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1.
Virology ; 524: 106-113, 2018 11.
Article in English | MEDLINE | ID: mdl-30170240

ABSTRACT

The major capsid protein of HPV, L1, assembles into pentamers that form a T = 7 icosahedral particle, but the location of the co-assembled minor capsid protein, L2, remains controversial. Several researchers have developed useful monoclonal antibodies targeting L2, but most react with linear epitopes toward the N-terminus. As a means to better define the virus capsid and better assess the localization and exposure of L2 epitopes in the context of assembled HPV, we have developed a panel of 30 monoclonal antibodies (mAbs) which target the N-terminus of L2 amino acids 11-200, previously defined as a broadly protective immunogen. Select mAbs were processed with enzymes and anti-L2 Fabs were generated. These new mAb/Fab probes will be beneficial in future studies to unravel the placement of L2 and to help better define the role of L2 in the HPV lifecycle and the nature of the broadly protective epitopes.


Subject(s)
Alphapapillomavirus/immunology , Antibodies, Viral/immunology , Capsid Proteins/immunology , Oncogene Proteins, Viral/immunology , Papillomavirus Infections/virology , Animals , Antibodies, Monoclonal/immunology , Cross Reactions , Epitopes/immunology , Humans , Mice , Mice, Inbred BALB C , Virion
3.
Clin Vaccine Immunol ; 13(8): 845-53, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16893983

ABSTRACT

The native cottontail rabbit papillomavirus (CRPV) L1 capsid protein gene was expressed transgenically via Agrobacterium tumefaciens transformation and transiently via a tobacco mosaic virus (TMV) vector in Nicotiana spp. L1 protein was detected in concentrated plant extracts at concentrations up to 1.0 mg/kg in transgenic plants and up to 0.4 mg/kg in TMV-infected plants. The protein did not detectably assemble into viruslike particles; however, immunoelectron microscopy showed presumptive pentamer aggregates, and extracted protein reacted with conformation-specific and neutralizing monoclonal antibodies. Rabbits were injected with concentrated protein extract with Freund's incomplete adjuvant. All sera reacted with baculovirus-produced CRPV L1; however, they did not detectably neutralize infectivity in an in vitro assay. Vaccinated rabbits were, however, protected against wart development on subsequent challenge with live virus. This is the first evidence that a plant-derived papillomavirus vaccine is protective in an animal model and is a proof of concept for human papillomavirus vaccines produced in plants.


Subject(s)
Agrobacterium tumefaciens/genetics , Antigens, Viral , Immunization , Vaccines/therapeutic use , Viral Structural Proteins , Animals , Antigens, Viral/genetics , Antigens, Viral/immunology , Antigens, Viral/therapeutic use , Base Sequence , Cloning, Molecular , Gene Transfer Techniques , Molecular Sequence Data , Plants, Genetically Modified , RNA/biosynthesis , Rabbits , Reverse Transcriptase Polymerase Chain Reaction , Nicotiana/genetics , Nicotiana/metabolism , Nicotiana/virology , Tobacco Mosaic Virus/genetics , Vaccines/genetics , Vaccines/immunology , Viral Structural Proteins/genetics , Viral Structural Proteins/immunology , Viral Structural Proteins/therapeutic use
4.
Vaccine ; 24(12): 2087-93, 2006 Mar 15.
Article in English | MEDLINE | ID: mdl-16343704

ABSTRACT

Recombinant Bacille Calmette-Guerin (rBCG) could potentially be the vaccine vehicle of choice to deliver foreign antigens from multiple pathogens. In this study we have used the cottontail rabbit papillomavirus (CRPV) rabbit model to provide a "proof of concept" that immunisation with rBCG expressing the CRPV major capsid protein, L1 (rBCG/CRPVL1), will protect outbred New Zealand White rabbits against CRPV challenge. Rabbits immunised with rBCG/CRPVL1 (10(7) cfu/ml) were protected 5 weeks post-CRPV challenge. Rabbits immunised with rBCG/CRPVL1 (10(5) cfu/ml) had papillomas, which were smaller and took longer to appear than the control rabbits. None of the negative control rabbits vaccinated with rBCG expressing an irrelevant gene or PBS were protected from CRPV challenge. Sera from rabbits immunised with rBCG/CRPVL1 (10(7) cfu/ml) were able to neutralise 54.5% of CRPV at serum dilutions of 1:200. These results provide evidence that BCG could potentially be used as a vaccine delivery vehicle for human papillomavirus proteins as a possible prophylactic vaccine.


