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Lynn R Budgeon

Publications and source records attributed to Lynn R Budgeon.

18 recordsLinked to original sources
PubMed2006

An HLA-A2.1-transgenic rabbit model to study immunity to papillomavirus infection.

We have established several HLA-A2.1-transgenic rabbit lines to provide a host to study CD8(+) T cell responses during virus infections. HLA-A2.1 protein expression was detected on cell surfaces within various organ tissues. Continuous cultured cells from these transgenic rabbits were capable of presenting both endogenous and exogenous HLA-A2.1-restricted epitopes to an HLA-A2.1-restricted epitope-specific CTL clone. A DNA vaccine containing an HLA-A2.1-restricted human papillomavirus type 16 E7 epitope (amino acid residues 82-90) stimulated epitope-specific CTLs in both PBLs and spleen cells of transgenic rabbits. In addition, vaccinated transgenic rabbits were protected against infection with a mutant cottontail rabbit papillomavirus DNA containing an embedded human papillomavirus type 16 E7/82-90 epitope. Complete protection was achieved using a multivalent epitope DNA vaccine based on epitope selection from cottontail rabbit papillomavirus E1 using MHC class I epitope prediction software. HLA-A2.1-transgenic rabbits will be an important preclinical animal model system to study virus-host interactions and to assess specific targets for immunotherapy.

Amino Acid Sequence↗
PubMed2006

Preclinical model to test human papillomavirus virus (HPV) capsid vaccines in vivo using infectious HPV/cottontail rabbit papillomavirus chimeric papillomavirus particles.

A human papillomavirus (HPV) vaccine consisting of virus-like particles (VLPs) was recently approved for human use. It is generally assumed that VLP vaccines protect by inducing type-specific neutralizing antibodies. Preclinical animal models cannot be used to test for protection against HPV infections due to species restriction. We developed a model using chimeric HPV capsid/cottontail rabbit papillomavirus (CRPV) genome particles to permit the direct testing of HPV VLP vaccines in rabbits. Animals vaccinated with CRPV, HPV type 16 (HPV-16), or HPV-11 VLPs were challenged with both homologous (CRPV capsid) and chimeric (HPV-16 capsid) particles. Strong type-specific protection was observed, demonstrating the potential application of this approach.

Animals↗
PubMed2006

Papillomavirus particles assembled in 293TT cells are infectious in vivo.

Papillomaviruses (PVs) demonstrate both tissue and species tropisms. Because PVs replicate only in terminally differentiating epithelium, the recent production of infectious PV particles in 293 cells marks an important breakthrough. In this article, we demonstrate that infectious PV particles produced in 293TT cells can cause papillomatous growths in the natural host animal. Moreover, we show that species-matched PV genomes can be successfully delivered in vivo by a heterologous, species-mismatched PV capsid. Additionally, our results indicate that the addition of the simian virus 40 origin of replication to the papillomavirus genome increases the production of infectious papillomavirus particles by increasing genome amplification in the transfected 293TT cells.

Animals↗
PubMed2006

High-throughput zebrafish histology.

The morphological effects of mutation and disease are often critical to our understanding of normal and abnormal function. The power and popularity of zebrafish as a forward and reverse genetic vertebrate model system, combined with its small size, have made it an ideal model in which to study the genetics of histologically scorable phenotypes. The presence of multiple tissue types in this organism's small larvae also makes it a potentially important model for toxicological analysis. Studying histological phenotypes is greatly enhanced by high-throughput methods of histology. Here, we describe details of high-throughput histology of the zebrafish using larval arrays, along with recent advances in mold design and discussion of work in progress that will lead to easier ways for people in the field to more rapidly score phenotypes in arrays. These detailed descriptions, together with the troubleshooting guide, should enable any laboratory with ties to a histology facility to perform high-throughput histology of zebrafish.

