Michal Smahel

Immunisation with modified HPV16 E7 genes against mouse oncogenic TC-1 cell sublines with downregulated expression of MHC class I molecules

Human papillomavirus type 16 (HPV16)-transformed mouse TC-1 cells are extensively used in the evaluation of efficacy of experimental vaccines against tumours induced by HPVs. As these cells strongly express MHC class I molecules and downregulation of MHC class I surface expression is one of the important mechanisms that enable tumour escape from the host immune system, we undertook to derive TC-1 clones with reduced expression of MHC class I antigens. TC-1 cells were inoculated into mice preimmunised with an E7 gene-based DNA vaccine and from tumours developing in a portion of the animals, cell clones with downregulated MHC class I surface expression were isolated. Treatment with IFN-gamma resulted in an upregulation of MHC class I molecules in these cells, but after IFN-gamma removal, their expression gradually dropped again. When the expression of some components of the antigen-processing machinery (APM; LMP-2, TAP-1, and TAP-2) was tested, a reduced TAP-1 production was detected in cell lines with downregulated MHC class I expression. An enhanced immunoresistance of TC-1-derived clones with reduced MHC class I expression was observed in animals immunised with plasmids carrying modified E7 genes. Apart from the previously described fusion gene Sig/E7/LAMP-1, a new construct, Sig/E7GGG/LAMP-1, with a mutated Rb-binding site, was also used for immunisation. No significant change of immunogenicity was recorded for Sig/E7GGG/LAMP-1. Cell lines with downregulated MHC class I expression derived from TC-1 cells may represent a useful model for testing therapeutic anti-HPV vaccines in settings more relevant to clinical requirements.

Enhancement of immunogenicity of HPV16 E7 oncogene by fusion with E. coli β-glucuronidase

Human papillomavirus type 16 (HPV16) E7 is an unstable oncoprotein with low immunogenicity. In previous work, we prepared the E7GGG gene containing point mutations resulting in substitution of three amino acids in the pRb‐binding site of the HPV16 E7 protein.

PREVALENCE OF ANTIBODIES TO HUMAN PAPILLOMAVIRUSES IN THE GENERAL POPULATION OF THE CZECH REPUBLIC

Sera from 450 individuals between the age of 1 and 80 years, representing the general population of the Czech Republic, were tested for the presence of antibodies to human‐papillomavirus(HPV)‐derived antigens. The following antigens were used: (i) HPV1 virions; (ii) HPV16, ‐18 and ‐33‐virus‐like particles (VLP); (iii) peptides derived from L2 open reading frames (ORFs) of HPV16 and HPV6/11; (iv) peptides derived from HPV16 E2, E4 and E7 ORFs of HPV16. The prevalence of antibodies reactive with the capsid‐derived antigens was age‐dependent, while no clear age dependence was observed in the distribution of antibodies to peptides derived from HPV16 early proteins. In individual sera, high correlations between the presence of antibodies reactive with the 2 L2 peptides, also between the antibodies reactive with different VLPs, were found. While the simultaneous presence of the 2 L2 antibodies was frequently detected in individual sera in all age groups, the simultaneous occurrence of VLP antibodies was detected mostly in subjects older than 20 years. There were no significant differences in HPV‐antibody distribution between men and women. Int. J. Cancer 77:689–694, 1998.

Vaccination with human papillomavirus type 16-derived peptides using a tattoo device

Tattooing has been shown to be very efficient at inducing immunity by vaccination with DNA vaccines. In this study, we examined the usability of tattooing for delivery of peptide vaccines. We compared tattooing with subcutaneous (s.c.) needle injection using peptides derived from human papillomavirus type 16 (HPV16) proteins. We observed that higher peptide-specific immune responses were elicited after vaccination with the simple peptides (E7(44-62) and E7(49-57)) and keyhole limpet hemocyanin-(KLH)-conjugated peptides (E7(49-57), L2(18-38) and L2(108-120)) with a tattoo device compared to s.c. inoculation. The administration of the synthetic oligonucleotide containing immunostimulatory CpG motifs (ODN1826) enhanced the immune responses developed after s.c. injection of some peptides (E7(44-62), KLH-conjugated L2(18-38) and L2(108-120)) to levels close to or even comparable to those after tattoo delivery of identical peptides with ODN1826. The highest efficacy of tattooing was observed in combination with ODN1826 for the vaccination with the less immunogenic E6(48-57) peptide and KLH-conjugated and non-conjugated E7(49-57) peptides which form the visible aggregates that could negatively influence the development of immune responses after s.c. injection but probably not after tattooing. In summary, we first evidenced that tattoo administration of peptide vaccines that might be useful in some cases efficiently induced both humoral and cell-mediated immune responses.