Subject(s)
Antigens, Viral/immunology , BCG Vaccine/administration & dosage , Cottontail rabbit papillomavirus/immunology , Papillomavirus Infections/prevention & control , Viral Structural Proteins/immunology , Viral Vaccines/administration & dosage , Animals , Antigens, Viral/genetics , Antigens, Viral/metabolism , BCG Vaccine/immunology , Cottontail rabbit papillomavirus/genetics , Cottontail rabbit papillomavirus/metabolism , Drug Delivery Systems , Immunization , Neutralization Tests , Rabbits , Recombinant Proteins/immunology , Viral Structural Proteins/genetics , Viral Structural Proteins/metabolism , Viral Vaccines/immunology
5.
Gene Ther ; 11(17): 1331-41, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15229628

ABSTRACT

Human papillomaviruses (HPVs) are recognized as important human pathogens, causing a spectrum of hyperproliferative lesions from benign warts to cervical dysplasias/carcinomas. HPV-associated lesions require continued production of the oncogenic E6/E7 proteins, which are encoded by either bicistronic or overlapping mRNAs. Here we targeted the E6/E7 mRNA of HPV11, a type implicated in causation of genital warts, using molecular reagents. Accessible sites in the HPV11(E6/E7) RNA were identified using library selection protocols, and nucleic acids (DNAzymes, antisense oligonucleotides) targeted to these sites were constructed, and tested in cell culture and on human foreskin grafts. While DNAzymes were at least equally effective in cell culture, antisense oligonucleotides targeted to the region surrounding one of the library-selected sites (ASO(407)) proved most effective in blocking progression of HPV11-induced papillomas in human foreskin grafts on immunodeficient mice. In total, 11 papillomas were treated with ASO(407). Of these, four of seven small papillomas treated with ASO(407) showed loss of detectable virus by in situ hybridization (ISH), and in all four of these, papillomas were no longer evident grossly or histologically after treatment. When larger papillomas were treated, one of four showed loss of virus by ISH, associated with a minor decrease in papilloma size. Considering all 11 papillomas treated with ASO(407), loss of viral staining by ISH was significantly different from that observed in controls (P<0.016), as was true for the seven small treated papillomas (P<0.012). DNAzymes targeted to the same site (or other library selected sites) did not produce statistically significant differences in ISH staining (P<0.15). Our results with ASO(407) appear to represent the first specific molecular therapy against a bona fide HPV infection, and provide a rational proof-of-principle strategy for development of molecular therapeutics targeting other HPV-associated lesions.


Subject(s)
Condylomata Acuminata/therapy , Genetic Therapy/methods , Oligonucleotides, Antisense/administration & dosage , Oncogene Proteins, Viral/genetics , Papillomaviridae/genetics , Papillomavirus Infections/therapy , Animals , Cells, Cultured , DNA, Catalytic/administration & dosage , Humans , In Situ Hybridization , Male , Mice , Mice, SCID , RNA, Messenger/genetics , Skin Transplantation , Transplantation, Heterologous
6.
Antimicrob Agents Chemother ; 45(12): 3427-32, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11709319

ABSTRACT

The high-molecular-weight sulfated or sulfonated polysaccharides or polymers cellulose sulfate, dextran sulfate, and polystyrene sulfonate were tested for microbicidal activity against bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 11 (HPV-11) and type 40 (HPV-40). In vitro assays included the BPV-1-induced focus-forming assay and transient infection of human A431 cells with HPVs. The compounds were tested for microbicidal activity directly by preincubation with virus prior to addition to cell cultures and indirectly by addition of virus to compound-treated cells and to virus-coated cells to test inactivation of the virus after virus-cell binding. The data indicated that all three compounds showed direct microbicidal activity with 50% effective concentrations between 10 to 100 microg/ml. These concentrations were nontoxic to cell cultures for both assays. When a clone of C127 cells was tested for microbicidal activity, approximately 10-fold-less compound was required to achieve a 50% reduction in BPV-1-induced foci than for the uncloned parental C127 cells. Pretreatment of cells with compound prior to addition of virus also demonstrated strong microbicidal activity with dextran sulfate and polystyrene sulfonate, but cellulose sulfate required several orders of magnitude more compound for virus inactivation. Polystyrene sulfonate prevented subsequent infection of HPV-11 after virus-cell binding, and this inactivation was observed up to 4 h after addition of virus. These data indicate that the polysulfated and polysulfonated compounds may be useful nontoxic microbicidal compounds that are active against a variety of sexually transmitted disease agents including papillomaviruses.


