Regression of papillomas induced by cottontail rabbit papillomavirus is associated with infiltration of CD8+ cells and persistence of viral DNA after regression.

Cottontail rabbit papillomavirus (CRPV) is a highly oncogenic papillomavirus and has been successfully used as a model to develop protective vaccines against papillomaviruses. Papillomas induced by the virus may spontaneously regress, suggesting that CRPV can also serve as a model to develop therapeutic vaccines. As a first step toward this goal, we have analyzed immunologic and viral aspects associated with papilloma regression and have identified several features unique to regression. Immunohistochemical staining of biopsies from growing and regressing papillomas and from sites after complete regression showed infiltration of CD8+ cells into the basal and suprabasal layers of the epidermis only during active regression. In situ hybridizations with mRNA-specific probes were strongly positive for E6 and E7 mRNAs during regression, but no late mRNA was present. Viral DNA was detected by in situ hybridization during regression but not after regression. However, analysis by PCR revealed persistence of viral DNA for several months at the majority of regression sites. The results suggest that stimulation of a strong CD8+ response to virus-infected cells is important for an effective therapeutic vaccine and that special attention should be given to the suppression of latent infection.     

Identification of three transforming proteins encoded by cottontail rabbit papillomavirus.

Cottontail rabbit papillomavirus (CRPV) provides an animal model for human papillomaviruses associated with a high risk of cancer development. So far, nothing is known about the transforming functions of CRPV genes because of the lack of an assay system. We have recently developed two systems to assay for CRPV transforming functions. One is based on the finding that transformation of NIH 3T3 cells by CRPV is considerably increased by deleting sequences in open reading frame L2. The second one is based on the use of a cottontail rabbit skin epithelial cell line, sf1Ep (C. Meyers and F. O. Wettstein, Virology 181:637-646, 1991). Mutations were introduced which abolished expression of the full-length E6 protein (LE6), the short E6 protein (SE6) initiated at the second ATG of E6, the E7 protein, or the E5 protein. Mutations affecting LE6 or E7, but not SE6, reduced transformation of NIH 3T3 and sf1Ep cells. Transformed NIH 3T3 cell lines with mutations in LE6 and E7 did not grow in soft agar, while those with mutations in SE6 and E5 grew with a reduced efficiency. The cell lines with mutations in LE6, SE6, or E7 still did induce tumors in nude mice. These mutations, however, abolished the ability to induce papillomas in rabbits. When expressed individually with a retroviral vector, LE6, SE6, or E7, but not E5, conferred anchorage-independent growth. The level of viral protein expression in these cell lines was generally low, and a comparison of the abundance of virus-specific mRNA showed that cell lines contained 20 to 50 times less mRNA than a cottontail rabbit papilloma. These data demonstrate that CRPV encodes at least three transforming proteins.     

Immunization with nonstructural proteins E1 and E2 of cottontail rabbit papillomavirus stimulates regression of virus-induced papillomas.

Cottontail rabbit papillomavirus is the major animal model for cancer-associated papillomaviruses. Here we show that vaccination with the nonstructural proteins E1 and E2 induces the regression of virus-induced papillomas and that vaccination is equally effective when proteins are given with and without adjuvant. There was no correlation between antibody levels and regression, suggesting that tumor regression may be due to a cell-mediated response.     

Amino acid residues in the carboxy-terminal region of cottontail rabbit papillomavirus E6 influence spontaneous regression of cutaneous papillomas

Previous studies have identified two different strains of cottontail rabbit papillomavirus (CRPV) that differ by approximately 5% in base pair sequence and that perform quite differently when used to challenge New Zealand White (NZW) rabbit skin. One strain caused persistent lesions (progressor strain), and the other induced papillomas that spontaneously regressed (regressor strain) at high frequencies (J. Salmon, M. Nonnenmacher, S. Caze, P. Flamant, O. Croissant, G. Orth, and F. Breitburd, J. Virol. 74:10766-10777, 2000; J. Salmon, N. Ramoz, P. Cassonnet, G. Orth, and F. Breitburd, Virology 235:228-234, 1997). We generated a panel of CRPV genomes that contained chimeric and mutant progressor and regressor strain E6 genes and assessed the outcome upon infection of both outbred and EIII/JC inbred NZW rabbits. The carboxy-terminal 77-amino-acid region of the regressor CRPV strain E6, which contained 15 amino acid residues that are different from those of the equivalent region of the persistent CRPV strain E6, played a dominant role in the conversion of the persistent CRPV strain to one showing high rates of spontaneous regressions. In addition, a single amino acid change (G252E) in the E6 protein of the CRPV progressor strain led to high frequencies of spontaneous regressions in inbred rabbits. These observations imply that small changes in the amino acid sequences of papillomavirus proteins can dramatically impact the outcome of natural host immune responses to these viral infections. The data imply that intrastrain differences between separate isolates of a single papillomavirus type (such as human papillomavirus type 16) may contribute to a collective variability in host immune responses in outbred human populations.     

