John W. Kreider

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.

Baculovirus-expressed human papillomavirus type 11 (HPV-11) major capsid protein (L1) virus-like particles (VLPs) were produced in insect cells and purified on CsCl density gradients. The VLPs retained conformational neutralizing epitopes that were detected by a series of HPV-11-neutralizing monoclonal antibodies. Electron microscopy determined that the HPV-11 L1 VLPs were variable in size with a surface topography similar to that of infectious HPV-11. The VLPs were very antigenic, and induced high titres of neutralizing antibodies in rabbits and mice when used as an immunogen without commercial preparations of adjuvant. These VLP reagents may be effective vaccines for protection against HPV infections.

The open reading frame L2 of cottontail rabbit papillomavirus contains antibody-inducing neutralizing epitopes

Polyclonal antisera were generated against bacterially derived fusion proteins of the open reading frames (ORFs) of the capsid proteins of cottontail rabbit papillomavirus (CRPV). The carboxy-terminal two-thirds of CRPV L1 and the carboxy-terminal half of CRPV L2 were cloned into a bacterial expression vector and induced proteins were used as antigen and immunogen. The polyclonal antisera were tested in a series of immunological assays, including ELISA, Western blot, and neutralization of CRPV. ELISA demonstrated that the polyclonal antisera raised against expressed L1 proteins reacted strongly to disrupted CRPV virion antigen and weakly both to intact CRPV virion and disrupted BPV-1 virion. Anti-CRPV L2 antisera reacted strongly only to intact and disrupted CRPV virion antigen. Viral capsid proteins of CRPV were detected in Western blots of HPV-11, BPV-1, and CRPV virus particles by these polyclonal antisera. The anti-L1 sera recognized the major capsid protein (60 kDa) and the anti-L2 sera identified a 76-kDa viral protein of CRPV. Only the antisera generated against expressed L2 neutralized CRPV. The neutralizing titer of the anti-L2 sera, however, was several orders of magnitude lower than the titer of a neutralizing polyclonal antiserum that was generated by immunizations with intact CRPV virions.

The Shope papilloma-carcinoma complex of rabbits: a model system of neoplastic progression and spontaneous regression.

Publisher Summary The majority of domestic rabbits developed invasive, metastatic, and, ultimately, lethal epidermoid carcinomas. The Shope papilloma has a restricted geographic range, mostly confined to the high plains of the western United States. SPV (Shope papilloma virus) produced papillomas with equal facility on the skins of laboratory-infected jackrabbits, snowshoe hares , domestic rabbits, and cottontails. Papillomas cannot be induced in fetal, neonatal, suckling, or adult rat skin by direct inoculation of SPV suspensions or infectious DNA. Susceptibility to SPV infection is also determined by factors related to cell phenotype. Rabbit epidermal cell transformation by SPV requires interaction with mesenchyme. Vitamin A deficiency or excess can produce striking alterations in the differentiation of epithelia of various types. SPV replication in cottontail papillomas is also modulated by phenotypic factors—namely, epidermal maturation and keratinization. Independent studies have confirmed the presence of arginase in Shope papilloma but have not supported the assertion that the enzyme is encoded by the SPV genome. The Shope papilloma-carcinoma complex is an excellent model of neoplastic progression. Papillomas that become malignant undergo a characteristic series of gross morphological changes.

Detection of human serum antibodies that neutralize infectious human papillomavirus type 11 virions

A selection of human sera were tested for the presence of antibodies that neutralized infectious human papillomavirus (HPV) type 11. Neutralizing antibodies were detected by prevention of HPV-11-induced condylomatous transformation of human foreskin chips transplanted subrenally into athymic mice. Test sera were obtained from 21 female patients with genital condylomas and eight patients with laryngeal papillomas. Control patients consisted of 57 adult random blood donors and five asymptomatic children. ELISAs demonstrated that all sera from patients with genital papillomas were strongly reactive to disrupted papillomavirus (PV) antigens of HPV-11, bovine PV type 1 and cottontail rabbit PV, but only two were weakly reactive to intact HPV-11. None of the eight sera from the laryngeal papilloma bearers reacted significantly to disrupted PV antigens, but four of the eight showed strong specific responses to intact HPV-11 only. The majority of the sera that were reactive to intact HPV-11 by ELISA neutralized HPV-11 infectivity in the athymic mouse xenograft system. The data indicated that ELISA reactivity to intact HPV-11 virions was a good predictor for the presence of HPV-11 neutralizing antibodies.

Tumorigenicity of simian virus 40-hepatocyte cell lines: effect of in vitro and in vivo passage on expression of liver-specific genes and oncogenes.