Subject(s)
Anti-Infective Agents/pharmacology , Antiviral Agents/pharmacology , Cellulose/analogs & derivatives , Cellulose/pharmacology , Dextran Sulfate/pharmacology , Papillomaviridae/drug effects , Polystyrenes/pharmacology , Animals , Bovine papillomavirus 1/drug effects , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Humans , Mice , Papillomavirus Infections/drug therapy , Papillomavirus Infections/virology , Reverse Transcriptase Polymerase Chain Reaction
7.
Virology ; 289(2): 262-8, 2001 Oct 25.
Article in English | MEDLINE | ID: mdl-11689049

ABSTRACT

We have mapped the binding sites on human papillomavirus (HPV) type 6 for three HPV 6-specific neutralizing monoclonal antibodies (mAbs). The critical binding residues were first identified by making HPV 11-like amino acid substitutions in the HPV 6 major capsid protein L1 and assaying the resulting virus-like particles (VLPs) for reactivity with the mAbs. To confirm the relevance of these residues for mAb binding, we demonstrated that HPV 6 type-specificity could be transferred to HPV 11 VLPs by making the appropriate HPV 6-like amino acid substitutions in the HPV 11 L1. Two binding regions were found. For one mAb, all critical residues are centered at residue 53, while for the other two mAbs, type-specific binding also requires a second site located more than 100 residues distal to the first. Both binding sites coincide with regions of L1 where the sequences of the closely related HPV 6 and 11 diverge. These regions are where the L1 sequences are the least well conserved among all HPV types and they have been implicated in type-specific binding for other HPV types. This suggests that clusters of diverged residues, surrounded by conserved L1 sequences, are presented on the surface of assembled particles and are responsible for eliciting critical humoral immune responses to the virus.


Subject(s)
Capsid Proteins , Capsid/immunology , Genome, Viral , Oncogene Proteins, Viral/immunology , Papillomaviridae/immunology , Amino Acid Sequence , Antibodies, Monoclonal , Antibodies, Viral , Binding Sites , Capsid/genetics , Consensus Sequence , Epitope Mapping , Molecular Sequence Data , Mutation , Neutralization Tests , Oncogene Proteins, Viral/genetics , Papillomaviridae/genetics , Viral Proteins
8.
Antimicrob Agents Chemother ; 45(4): 1201-9, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11257035

ABSTRACT

We used the cottontail rabbit papillomavirus (CRPV) New Zealand White rabbit model to test a combination treatment of large established papillomas with intralesional cidofovir and DNA vaccination to cure sites and reduce recurrences. Intralesional 1% (wt/vol) (0.036 M) cidofovir treatment of rabbit papillomas led to elimination, or "cure," of the papillomas over a 6- to 8-week treatment period (N. D. Christenson, M. D. Pickel, L. R. Budgeon, and J. W. Kreider, Antivir. Res. 48:131-142, 2000). However, recurrences at periods from 1 to 8 weeks after treatment cessation were observed at approximately 50% of cured sites. DNA vaccinations with CRPV E1, E2, E6, and E7 were initiated either after or at the time of intralesional treatments, and the recurrence rates were observed. When DNA vaccinations were started after intralesional cures, recurrence rates were similar to those of vector-vaccinated rabbits. A small proportion of recurrent sites subsequently regressed (4 out of 10, or 40%) in the vaccinated group versus no regression of recurrences in the vector-immunized group (0 out of 19, or 0%), indicating partial effectiveness. In contrast, when DNA vaccinations were conducted during intralesional treatments, a significant reduction of recurrences (from 10 out of 19, or 53%, of sites in vector-immunized rabbits to 3 out of 20, or 15%, of sites in viral-DNA-immunized rabbits) was observed. DNA vaccination without intralesional treatments had a minimal effect on preexisting papillomas. These data indicated that treatment with a combination of antiviral compounds and specific immune stimulation may lead to long-term cures of lesions without the ensuing problem of papilloma recurrence.


Subject(s)
Antineoplastic Agents/administration & dosage , Antiviral Agents/administration & dosage , Cottontail rabbit papillomavirus , Cytosine/administration & dosage , Organophosphonates , Organophosphorus Compounds/administration & dosage , Viral Vaccines/therapeutic use , Warts/drug therapy , Animals , Cidofovir , Combined Modality Therapy , Cytosine/analogs & derivatives , Female , Genes, Viral , Injections, Intralesional , Male , Neoplasm Recurrence, Local/prevention & control , Oncogene Proteins, Viral/genetics , Rabbits , Time Factors , Vaccines, DNA/therapeutic use
9.
Vaccine ; 19(13-14): 1783-93, 2001 Feb 08.
Article in English | MEDLINE | ID: mdl-11166904