High efficiency, long-term clinical expression of cottontail rabbit papillomavirus (CRPV) DNA in rabbit skin following particle-mediated DNA transfer.

The ability of skin to support long lasting expression of genes delivered with a particle-mediated system was evaluated in rabbits inoculated with cottontail rabbit papillomavirus (CRPV) DNA. The optimal delivery force for maximal gene expression in rabbit skin was determined in transient beta-galactosidase assays. Forty-five sites in four rabbits were then inoculated at 350-400 p.s.i. with CRPV DNA. All sites (100%) formed papillomas with multiple papillomas at most sites. These results support the feasibility of using a particle-mediated delivery system for gene therapy and suggest that some papillomavirus features, such an origin of replication, may be well suited for use in vectors to target long term expression to skin.     

T-cell response to cottontail rabbit papillomavirus structural proteins in infected rabbits.

Cottontail rabbit papillomavirus (CRPV)-induced papillomas progress at a high frequency to carcinomas and thus can serve as a model for high-cancer-risk human papillomavirus infection. Previously, we have shown that antibodies to nonstructural and structural proteins are detected in only a fraction of papilloma-bearing animals. However, the antibody response to structural proteins drastically increases as papillomas progress to carcinoma (Y.-L. Lin, L. A. Borenstein, R. Selvakumar, R. Ahmed, and F. O. Wettstein, J. Virol. 67:382-389, 1993). Here we have monitored the cellular immune response to viral proteins during the course of infection and particularly during progression from papilloma to carcinoma. This was done by measuring the in vitro proliferation response of peripheral blood mononuclear cells (PBMCs) to CRPV structural proteins L1 and L2. The proliferating cells were identified as T cells by selective removal of B or T cells. In general, the T-cell response was low for rabbits at the papilloma stage and none responded to L2. Lymphocytes from animals with carcinomas more frequently and more strongly responded to L1, and more than half also responded to L2. In addition to stimulation of PBMCs, L1- and L2-specific proliferation could also be demonstrated with lymph node and spleen cells. Overall, our data show that progression of papilloma to carcinoma is associated with an increased T-cell response to CRPV structural proteins in addition to an increased humoral response. This greater immune reactivity, however, was not associated with a selectively increased expression of structural proteins, since RNA isolated from papillomas and carcinomas contained similar relative levels of late and early RNA as shown by dot blot analysis. Thus, the heightened immune reactivity seen in carcinoma-bearing rabbits most likely reflects greater stimulation of the immune system owing to dissemination of the tumor. These findings suggest that increased immune responses to papillomavirus proteins may be prognostic of progression to carcinoma and particularly of the development of metastases.     

Intracutaneous DNA vaccination with the E8 gene of cottontail rabbit papillomavirus induces protective immunity against virus challenge in rabbits

The cottontail rabbit papillomavirus (CRPV)-rabbit model has been used in several studies for testing prophylactic and therapeutic papillomavirus vaccines. Earlier observations had shown that the CRPV nonstructural genes E1, E2, and E6 induced strong to partial protective immunity against CRPV infection. In this study, we found that CRPV E8 immunization eliminated virus-induced papillomas in EIII/JC inbred rabbits (100%) and provided partial protection (55%) against virus challenge in outbred New Zealand White rabbits. CRPV-E8 is a small open reading frame, coding for a 50-amino-acid protein, that is colinear with the CRPV E6 gene and has features similar to those of the bovine papillomavirus and human papillomavirus E5 genes. Papillomas that grew on E8-vaccinated outbred rabbits were significantly smaller than those on vector-vaccinated rabbits (P < 0.01; t test). Delayed-type hypersensitivity skin tests showed that some of the E8-vaccinated rabbits had positive responses to E8-specific peptides.     

Pathogenicity of molecularly cloned bovine leukemia virus.