Five simian virus 40 (SV40)-hepatocyte cell lines were examined for tumorigenicity and the effect of in vitro passage on the expression of four liver-specific genes (albumin, transferrin, alpha 1-antitrypsin, and phosphoenolpyruvate carboxykinase), two oncogenes (c-Ha-ras and c-raf), and two genes associated with hepatocarcinogenesis (alpha-fetoprotein and placental-type glutathione-S-transferase). At low passage (12 to 22), all five cell lines expressed the four liver-specific genes at levels similar to those in the liver and were not tumorigenic or were weakly tumorigenic. At high passage (33 to 61), the cell lines formed carcinomas, and four out of five cell lines produced primary tumors that metastasized. At least two cell lines produced well-differentiated hepatocellular carcinomas that expressed liver-specific RNAs. Levels of expression of liver-specific genes changed with time in culture. Some of the changes in liver-specific gene expression in the tumor tissue (such as for the phosphoenolpyruvate carboxykinase gene) paralleled those that occurred with in vitro passage, while other changes (such as for the albumin gene) did not parallel those that occurred with in vitro passage. Correlations between enhanced expression of c-Ha-ras and tumorigenic potential and between the process of SV40 immortalization and induced expression of c-raf and glutathione-S-transferase-P were observed. Induction of alpha-fetoprotein was detected with in vitro and in vivo passage only in the CWSV14 cell line and was paralleled by diminished albumin expression. In conclusion, we developed a model system with five SV40-hepatocyte cell lines, tumors induced by them, and tumor cell lines to examine changes in gene expression that accompany the progression from a normal cell to a hepatocellular carcinoma. Because the SV40-hepatocyte cell lines and tumor cell lines remain highly differentiated and vary in the magnitude of expression of specific genes, they can be used to study the molecular mechanisms regulating gene expression, in particular those regulating specific genes associated with differentiation.

Relationship of tumor leucocytic infiltration to host defense mechanisms and prognosis.

SummaryThe interface between the tumor and the host is often the site of leucocytic infiltration. We will examine the dea that the infiltrating leucocytes of human and experimental tumors are components of the host ammunological defense against the tumor, and that the presence of the infiltrate is a marker of favorable prognosis. Leucocytes could infiltrate tumors because of an active immune response, either nonspecific or specifically directed to tumor-associated antigens. Leucocyte influx may also occur because of chemotactic factors secreted by the tumor cells. Some tumors release factors which enhance vascular permeability and permit improved access by leucocytes to the tumor focus. The consequences of leucocytic infiltration include tumor cell cytolysis, cytostasis, or stimulation of proliferation. The present state of our knowledge of the interactions between tumor cells and infiltrating leucocytes precludes broad generalization of mechanisms. Further study will probably reveal that the mechanisms are diverse, and that there are some systems in which immune interactions occur at this interface and others in which they do not.

Quantitative assay of melanin in melanoma cells in culture and in tumors

Abstract A simple method has been developed for the quantitative fluorimetric determination of melanin. In these studies, we have used synthetic melanin prepared from l -dopa by mushroom tyrosinase and natural melanin present in B16 mouse melanoma cells grown in culture or as a solid tumor. In the assay procedure, the melanin is oxidatively degraded into soluble products by heating in an alkaline, dilute hydrogen peroxide solution under standard conditions. The resulting solution has a characteristic fluorescence which is quantitatively related to the melanin initially present. The assay is very sensitive and can be applied directly to tissues or cell cultures, without prior isolation of the melanin.

Treatment of latent rabbit and human papillomavirus infections with 9-(2-phosphonylmethoxy)ethylguanine (PMEG)

The acyclic nucleotide PMEG was studied for effectiveness against Shope papillomavirus (CRPV) infection of rabbits and human papillomavirus type 11 (HPV-11) infections of human foreskin xenografts in athymic mice. PMEG given in the latent period strongly suppressed the subsequent growth rates of Shope papillomas. PMEG starting in the latent period and continuing for the duration of the experiment, inhibited HPV-11 infections of human skin, including condyloma growth, and synthesis of viral DNA and capsid antigen. Drug toxicity paralleled the therapeutic effects in rabbits but there was much less toxicity in athymic mice.

In vivo anti-papillomavirus activity of nucleoside analogues including cidofovir on CRPV-induced rabbit papillomas

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.

Neutralization of CRPV infectivity by monoclonal antibodies that identify conformational epitopes on intact virions

Monoclonal antibodies were generated against cottontail rabbit papillomavirus (CRPV) and tested for neutralization of CRPV-induced papillomas on domestic NZW rabbits. Intact CRPV was semi-purified on CsCl gradients and used to immunize BALB/c mice. Hybridomas were prepared from a fusion with lymph node cells, and supernatants from growing hybridomas were analyzed by enzyme-linked immunosorbent assay (ELISA) for reactivity to both intact and disrupted CRPV virion antigen. Supernatants from 22 cultures were initially selected that were responsive to CRPV. Ten were reactive to intact CRPV alone, 4 were reactive only to disrupted CRPV, and 8 were reactive to both intact and disrupted CRPV virion antigen. None of these supernatants contained antibodies which recognized epitopes on CRPV capsid proteins (L1 and L2) that were separated on Western blots. Five hybridomas which produced antibodies that bound to intact CRPV, and did not react to intact HPV-11 or BPV-1 were selected and tested for antibody-mediated neutralization of CRPV infectivity. All five monoclonal antibodies were neutralizing, and identified epitopes on intact CRPV virions which were non-linear and conformational in nature. The five neutralizing monoclonal antibodies appeared to recognize a similar epitope or epitope cluster on the intact CRPV virion as determined by competition ELISA.