ABSTRACT

Recombinant human papillomavirus (HPV) virus-like particles (VLPs) are promising vaccine candidates for controlling anogenital HPV disease. Questions remain, however, concerning the extent of capsid antigenic similarity between closely related virus genotypes. To investigate this issue, we produced VLPs and corresponding polyclonal immune sera from several anogenital HPV types, and examined these reagents in enzyme-linked immunosorbent assays (ELISAs) and in cross-neutralization studies. Despite varying degrees of L1 genetic sequence relatedness, VLPs of each type examined induced high-titer serum polyclonal antibody responses that were entirely genotype-specific. In an in vitro infectivity assay, only cognate VLP antisera were able to neutralize pseudovirions of HPV-16, HPV-18 and HPV-33, with two exceptions: HPV-31 and HPV-45 VLP post-immune sera demonstrated low levels of neutralizing activity against pseudovirions of HPV-33 and HPV-18, respectively. In other experiments, epitopes shared between closely related types were found to be less immunogenic than, and antigenically distinct from, primary type-specific B-cell determinants of the viral capsid. In addition, results from epitope blocking experiments suggested a close correlation between primary type-specific capsid antigenic sites and virion neutralization. These findings support the view that papillomavirus genotypes denote unique viral serotypes, and suggest that a successful vaccine for these viruses will likely require the inclusion of VLPs of each serotype for which protection is desired.


Subject(s)
Antibody Specificity/immunology , Capsid/genetics , Capsid/immunology , Papillomaviridae/immunology , Absorption , Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Antigens, Viral/genetics , Antigens, Viral/immunology , Capsid/chemistry , Cross Reactions/immunology , Enzyme-Linked Immunosorbent Assay , Epitopes/immunology , Genotype , Humans , Immune Sera/immunology , Neutralization Tests , Papillomaviridae/classification , Papillomaviridae/genetics , Papillomaviridae/physiology , Protein Conformation , Protein Denaturation , Serotyping , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , Vaccinia virus/genetics , Viral Vaccines/immunology
10.
Virology ; 291(2): 324-34, 2001 Dec 20.
Article in English | MEDLINE | ID: mdl-11878901

ABSTRACT

Human papillomavirus (HPV) hybrid virus-like particles (VLPs) were prepared using complementary regions of the major capsid L1 proteins of HPV-11 and -16. These hybrid L1 proteins were tested for assembly into VLPs, for presentation and mapping of conformational neutralizing epitopes, and as immunogens in rabbits and mice. Two small noncontiguous hypervariable regions of HPV-16 L1, when replaced into the HPV-11 L1 backbone, produced an assembly-positive hybrid L1 which was recognized by the type-specific, conformationally dependent HPV-16 neutralizing monoclonal antibody (N-MAb) H16.V5. Several new N-MAbs that were generated following immunization of mice with wild-type HPV-16 L1 VLPs also recognized this reconstructed VLP, demonstrating that these two hypervariable regions collectively constituted an immunodominant epitope. When a set of hybrid VLPs was tested as immunogens in rabbits, antibodies to both HPV-11 and -16 wild-type L1 VLPs were obtained. One of the hybrid VLPs containing hypervariable FG and HI loops of HPV-16 L1 replaced into an HPV-11 L1 background provoked neutralizing activity against both HPV-11 and HPV-16. In addition, conformationally dependent and type-specific MAbs to both HPV-11 and HPV-16 L1 VLP were obtained from mice immunized with hybrid L1 VLPs. These data indicated that hybrid L1 proteins can be constructed that retain VLP-assembly properties, retain type-specific conformational neutralizing epitopes, can map noncontiguous regions of L1 which constitute type-specific conformational neutralizing epitopes recognized by N-MAbs, and trigger polyclonal antibodies which can neutralize antigenically unrelated HPV types.


Subject(s)
Capsid Proteins , Epitopes, B-Lymphocyte/immunology , Oncogene Proteins, Viral/immunology , Papillomaviridae/immunology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Viral/biosynthesis , Enzyme-Linked Immunosorbent Assay , Epitope Mapping , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/genetics , Humans , Mice , Neutralization Tests , Oncogene Proteins, Viral/chemistry , Oncogene Proteins, Viral/genetics , Papillomaviridae/genetics , Protein Conformation , Rabbits , Virion
11.
Antiviral Res ; 48(2): 131-42, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11114415