To delineate the mechanisms of bovine leukemia virus (BLV) pathogenesis, four full-length BLV clones, 1, 8, 9, and 13, derived from the transformed cell line FLK-BLV and a clone construct, pBLV913, were introduced into bovine spleen cells by microinjection. Microinjected cells exhibited cytopathic effects and produced BLV p24 and gp51 antigens and infectious virus. The construct, pBLV913, was selected for infection of two sheep by inoculation of microinjected cells. After 15 months, peripheral blood mononuclear cells from these sheep served as inocula for the transfer of infection to four additional sheep. All six infected sheep seroconverted to BLV and had detectable BLV DNA in peripheral blood mononuclear cells after amplification by polymerase chain reaction. Four of the six sheep developed altered B/T-lymphocyte ratios between 33 and 53 months postinfection. One sheep died of unrelated causes, and one remained hematologically normal. Two of the affected sheep developed B lymphocytosis comparable to that observed in animals inoculated with peripheral blood mononuclear cells from BLV-infected cattle. This expanded B-lymphocyte population was characterized by elevated expression of B-cell surface markers, spontaneous blastogenesis, virus expression in vitro, and increased, polyclonally integrated provirus. One of these two sheep developed lymphocytic leukemia-lymphoma at 57 months postinfection. Leukemic cells had the same phenotype and harbored a single, monoclonally integrated provirus but produced no virus after in vitro cultivation. The range in clinical response to in vivo infection with cloned BLV suggests an important role for host immune response in the progression of virus replication and pathogenesis.     

Ubiquitin-fused and/or multiple early genes from cottontail rabbit papillomavirus as DNA vaccines

Human papillomavirus (HPV) vaccines have the potential to prevent cervical cancer by preventing HPV infection or treating premalignant disease. We previously showed that DNA vaccination with the cottontail rabbit papillomavirus (CRPV) E6 gene induced partial protection against CRPV challenge and that the vaccine's effects were greatly enhanced by priming with granulocyte-macrophage colony-stimulating factor (GM-CSF). In the present study, two additional strategies for augmenting the clinical efficacy of CRPV E6 vaccination were evaluated. The first was to fuse a ubiquitin monomer to the CRPV E6 protein to enhance antigen processing and presentation through the major histocompatibility complex class I pathway. Rabbits vaccinated with the wild-type E6 gene plus GM-CSF or with the ubiquitin-fused E6 gene formed significantly fewer papillomas than the controls. The papillomas also required a longer time to appear and grew more slowly. Finally, a significant proportion of the papillomas subsequently regressed. The ubiquitin-fused E6 vaccine was significantly more effective than the wild-type E6 vaccine plus GM-CSF priming. The second strategy was to vaccinate with multiple CRPV early genes to increase the breadth of the CRPV-specific response. DNA vaccines encoding the wild-type CRPV E1-E2, E6, or E7 protein were tested alone and in all possible combinations. All vaccines and combinations suppressed papilloma formation, slowed papilloma growth, and stimulated subsequent papilloma regression. Finally, the two strategies were merged and a combination DNA vaccine containing ubiquitin-fused versions of the CRPV E1, E2, and E7 genes was tested. This last vaccine prevented papilloma formation at all challenge sites in all rabbits, demonstrating complete protection.     

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.     

Transfection of molecularly cloned Friend murine leukemia virus DNA yields a highly leukemogenic helper-independent type C virus.

Unintegrated viral DNA was isolated via the Hirt procedure from mouse fibroblasts newly infected with Friend murine leukemia virus (F-MuLV) clone 201, a biologically cloned helper virus isolated from stocks of F-MuLV complex. A physical map of the unintegrated in vivo linear viral DNA was generated for several restriction endonucleases. The supercoiled viral DNA was digested with EcoRI, which cleaved the viral DNA at a unique site. The linearized viral DNA was then inserted into lambda gtWES.lambda B at the EcoRI site and cloned in an approved EK2 host. Eight independent lambda-mouse recombinants were identified as containing F-MuLV DNA inserts by hybridization with F-MuLV 32P-labeled complementary DNA. One of the F-MuLV DNA inserts was 9.1 kilobases (kb) and had the same restriction enzyme sites as the unintegrated linear F-MuLV DNA. Six inserts were 8.5 kb; each lacked a single copy of the terminally redundant sequences of the unintegrated linear viral DNA. One insert was 8.2 kb and contained a 0.9-kb deletion. After digestion with EcoRI, one recombinant DNA preparation containing an 8.5-kb insert was infectious for NIH 3T3 cells. Undigested recombinant DNA was not infectious. The infectivity of the EcoRI-digested DNA followed multihit kinetics, indicating that more than one molecule was required to register as an infectious unit. The virus isolated from this transfection (F-MuLV-57) was NB-ecotropic, helper-independent, and formed XC plaques. Inoculation of this virus into newborn NIH Swiss mice induced leukemia and splenomegaly in greater than 90% of animals within 3 to 4 weeks. The gross and microscopic abnormalities induced by F-MuLV clone 57 were identical to those seen with the original parent stocks of F-MuLV clone 201. These results indicate that this helper-independent F-MuLV can induce a rapid nonthymic leukemia in the absence of the spleen focus-forming virus.     