Animals↗
PubMed2006

Protective cell-mediated immunity by DNA vaccination against Papillomavirus L1 capsid protein in the Cottontail Rabbit Papillomavirus model.

Papillomavirus major capsid protein L1 has successfully stimulated protective immunity against virus infection by induction of neutralizing antibodies in animal models and in clinical trials. However, the potential impact of L1-induced protective cell-mediated immune (CMI) responses is difficult to measure in vivo because of the coincidence of anti-L1 antibody. In this study, we tested the hypothesis that L1 could activate CMI, using the Cottontail Rabbit Papillomavirus (CRPV)-rabbit model. A unique property of this model is that infections can be initiated with viral DNA, thus bypassing all contributions to protection via neutralizing anti-L1 antibody. DNA vaccines containing either CRPV L1, or subfragments of L1 (amino-terminal two-thirds of L1 [L1N] and the carboxylterminal two-thirds of L1 [L1C]), were delivered intracutaneously into rabbits, using a gene gun. After three booster immunizations, the rabbits were challenged with several viral DNA constructs: wild-type CRPV, CRPV L1ATGko (an L1 ATG knockout mutation), and CRPV-ROPV hybrid (CRPV with a replacement L1 from Rabbit Oral Papillomavirus). Challenge of L1 DNA-vaccinated rabbits with wild-type CRPV resulted in significantly fewer papillomas when compared with challenge with CRPV L1ATGko DNA. Significantly smaller papillomas were found in CRPV L1-, L1N-, and L1C-vaccinated rabbits. In addition, rabbits vaccinated with either L1 or L1N grew significantly fewer and smaller papillomas when challenged with CRPV-ROPV hybrid DNA. Therefore, CRPV L1 DNA vaccination induced CMI responses to CRPV DNA infections that can contribute to protective immunity. Cross-protective immunity against CRPV L1 and ROPV L1 was elicited in these CRPV L1- and subfragment-vaccinated rabbits.

Animals↗
PubMed2006

Keratinocyte-secreted laminin 5 can function as a transient receptor for human papillomaviruses by binding virions and transferring them to adjacent cells.

Human papillomaviruses (HPVs) replicate only in the terminally differentiating epithelium of the skin and mucosa. While infection of basal keratinocytes is considered a requirement for permissive infection, it remains unclear whether virions can specifically target basal cells for adsorption and uptake following epithelial wounding. We present evidence that HPV binds specifically to laminin 5 (LN5), a component of the extracellular matrix (ECM) secreted by migrating and basal keratinocytes. HPV type 11 capsids colocalized with LN5 in the ECM secreted by vaginal keratinocytes. Binding of both virions and virus-like particles to purified LN5 and to the LN5-rich ECM secreted by cultured keratinocytes was effectively blocked by pretreatment with anti-LN5 antibodies. HPV capsid binding to human cervical mucosa sections included the basement membrane which contains LN5. Cultured keratinocytes expressing alpha6 integrin, a transmembrane protein known to bind LN5, were readily infected by virions preadsorbed to LN5-containing substrates, whereas mutant keratinocytes lacking alpha6 integrin were relatively resistant to infection via this route. These findings suggest a model of natural HPV infection in which proliferating keratinocytes expressing alpha6 integrin at the site of epithelial wounding might be targeted by virions adsorbed transiently to LN5 secreted by migrating keratinocytes.

Animals↗
PubMed2005

Human papillomaviruses bind a basal extracellular matrix component secreted by keratinocytes which is distinct from a membrane-associated receptor.