ABSTRACT

A series of nucleoside analogues were tested for in vivo anti-papillomavirus activity using the cottontail rabbit papillomavirus (CRPV) domestic rabbit model. Compounds were delivered either topically, injected into growing papillomas, or delivered subcutaneously at a site remote from the papillomas. Compounds tested included cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine] (HPMPC); cyclic HPMPC (cHPMPC); cyclopentenylcytosine (CPE-C); lobucavir [1R(1alpha,2beta,3alpha)]-9-[2, 3-bis(hydroxymethyl)cyclobutyl]guanine; 9-((2-phosphonylmethoxy)propyl)adenine (PMPA); adefovir 9-((2-phosphonylmethoxy)ethyl)adenine(PMEA) and cyclopropyl 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (cyclopropylPMEDAP). Dose response curves and time-course treatments were included for most compounds tested. Strong anti-viral activity was detected using cidofovir and cHPMPC when delivered either topically or by the intralesional route. Complete cures were obtained using 1% (w/v) topical cidofovir at dosing schedules of twice daily for 8 weeks beginning at 4 weeks after CRPV infection, which represents a time when papillomas were clearly visible. Complete cures of large established papillomas were obtained by intralesional injection of 1% cidofovir three times per week for 8 weeks. Topical treatments with adefovir had strong anti-viral activity, cyclopropyl PMEDAP had moderate anti-viral activity, and CPE-C, PMPA and lobucavir showed no effects. These data indicate that certain nucleoside analogues have strong in vivo anti-papillomavirus activity and that the CRPV/rabbit model is a good model for assessing clinical responses of anti-viral treatments for patients with HPV disease.


Subject(s)
Antiviral Agents/therapeutic use , Cottontail rabbit papillomavirus , Cytosine/therapeutic use , Organophosphonates , Organophosphorus Compounds/therapeutic use , Papilloma/drug therapy , Papillomavirus Infections/drug therapy , Animals , Cidofovir , Cottontail rabbit papillomavirus/drug effects , Cottontail rabbit papillomavirus/pathogenicity , Cytosine/analogs & derivatives , Disease Models, Animal , Humans , Nucleosides/chemistry , Nucleosides/therapeutic use , Papilloma/virology , Papillomavirus Infections/virology , Rabbits , Treatment Outcome , Tumor Virus Infections/diagnosis , Tumor Virus Infections/drug therapy
12.
Virology ; 278(2): 570-7, 2000 Dec 20.
Article in English | MEDLINE | ID: mdl-11118379

ABSTRACT

The development of vaccines against human papillomaviruses (HPVs) has long been hampered by the inability to grow HPVs in tissue culture and the lack of an efficient neutralization assay. To date, less than 10% of more than 100 different HPV types can be grown in athymic and "SCID" mouse xenograft systems or raft culture systems. Recently, the in vitro generation of HPV pseudovirions and their use in neutralization assays were demonstrated. The major shortcomings of the current approaches to HPV neutralization are the lack of HPV virions for most types for the xenograft methods and the time-consuming and inefficient generation of infective pseudovirions for the latter methods, which precludes their use in large-scale HPV clinical trials or epidemiological studies. We describe here a novel and efficient approach to generating pseudovirions in which HPV virus-like particles (VLPs) are coupled to the beta-lactamase gene as a reporter. We show that it is not necessary to encapsidate the reporter gene constructs into the pseudovirions. Using sera from human volunteers immunized with HPV-11 VLPs expressed in yeast, we demonstrate that our novel neutralization assay compares favorably with the athymic mouse neutralization assay. Furthermore, our assay was used to define neutralizing monoclonal antibodies to HPV-6, which were previously unknown.


Subject(s)
Antibodies, Viral/blood , Papillomaviridae/immunology , Virion/immunology , Animals , Cells, Cultured , Female , Genes, Reporter , Genetic Vectors , Humans , Male , Mice , Mice, Nude , Mice, SCID , Neutralization Tests , Papillomaviridae/genetics , Papillomaviridae/growth & development , Radioimmunoassay/methods , Skin Transplantation/immunology , Transplantation, Heterologous , Tumor Cells, Cultured , Uterine Cervical Neoplasms , beta-Lactamases/genetics
13.
J Virol ; 74(20): 9712-6, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11000243

ABSTRACT

Malignant progression is a life-threatening consequence of human papillomavirus-associated lesions. In this study, we tested the efficacy of papillomavirus early-gene-based vaccines for prevention of carcinoma development of papillomavirus-induced skin papillomas on rabbits. Rabbit skin papillomas were initiated by infection with cottontail rabbit papillomavirus (CRPV). The papillomas were allowed to grow for 3 months without any treatment intervention. Rabbits were then immunized by gene gun-mediated intracutaneous administration of four DNA plasmids encoding CRPV E1, E2, E6, and E7 genes, respectively. All eight control rabbits receiving vector alone developed invasive carcinoma within 8 to 13 months. In contrast, only two of eight vaccinated rabbits developed carcinoma at 12 and 15 months, respectively. Papilloma growth was suppressed in the majority of vaccinated rabbits but not completely eradicated. These results indicate that gene gun-mediated immunization with papillomavirus early genes may be a promising strategy for prevention of malignant progression of human papillomavirus-associated lesions in humans.