Evolution of replication efficiency following infection with a molecularly cloned feline immunodeficiency virus of low virulence

The development of an effective vaccine against human immunodeficiency virus is considered to be the most practicable means of controlling the advancing global AIDS epidemic. Studies with the domestic cat have demonstrated that vaccinal immunity to infection can be induced against feline immunodeficiency virus (FIV); however, protection is largely restricted to laboratory strains of FIV and does not extend to primary strains of the virus. We compared the pathogenicity of two prototypic vaccine challenge strains of FIV derived from molecular clones; the laboratory strain PET(F14) and the primary strain GL8(414). PET(F14) established a low viral load and had no effect on CD4(+)- or CD8(+)-lymphocyte subsets. In contrast, GL8(414) established a high viral load and induced a significant reduction in the ratio of CD4(+) to CD8(+) lymphocytes by 15 weeks postinfection, suggesting that PET(F14) may be a low-virulence-challenge virus. However, during long-term monitoring of the PET(F14)-infected cats, we observed the emergence of variant viruses in two of three cats. Concomitant with the appearance of the variant viruses, designated 627(W135) and 628(W135,) we observed an expansion of CD8(+)-lymphocyte subpopulations expressing reduced CD8 beta-chain, a phenotype consistent with activation. The variant viruses both carried mutations that reduced the net charge of the V3 loop (K409Q and K409E), giving rise to a reduced ability of the Env proteins to both induce fusion and to establish productive infection in CXCR4-expressing cells. Further, following subsequent challenge of na?ve cats with the mutant viruses, the viruses established higher viral loads and induced more marked alterations in CD8(+)-lymphocyte subpopulations than did the parent F14 strain of virus, suggesting that the E409K mutation in the PET(F14) strain contributes to the attenuation of the virus.     

Cellular factors required for papillomavirus DNA replication.

In vitro replication of papillomavirus DNA has been carried out with a combination of purified proteins and partially purified extracts made from human cells. DNA synthesis requires the viral E1 protein and the papillomavirus origin of replication. The E2 protein stimulates DNA synthesis in a binding site-independent manner. Papillomavirus DNA replication is also dependent on the cellular factors replication protein A, replication factor C, and proliferating-cell nuclear antigen as well as a phosphocellulose column fraction (IIA). Fraction IIA contains DNA polymerase alpha-primase and DNA polymerase delta. Both of these polymerases are essential for papillomavirus DNA replication in vitro. However, unlike the case with T-antigen-dependent replication from the simian virus 40 origin, purified DNA polymerase alpha-primase and delta cannot efficiently replace fraction IIA in the replication reaction. Hence, additional cellular factors seem to be required for papillomavirus DNA replication. Interestingly, replication factor C and proliferating-cell nuclear antigen are more stringently required for DNA synthesis in the papillomavirus system than in the simian virus 40 in vitro system. These distinctions indicate that there must be mechanistic differences between the DNA replication systems of papillomavirus and simian virus 40.     

Growth and DNA replication in rabbit blastocysts.

DNA content and DNA polymerase activity were measured on rabbit blastocysts removed from the uterus at 24-hr intervals over the period of days 4-7 postcoitum (pc). Median DNA content increased 53 times over the 72-hr period, from 25.3 ng on day 4 to 1,360 ng on day 7. Median DNA polymerase activity (fmole of radiolabeled nucleotide incorporated in 30 min at 37 degrees C) increased 393-fold from day 4 to day 7: 32.8 to 12,900. These embryos also increased in surface area and volume by 334-fold and 6,078-fold, respectively. Litters containing individuals with high DNA content also tended to have similar individuals with high DNA polymerase activity. Therefore, DNA polymerase activity may be a useful measure of the potential for the next cell division. A large amount of variation existed between blastocysts in all parameters measured. An analysis of variance, conducted to partition variation between litters and within litters, determined that within-litter variation was actually greater than that between litters, resulting in intraclass correlation coefficients less than 0.5. There was also a positive regression of DNA content and DNA polymerase activity on surface area in 6- and 7-day-old blastocysts after eliminating variation attributable to litters. The developmental pattern of DNA polymerase activity in the rabbit may be quantitatively different from that described in the mouse. The pattern in mammals is very different from that described in several nonmammalian species.     