Human papillomaviruses (HPVs) have previously been shown to adsorb to cultured cells via membrane-associated heparan sulfate (HS) and alpha6 integrin. We demonstrate that cultured keratinocytes uniquely secrete a component into the basal extracellular matrix (ECM) which can function to adsorb HPV particles which can then be internalized by adherent cells. This uncharacterized basal ECM adsorption receptor was secreted by normal human epidermal keratinocytes (NHEK) and by each of the four keratinocyte-derived cell lines we examined, but not by non-keratinocyte cell lines. Multiple HPV types bound preferentially to this keratinocyte-specific receptor over the membrane-associated receptor, and binding to the basal ECM adsorption receptor was refractory to inhibition by heparin. Like the membrane-associated receptor, this basal ECM component was functional as an adsorption receptor in our in vitro infection model using HPV-11. Unlike particle adsorption, however, successful infection with HPV-11 virions remained sensitive to the pretreatment of virions with heparin. The secreted basal ECM receptor did not colocalize with antibodies against HS, perlecan, or alpha6 integrin, but colocalized with antibody against laminin-5, a marker of keratinocyte ECM and an abundant component of the basement membrane in mucosa and skin. These findings suggest a model for natural infections in which HPV virions, nonspecifically adsorbed to HS on suprabasal keratinocytes throughout an epithelial wound, might be transferred to mitotically active migrating keratinocytes via an intermediate association with the ECM secreted by these cells as they reestablish the basement membrane.

Adsorption↗
PubMed2005

Comparative safety evaluation of the candidate vaginal microbicide C31G.

C31G is currently the focus of clinical trials designed to evaluate this agent as a microbicidal and spermicidal agent. In the following studies, the in vivo safety of C31G was assessed with a Swiss Webster mouse model of cervicovaginal toxicity and correlated with results from in vitro cytotoxicity experiments and published clinical observations. A single exposure of unformulated 1% C31G resulted in mild-to-moderate epithelial disruption and inflammation at 2 and 4 h postapplication. The columnar epithelium of the cervix was the primary site of damage, while no perturbation of the vaginal mucosa was observed. In contrast, application of unformulated 1.7% C31G resulted in greater levels of inflammation in the cervical epithelium at 2 h postapplication and severe epithelial disruption that persisted to 8 h postapplication. Application of a nonionic aqueous gel formulation containing 1% C31G resulted in no apparent cervicovaginal toxicity at any time point evaluated. However, formulation of 1.7% C31G did not substantially reduce the toxicity associated with unformulated C31G at that concentration. These observations correlate with findings gathered during a recent clinical trial, in which once-daily applications resulted in no adverse events in women receiving the formulation containing 1% C31G, compared to moderate-to-severe adverse events in 30% of women receiving the 1.7% C31G formulation. The Swiss Webster mouse model was able to effectively discriminate between concentrations and formulations of C31G that produced distinct clinical effects in human trials. The Swiss Webster animal model may be a highly valuable tool for preclinical evaluation of candidate vaginal microbicides.

Administration, Intravaginal↗
PubMed2005

Characterization of immune responses during regression of rabbit oral papillomavirus infections.

Rabbit oral papillomavirus (ROPV) is a mucosatropic papillomavirus that causes small benign discrete papillomas within the oral cavity of domestic rabbits. The goal of this study was to characterize the immune cell infiltrate over the course of regression of oral papillomas. ROPV-infected oral tissues were harvested at various time points after infection and analyzed by immunohistochemistry for papilloma morphology, viral capsid proteins, and associated immune infiltrates. The results of this study indicated that the L1 and L2 viral capsid proteins were lost rapidly at a time that coincided with an inflammatory response from the rabbit. This inflammatory response began with a rapid rise in numbers of CD11c+ cells at early regression. CD11c+ cells continued to increase in frequency through mid-regression and remained the most-represented cell through late regression. The initial rise in CD11c+ cells was followed by an infiltrate containing increased numbers of activated T cells, including CD4+ and CD25+ cells, during mid-regression. Mid-regression coincided spatially with a loss of viral capsid stain, suggesting that immune cells or cytokines or both were playing a key role in clearance of the papillomas. CD8+ cells increased at the lowest rate and were at low levels in the papilloma epidermis even at mid-regression. All cell types decreased by late regression. CD11c+ and major histocompatibility class II+ cells were the last populations of cells to decrease in number.