Subject(s)
Cancer Vaccines/immunology , Cottontail rabbit papillomavirus/immunology , Papilloma/prevention & control , Papillomavirus Infections/prevention & control , Skin Neoplasms/prevention & control , Tumor Virus Infections/prevention & control , Vaccines, DNA/immunology , Animals , Biolistics , Cottontail rabbit papillomavirus/genetics , Papilloma/pathology , Rabbits , Skin Neoplasms/pathology , T-Lymphocytes, Cytotoxic/immunology , Vaccination
14.
Vaccine ; 18(26): 2937-44, 2000 Jul 01.
Article in English | MEDLINE | ID: mdl-10825594

ABSTRACT

We previously demonstrated that gene gun-based intracutaneous vaccination of rabbits with a combination of, but not with individual papillomavirus E1, E2, E6 and E7 genes provided complete protection against cottontail rabbit papillomavirus (CRPV) infection. In the present study, we tested whether vaccination of inbred and outbred rabbits with a combination of CRPV E1 and E2 genes could provide complete protection against virus infection. In the first experiment, gene gun-based intracutaneous vaccination with E1 and E2 genes prevented papilloma formation in the majority of inbred rabbits and promoted systemic papilloma regression in one non-protected rabbit. In contrast, needle-mediated intramuscular injection of E1 and E2 genes did not prevent papilloma formation nor promoted systemic papilloma regression, indicating an absence of strong protective immunity. In the second experiment, six outbred rabbits were immunized by gene gun-based intracutaneous administration of the E1 and E2 genes. Prevention of papilloma formation or systemic papilloma regression was observed in three vaccinated rabbits. Papillomas persisted on the remaining three rabbits, but were significantly smaller than that on control rabbits. These results suggested that gene gun-based intracutaneous vaccination with the combination of papillomavirus E1 and E2 genes induced strong protective antivirus immunity but may be insufficient for complete protection in an outbred population.


Subject(s)
Biolistics , Cottontail rabbit papillomavirus/immunology , Oncogene Proteins, Viral/immunology , Vaccines, DNA/immunology , Viral Vaccines/immunology , Administration, Cutaneous , Animals , Immunization , Injections, Intramuscular , Major Histocompatibility Complex , Oncogene Proteins, Viral/genetics , Papilloma/prevention & control , Rabbits , T-Lymphocytes, Cytotoxic/immunology
15.
Virology ; 266(2): 237-45, 2000 Jan 20.
Article in English | MEDLINE | ID: mdl-10639310

ABSTRACT

Characterization of the regions of human papillomaviruses (HPVs) that elicit neutralizing immune responses supports studies on viral infectivity and provides insight for the development and evaluation of prophylactic vaccines. HPV11 is a major etiologic agent of genital warts and a likely vaccine candidate. A conformationally dependent epitope for the binding of three neutralizing monoclonal antibodies (mAbs) has been mapped to residues G(131)T(132) of the L1 major capsid protein. The mAbs bind L1 only when it is assembled into virions or into virus-like particles (VLPs) that mimic the capsid structure. We were interested in identifying other domains of L1 that elicit neutralizing responses. To this end, we have generated a panel of mAbs against VLPs derived from HPV11 L1 harboring a G131S substitution. The new mAbs are unlike the neutralizing mAbs previously mapped to residues G(131)T(132) in that they bind both prototype and HPV11:G131S mutant VLPs. Some of the new mAbs neutralized virus in vitro. We have mapped epitopes for three of these new mAbs, as well as a neutralizing mAb generated against HPV11 virions, by measuring binding to HPV6 VLPs substituted with HPV11-like amino acids. Two regions are critical: one defined by HPV11 L1 residues 263-290 and the other by residues 346-349. mAbs H11.H3 and H11.G131S.G3 bind HPV6 VLPs with substitutions derived from the 346-349 region; in addition, H11.G131S.G3 binds HPV6 VLPs with substitutions derived only from the 263-290 region. Although H11.H3 does not bind HPV6 VLPs with substitutions derived from the 263-290 region, binding to HPV6 VLPs is enhanced when both sets of substitutions are present. mAbs H11.G131S.I1 and H11.G131S.K5 bind HPV6 VLPs with the 263-290 substitutions, but show little binding to HPV6 VLPs with the 346-349 substitutions. However, binding to HPV6 VLPs is enhanced when substitutions at both regions are present. The 346-349 region has not previously been described as eliciting a neutralizing response for any HPV type. In addition, the work demonstrates a complex binding site contributed by two distinct regions of L1.