Specific detection of Plasmodium falciparum malaria by a molecularly cloned DNA probe

A highly repeated DNA sequence from Plasmodium falciparum was cloned and used as a probe in molecular hybridization to detect malaria. Our results indicate that the probe is specific to P. falciparum but not to other species of Plasmodium and is extremely sensitive. As little as a 20 pg parasite DNA, which is equivalent to about 1000 parasites can be detected. The cloned DNA can be used as a diagnostic tool to follow the course of infection of falciparum malaria.     

Immunization with viruslike particles induces long-term protection of rabbits against challenge with cottontail rabbit papillomavirus.

Rabbits were immunized with recombinant baculovirus-produced virus-like particles (VLPs) of cottontail rabbit papillomavirus (CRPV) to determine whether these antigens could induce long-term protection against experimental challenge with CRPV. Infectious CRPV and human papillomavirus type 11 L1 VLPs were used as positive and negative control immunogens, respectively. Three groups of immunized animals were challenged with 10-fold serial dilutions of infectious CRPV at 2 weeks, 6 months, and 12 months after immunizations. Antibody titers in serum reached 1:10,000 immediately after the final booster immunization and then decayed to 1:150 at 6 months and 1:100 at 12 months in unchallenged rabbits. Serum neutralization titers followed similar kinetics. Papillomas grew on control-immunized rabbits at sites challenged with 10(-1) (100% of sites), 10(-2) (96% of sites), 10(-3) (63% of sites), and 10(-4) (13% of sites) dilutions of virus. At 2 weeks after CRPV L1 VLP immunizations, the rabbits were completely protected against virus challenge. At both 6 and 12 months after CRPV L1 VLP immunizations, strong protection was also observed. In the last two groups, three of seven rabbits were completely protected and only 4 of 14 or 29% of sites challenged with 10(-1 dilution of virus grew papillomas. Papillomas growing at these four sites were also reduced in size (3.5 +/- 0.7 mm) at 50 days postchallenge compared with sites challenged with 10(-1) dilution on control-immunized rabbits (13.2 +/- 4.2 mm). The results demonstrate that strong and long-lasting protection against experimental challenge with papillomaviruses can be achieved with VLP immunogens.     

Investigation of the attenuation exhibited by a molecularly cloned chicken anemia virus isolate by utilizing a chimeric virus approach.

Molecular cloning of the Cux-1 isolate of chicken anemia virus (CAV), which had been passaged 173 times in cell culture, resulted in the isolation of an attenuated strain, designated cloned isolate 10, which reverted to virulence following 10 passages in young chicks (D. Todd, T. J. Connor, V. M. Calvert, J. L. Creelan, B. M. Meehan, and M. S. McNulty, Avian Pathol. 24:171-187, 1995). The attenuated cloned isolate 10 differs from the molecularly cloned pathogenic Cux-1 isolate in that it possesses a 21-nucleotide insertion within the nontranscribed region of the CAV genome and 17 individual nucleotide substitutions dispersed throughout the genome. Comparative analyses with other published CAV sequences indicated that cloned isolate 10 was unique at nine nucleotide positions and at five amino acid positions. The molecular basis of the attenuation exhibited by cloned isolate 10 was investigated by evaluating the pathogenicities of two sets of complementary chimeric viruses. These sets were produced by transfection with chimeric double-stranded replicative-form (RF) DNA equivalents that contained DNA sequences derived from cloned isolate 10 and the pathogenic cloned Cux-1 isolate. The construction of the chimeric RFs exploited the occurrence of unique EcoRI, PstI, and BamHI restriction sites, which allowed their respective circular CAV RFs to be manipulated as three restriction fragments of 0.58, 0.93, and 0.71 kbp. Examination of the levels of anemia and gross pathology in the thymuses and bone marrows of 14 day-old specific-pathogen-free chicks following infection of 1-day-old chicks with the chimeric and cloned parental isolates indicated that nucleotide changes in each of the three genomic regions contributed towards attenuation. The significance of this result to the development and use of live attenuated CAV vaccines is discussed.