Animals↗
PubMed2004

Characterization of three rabbit oral papillomavirus oncogenes.

Rabbit oral papillomavirus (ROPV) induces warts in mucosal tissues, and represents a useful model for understanding host-virus interactions that are reflected in mucosal/HPV infections. ROPV induces benign papillomas that regress in 100% of infected rabbits. We previously reported the complete genome sequence of ROPV. However, the oncogenic potential of this virus is unknown because of immunologically mediated regression. The purpose of this study was to characterize the transforming proteins of E6, E7, and E8 genes of ROPV. E6, E7, and E8 genes of ROPV were cloned into the expression vector PCR3. Two hybrid CRPV-ROPV E6 genes were also constructed and tested together with the three wild-type ROPV genes. Each construct was transfected into NIH3T3 cells and stable transfected cell lines were established. Transforming properties of ROPV E6, E7, and E8 were tested via anchorage-independent growth of cells in agar plates and tumor growth in athymic mice. Cells with ROPV E6, E7, or E8 formed colonies in agar and tumors in athymic mice. These observations suggest that ROPV E6, E7, and E8 are oncogenic.

Amino Acid Sequence↗
PubMed2004

Differential antibody responses to a distinct region of human papillomavirus minor capsid proteins.

A peptide derived from the human papillomavirus type 16 (HPV-16) minor capsid protein, L2, has previously been reported to induce cross-neutralizing antibodies in mice. In this report, four HPV L2 peptides, including the HPV-16 peptide and its HPV type 6 and 11 homologues, along with extended peptides containing a conserved set of amino acids, were used to immunize rabbits and mice. Antibody responses were evaluated for specificity and ability to neutralize viral infection in vitro with a quantitative reverse transcriptase (RT)-polymerase chain reaction (PCR) assay. All peptide immunizations resulted in cognate and cross-peptide reactivity, but this did not translate equally into recognition of full-length protein, VLP, or neutralization of virus in vitro. This report provides the first evidence of cross-neutralization of authentic HPV by antiserum to L2 peptides. Comparison of the anti-peptide serum reactivity, especially with regard to neutralization of virus, indicates that the extended peptides may offer more potential to induce adequate responses for cross-protective immunity.

Animals↗
PubMed2004

A small molecule antagonist of the alpha(v)beta3 integrin suppresses MDA-MB-435 skeletal metastasis.

INTRODUCTION: Breast cancer is one of the most common malignancies affecting women in the United States and Europe. Approximately three out of every four women with breast cancer develop metastases in bone which, in turn, diminishes quality of life. The alpha(v)beta3 integrin has previously been implicated in multiple aspects of tumor progression, metastasis and osteoclast bone resorption. Therefore, we hypothesized that the alpha(v)beta3-selective inhibitor, S247, would decrease the development of osteolytic breast cancer metastases. MATERIALS AND METHODS: Cells were treated in vitro with S247 and assessed for viability and adhesion to matrix components. Athymic mice received intracardiac (left ventricle) injections of human MDA-MB-435 breast carcinoma cells expressing enhanced green-fluorescent protein. Mice were treated with vehicle (saline) or S247 (1, 10, or 100 mg/kg/d) using osmotic pumps beginning either one week before or one week after tumor cell inoculation. Bones were removed and examined by fluorescence microscopy and histology. The location and size of metastases were recorded. RESULTS AND CONCLUSIONS: IC50 for S247 adhesion to alpha(v)beta3 or alpha(IIB)beta3a substrates was 0.2 nM vs. 244 nM, respectively. Likewise, S247 was not toxic at doses up to 1000 microM. However, osteoclast cultures treated with S247 exhibited marked morphological changes and impaired formation of the actin sealing zone. When S247 was administered prior to tumor cells, there was a significant, dose-dependent reduction (25-50% of vehicle-only-treated mice; P = 0.002) in osseous metastasis. Mice receiving S247 after tumor cell inoculation also developed fewer bone metastases, but the difference was not statistically significant. These data suggest that, in the MDA-MB-435 model, the alpha(v)beta3 integrin plays an important role in early events (e.g., arrest of tumor cells) in bone metastasis. Furthermore, the data suggest that alpha(v)beta3 inhibitors may be useful in the treatment and/or prevention of breast cancer metastases in bone.