Subject(s)
Papillomaviridae/genetics , Papillomaviridae/immunology , Animals , Antibodies, Monoclonal , Antibodies, Viral , Antigens, Viral/genetics , Base Sequence , Binding Sites/genetics , Cell Line , DNA Primers/genetics , Epitope Mapping , Humans , Immunization , Mice , Mutation , Neutralization Tests , Papillomaviridae/classification , Spodoptera , Viral Vaccines/immunology
16.
Clin Exp Metastasis ; 18(8): 683-93, 2000.
Article in English | MEDLINE | ID: mdl-11827072

ABSTRACT

Introduction of normal, neomycin-tagged human chromosome 11 (neo11) reduces the metastatic capacity of MDA-MB-435 human breast carcinoma cells by 70-90% without affecting tumorigenicity. Differential display comparing MDA-MB-435 and neo11/435 led to the discovery of a human breast carcinoma metastasis suppressor gene, BRMS1, which maps to chromosome 11q13.1-q13.2. Stable transfectants of MDA-MB-435 and MDA-MB-231 breast carcinoma cells with BRMS1 cDNA still form progressively growing, locally invasive tumors when injected in mammary fat pads of athymic mice but exhibit significantly lower metastatic potential (50-90% inhibition) to lungs and regional lymph nodes. To begin elucidating the mechanism(s) of action, we measured the ability of BRMS1 to perturb individual steps of the metastatic cascade modeled in vitro. Consistent differences were not observed for adhesion to extracellular matrix components (laminin, fibronectin, type IV collagen, type I collagen, Matrigel); growth rates in vitro or in vivo; expression of matrix metalloproteinases, heparanase, or invasion. Likewise. BRMS1 expression did not up regulate expression of other metastasis suppressors, such as NM23, Kai1, KiSS1 or E-cadherin. Motility of BRMS1 transfectants was modestly inhibited (30-60%) compared to parental and vector-only transfectants. Ability to grow in soft agar was also decreased in MDA-MB-435 cells by 80-89%, but the decrease for MDA-MB-231 was less (13-15% reduction). Also, transfection and re-expression of BRMS1 restored the ability of human breast carcinoma cells to form functional homotypic gap junctions. Collectively, these data suggest that BRMS1 suppresses metastasis of human breast carcinoma by complex, atypical mechanisms.


Subject(s)
Lung Neoplasms/prevention & control , Mammary Neoplasms, Experimental/prevention & control , Neoplasm Proteins , Proteins/physiology , Animals , Blotting, Northern , Blotting, Southern , DNA Primers/chemistry , Female , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/secondary , Mammary Neoplasms, Experimental/metabolism , Mammary Neoplasms, Experimental/pathology , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Mice , Mice, Nude , Phosphorylation , RNA, Messenger/metabolism , Repressor Proteins , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Tumor Cells, Cultured/metabolism
17.
Virology ; 269(2): 451-61, 2000 Apr 10.
Article in English | MEDLINE | ID: mdl-10753723

ABSTRACT

Rabbit oral papillomavirus (ROPV) infects mucosal tissues of domestic rabbits. The viral genomic sequence has been determined and the most related papillomavirus type was the cutaneous cottontail rabbit papillomavirus (CRPV). Homologies between the open reading frames (ORFs) of ROPV and CRPV, however, ranged from 68% amino acid identity for L1 to only 23% identity for E4. Shared features unique to the two rabbit viruses included a large E6 ORF and a small E8 ORF that overlapped the E6 ORF. Serological responses to ROPV L1 viruslike particles (VLPs) were detected in rabbits infected at either the genital or oral mucosa with ROPV. The antibody response was specific to intact ROPV L1 VLP antigen, was first detected at the time of late regression, and persisted at high levels for several months after complete regression. Both oral and genital lesions regressed spontaneously, accompanied by a heavy infiltrate of lymphocytes. ROPV infection of rabbit genital mucosa is a useful model to study host immunological responses to genital papillomavirus infections.


