies have indicated that different HPV types can be 
distinguished from one another using serologic techniques. For example, 
antibodies reactive with plantar wart virions were found much more 
commonly in sera from patients with plantar warts than in sera from 
patients with either common, flat, anogenital, or laryngeal warts (Pfister 
& zur Hausen, Int. J. Cancer 21:161-165 (1978); Kienzler et al., 1983, 
Brit. J. Dermatilogy 108:665-672 (1983); and Viac et al., J. Med. Virol. 
32:18-21 (1990), which are hereby incorporated by reference). Anisimova et 
al., 1990, also showed directly by immunoelectron microscopy that HPV-1 
and HPV-2 are antigenically distinct. However, it also appears that other 
HPV types are antigenically related. For example, the detection of 
antibodies which specifically recognize HPV-11 virions in sera from 
patients with documented HPV-6 infection was previously reported (Bonnez 
et al., J. Gen. Virol. 72:1343-1347 (1991); and Bonnez et al., Virol. 
188:384-387 (1992), which are hereby incorporated by reference). Due to 
the lack of available HPV virions from most HPV types, VLPs are at present 
the best tool available to explore antigenic relatedness among HPVs. 
Antigenic differences among HPV types are likely to reflect genetic 
diversity within the L1 coding sequence. Chan et al. constructed a 
papillomavirus phylogenetic tree that is based upon genetic divergence 
within a defined region of the papillomavirus L1 amino acid sequence (Chan 
et al, J. Virol., 66:5714-5725 (1992), which is hereby incorporated by 
reference). Their work shows the relatively close evolutionary 
relationship between HPV-6 and HPV-11, which is consistent with potential 
cross-reactivity between HPV-6 and -11 capsids. On the other hand, HPV-16 
and HPV-18, which have diverged extensively in their L1 sequences, are 
expected to have little antigenic cross-reactivity with each other or with 
HPV-11. Those predictions are consistent with the results of the present 
invention. 
The biologic relevance of HPV capsid antigenic variability is unknown, but 
diversity of the capsid protein could account for papillomavirus 
tissue-specificity. The availability of recombinant VLPs from a variety of 
papillomaviruses may prove useful in the identification of putative host- 
and tissue-specific cellular receptors. In addition, VLPs should play an 
important role in the delineation of the antigenic characteristics of 
HPVs, and in the conduct of studies of immune responses to these viruses. 
The present invention has been described in some detail by way of 
illustration and example for purposes of clarity of understanding, 
however, it will be obvious that certain changes and modifications may be 
practiced within the scope of the appended claims. 
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