Actins↗
PubMed2004

Mouse model of cervicovaginal toxicity and inflammation for preclinical evaluation of topical vaginal microbicides.

Clinical trials evaluating the efficacy of nonoxynol-9 (N-9) as a topical microbicide concluded that N-9 offers no in vivo protection against human immunodeficiency virus type 1 (HIV-1) infection, despite demonstrated in vitro inactivation of HIV-1 by N-9. These trials emphasize the need for better model systems to determine candidate microbicide effectiveness and safety in a preclinical setting. To that end, time-dependent in vitro cytotoxicity, as well as in vivo toxicity and inflammation, associated with N-9 exposure were characterized with the goal of validating a mouse model of microbicide toxicity. In vitro studies using submerged cell cultures indicated that human cervical epithelial cells were inherently more sensitive to N-9-mediated damage than human vaginal epithelial cells. These results correlated with in vivo findings obtained by using Swiss Webster mice in which intravaginal inoculation of 1% N-9 or Conceptrol gel (containing 4% N-9) resulted in selective and acute disruption of the cervical columnar epithelial cells 2 h postapplication accompanied by intense inflammatory infiltrates within the lamina propria. Although damage to the cervical epithelium was apparent out to 8 h postapplication, these tissues resembled control tissue by 24 h postapplication. In contrast, minimal damage and infiltration were associated with both short- and long-term exposure of the vaginal mucosa to either N-9 or Conceptrol. These analyses were extended to examine the relative toxicity of polyethylene hexamethylene biguanide (PEHMB), a polybiguanide compound under evaluation as a candidate topical microbicide. In similar studies, in vivo exposure to 1% PEHMB caused minimal damage and inflammation of the genital mucosa, a finding consistent with the demonstration that PEHMB was >350-fold less cytotoxic than N-9 in vitro. Collectively, these studies highlight the murine model of toxicity as a valuable tool for the preclinical assessment of toxicity and inflammation associated with exposure to candidate topical microbicides.

Administration, Intravaginal↗
PubMed2003

HIV-1 infection in a small animal human vaginal xenograft model.

A limitation in advancing the study of HIV-1 is the lack of a suitable small animal model system that allows for HIV-1 infection to be monitored within human target epithelium. Studies have demonstrated that HIV-1 can infect vaginal mucosa after sexual exposure; however, the primary target cells for HIV-1 in the vagina and interactions between these target cells are not completely defined. A mouse human vaginal xenograft model that recapitulates, histologically and cytochemically, the features of the human vaginal epithelial barrier has been developed in our laboratory. Results of experiments utilizing this system to characterize HIV-1 BaL and IIIB infections within human vaginal xenografts are reported here. HIV-1 RNA, spliced transcripts, and HIV-1 p24 core antigen protein were detected in the xenografts 7 days after infection. This unique system offers a small animal model for studying HIV-1 transmission and replication within the context of natural host tissue and for examining initial events and cell populations involved in the establishment of HIV-1 infection.

Acquired Immunodeficiency Syndrome↗
PubMed2003

Mucosally-derived HPV-40 can infect both human genital foreskin and cutaneous hand skin tissues grafted into athymic mice.