Subject(s)
Cottontail rabbit papillomavirus/genetics , Genome, Viral , Papillomavirus Infections/genetics , Tumor Virus Infections/genetics , Animals , Balanitis/immunology , Balanitis/virology , Base Sequence , Enzyme-Linked Immunosorbent Assay , Female , Male , Molecular Sequence Data , Open Reading Frames , Papillomavirus Infections/immunology , Rabbits , Stomatitis/immunology , Stomatitis/virology , Tumor Virus Infections/immunology , Vaginitis/immunology , Vaginitis/virology
18.
Toxicology ; 136(2-3): 67-77, 1999 Aug 31.
Article in English | MEDLINE | ID: mdl-10514000

ABSTRACT

Toxicity studies with drugs in animals are performed to establish the toxicity profile including the no-toxic-effect-level in the animals. One of the responses used in the search for effects is organ weight. Since organ weight is related to body weight of the animal, the analysis of organ weight has to be adjusted. A traditional analysis of covariance with body weight as covariate is not always appropriate since the body weight itself can be affected by the test compound. Hence, a multivariate analysis of variance is suggested, where the correlation between organ weight and body weight is taken into account in testing an overall treatment effect. Two dimensional plots of the contour curves are used to visualize the treatment effect on organ weight and body weight simultaneously. The test procedure suggested for comparing differences between control and treated groups of animals is: start by testing hypotheses of equality of covariance matrices for the different treatment groups and then, depending on the results from the first test, test equality of the mean vectors.


Subject(s)
Multivariate Analysis , Organ Size/drug effects , Toxicology/statistics & numerical data , Algorithms , Animals , Body Weight/drug effects , Female , Rats , Receptors, Estrogen/agonists , Research Design
19.
J Virol ; 73(8): 7039-43, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10400806

ABSTRACT

In this study, cottontail rabbit papillomavirus infection of domestic rabbits was used as an animal model to develop papillomavirus early gene-based vaccines. Groups of rabbits were intracutaneously vaccinated with single papillomavirus early genes E1, E2, E6, and E7 or with a combination of these four genes. Only a fraction of rabbits were protected from subsequent viral challenge when vaccinated with the E1 or E6 gene. Viral tumor growth in those rabbits vaccinated with the E1 or E2 gene was suppressed compared to that in controls. In contrast, seven of nine rabbits vaccinated with the combination of the E1, E2, E6, and E7 genes were completely protected against viral challenge. These data indicated that intracutaneous genetic vaccination with the combination of the E1, E2, E6, and E7 genes can be an effective strategy for immunoprophylaxis of papillomavirus infection.


Subject(s)
Cottontail rabbit papillomavirus/immunology , Genes, Viral , Papillomavirus Infections/prevention & control , Tumor Virus Infections/prevention & control , Vaccines, DNA/immunology , Viral Vaccines/immunology , Animals , Antibodies, Viral/blood , Antibodies, Viral/immunology , Cottontail rabbit papillomavirus/genetics , Cottontail rabbit papillomavirus/growth & development , Injections, Subcutaneous , Oncogene Proteins, Viral/genetics , Rabbits , T-Lymphocytes/immunology , Vaccination , Vaccines, DNA/administration & dosage , Vaccines, DNA/genetics , Viral Vaccines/administration & dosage , Viral Vaccines/genetics , Virion/immunology
20.
Vaccine ; 17(11-12): 1558-66, 1999 Mar 17.
Article in English | MEDLINE | ID: mdl-10195793

ABSTRACT

To test the efficacy of genetic vaccination against papillomavirus infection, plasmid DNA encoding cottontail rabbit papillomavirus (CRPV) E1, E2, E6, E7 or without insert were intramuscularly injected into five groups of rabbits. Peripheral blood mononuclear cells (PBMCs) showed specific proliferation upon in vitro stimulation with E1, E2, E6 or E7 proteins in a majority of vaccinated rabbits but Western blot analysis did not detect antibodies specific for these viral proteins in rabbit serum. All rabbits grew papillomas after virus challenge and none of the rabbits showed systemic papilloma regression. These observations showed that intramuscular injection of plasmid DNA encoding CRPV E1, E2, E6 or E7 induced CD4+ T cell-mediated but not humoral immune responses, and did not result in the protection of rabbits from virus infection.


Subject(s)
Antibodies, Viral/biosynthesis , Cottontail rabbit papillomavirus/genetics , DNA, Viral/administration & dosage , DNA, Viral/immunology , T-Lymphocytes/immunology , Vaccines, DNA , Viral Vaccines , Animals , Blotting, Western , Cottontail rabbit papillomavirus/immunology , Injections, Intramuscular , Leukocytes, Mononuclear/immunology , Papillomavirus Infections/prevention & control , Plasmids/genetics , Rabbits , T-Lymphocytes/virology , Tumor Virus Infections/prevention & control , Vaccines, DNA/administration & dosage , Viral Proteins/genetics , Viral Proteins/immunology , Viral Vaccines/administration & dosage
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