HPV-40 is a rare HPV type that has been detected only in genital mucosal tissues. This HPV type is very closely related to HPV-7, which has a predominantly cutaneous tissue tropism. We have shown, previously, that an isolate of HPV-40 (described here as HPV-40(Hershey) or HPV-40(H)) productively infected genital tissues. In this study, HPV-40(H) was tested for productive infection of cutaneous tissue. Fetal hand skin fragments were incubated with infectious HPV-40(H) and implanted subrenally into athymic mice. After 120 days, xenografts showed morphological changes consistent with HPV-40(H) infection and were HPV-40 DNA in situ positive and capsid antigen positive. The results demonstrated that hand skin can support HPV-40(H) infection thereby indicating that this viral type has the capacity to infect both genital mucosal and cutaneous tissues.

Amino Acid Sequence↗
PubMed2002

Gene gun-mediated intracutaneous vaccination with papillomavirus E7 gene delays cancer development of papillomavirus-induced skin papillomas on rabbits.

High-risk human papillomavirus (HPV) E6 and E7 viral oncogenes are expressed in HPV-associated cancers, and thus represent tumor-specific antigens. We used the cottontail rabbit papillomavirus (CRPV) rabbit model to test whether vaccination with either the E6 or E7 genes alone could prevent or delay carcinoma development. CRPV-induced papillomas on 24 rabbits were allowed to grow for 3 months without any treatment intervention. An immunization protocol using gene gun-mediated intracutaneous administration of DNA plasmids encoding the E6 or the E7 gene or vector only, respectively was initiated at this time point. Carcinoma development was followed up to 24 months after virus infection. Within this period, five rabbits died due to other causes but without carcinoma; one from the vector control group, and two each from the E6- and E7-vaccinated groups. The remaining seven rabbits from the vector control group developed carcinoma within 7-17 months. The remaining six E6-vaccinated rabbits developed cancer within 8-15 months. There was no delay in cancer development for the E6-vaccinated rabbits compared to the vector-injected rabbits. Some delay in cancer development in the remaining E7-vaccinated rabbits was observed; one developed cancer at month 23 and a second was without cancer at month 24. In addition, some E7-vaccinated rabbits with primary skin carcinomas had fewer lung metastases (<2) compared to vector-vaccinated controls (20+). These results suggested that gene gun-mediated intracutaneous immunization with papillomavirus early gene E7 but not E6 delayed carcinoma development of papillomavirus-induced lesions.

Animals↗
PubMed2002

Protective immunity to rabbit oral and cutaneous papillomaviruses by immunization with short peptides of L2, the minor capsid protein.

The papillomavirus minor capsid protein, L2, has been shown to exhibit immunogenicity, whereby a variety of B-cell epitopes, predominantly in the amino terminus of L2, have been deduced. However, immunity to L2 in vivo has not been examined extensively. Notably, a common neutralization epitope for human papillomavirus (HPV) types 6 and 16 was mapped to amino acids (aa) 108 to 120. The objectives of this study were to derive antisera from rabbits using the corresponding sequences from rabbit viruses and to assess the ability of these peptides to protect against infection. Synthetic peptides consisting of two overlapping sequences each in the region of aa 94 to 122 of the rabbit oral (ROPV) and cottontail rabbit (CRPV) papillomaviruses were used to immunize rabbits. Rabbits were then infected with both ROPV and CRPV and monitored for the development of oral and cutaneous papillomas, respectively. Serum derived from rabbits immunized with either of the two peptides was shown to (i) react to purified L2 from the cognate virus, (ii) specifically recognize L2 within virus-infected cells, and (iii) neutralize virus in vitro. Following viral challenge, cutaneous papilloma growth was completely absent in rabbits immunized with either CRPV peptide. Likewise, ROPV peptide-immunized rabbits were protected from oral papillomatosis. Challenge of CRPV peptide-immune rabbits with the viral genome resulted in efficient papilloma growth, suggesting a neutralizing antibody-mediated mechanism of protection. These results afford in vivo evidence for the immunogenicity provided by a distinct region of L2 and further support previous evidence for the ability of this region to elicit antiviral immunity.

Amino Acid Sequence↗