Antigens in chronic myeloid leukemia: implications for vaccine development

Treatment with imatinib mesylate and other tyrosine kinase inhibitors (TKI) revolutionized the therapy of chronic myeloid leukemia (CML). However, it alone does not cure this disease. Moreover, some patients develop resistance or adverse effects to this therapy. As successful treatment of a portion of CML patients by hematopoietic stem cell transplantation (HSCT) suggests the importance of immune mechanisms in the elimination of leukemic cells, including leukemia stem cells, TKI administration or HSCT might be combined with vaccination to cure CML patients. However, antigens implicated in the immune responses have not yet been sufficiently identified. Therefore, in this report, we compiled and characterized a list of 165 antigens associated with CML (CML-Ag165) and analyzed the expression of the corresponding genes in CML phases, subpopulations of leukemic cells, and CML-derived cell lines using available datasets from microarray transcriptional-profiling studies. From the CML-Ag165 list, we selected antigens most suitable for vaccine development and evaluated their appropriate characteristics.

Interleukin-2 and dendritic cells as adjuvants for surgical therapy of tumours associated with human papillomavirus type 16.

Moderately immunogenic HPV 16-associated tumours TC-1 (MHC class I(+), HPV 16 E6/E7(+), G12V Ha-ras(+)) and MK16/1/III ABC (MHC class I(-), HPV 16 E6/E7(+), G12V Ha-ras(+)), both of the H-2(b) haplotype and transplanted in syngeneic mice, were used to examine the adjuvant effects of IL-2 and dendritic cells for surgical therapy. Mice were inoculated s.c. with the respective tumour cells, and when the tumours reached 8-12 mm in diameter, they were extirpated. Three days after surgery, the experimental mice were treated with IL-2, IL-2 gene-modified tumour vaccines, or dendritic cells, injected s.c. to the site of previous surgery. It has been found in both, MHC class I(+) and MHC class I(-) tumours that the recombinant IL-2 and IL-2 gene-modified vaccines substantially reduced the tumour recurrence rate and inhibited growth of tumour recurrences. The dendritic cells were significantly effective only in mice with surgical minimal residual TC-1 (MHC class I(+)) tumour disease and when injected before they have reached the terminal stage of their differentiation.

Metastatic MHC class I-negative mouse cells derived by transformation with human papillomavirus type 16

In the endeavour to develop a model for studying gene therapy of cancers associated with human papillomaviruses (HPVs), mouse cells were transformed with the HPV type 16 (HPV16) and activated H-ras oncogenes. This was done by contransfection of plasmid p16HHMo, carrying the HPV16 E6/E7 oncogenes, and plasmid pEJ6.6, carrying the gene coding for human H-ras oncoprotein activated by G12V mutation, into secondary C57BL/6 mouse kidney cells. An oncogenic cell line, designated MK16/1/IIIABC, was derived. The epithelial origin of the cells was confirmed by their expression of cytokeratins. No MHC class I and class II molecules were detected on the surface of MK16/1/IIIABC cells. Spontaneous metastases were observed in lymphatic nodes and lungs after prolonged growth of MK16/1/IIIABC-induced subcutaneous tumours. Lethally irradiated MK16/1/IIIABC cells induced protection against challenge with 105 homologous cells, but not against a higher cell dose (5 × 105). Plasmids p16HHMo and pEJ6.6 were also used for preventive immunization of mice. In comparison with a control group injected with pBR322, they exhibited moderate protection, in terms of prolonged survival, against MK16/1/IIIABC challenge (P < 0.03). These data suggest that MK16/1/IIIABC cells may serve as a model for studying immune reactions against HPV16-associated human tumours.

Combined immunization with fusion genes of mutated E7 gene of human papillomavirus type 16 did not enhance antitumor effect

The E7 oncoprotein of human papillomavirus type 16 (HPV16) is frequently used as a model tumor‐associated antigen. Its immunogenicity has been substantially enhanced by fusion with several proteins of various origins and functions. Different mechanisms have been responsible for increased vaccination efficacy of fusion proteins.

Comparison of hCMV immediate early and CaMV 35S promoters in both plant and human cells

Cauliflower mosaic virus 35S promoter, widely used in transgenic crop plants, is known to be recognized in widely differing kinds of cells. Its activity in human cells may have impact on the risk assessment for the environmental release of genetically modified plants. In this study, transient expression of several constructs containing beta-glucuronidase (GUS) gene driven by cauliflower mosaic virus 35S promoter or by immediate early promoter of human cytomegalovirus (pCMV) was tested in both potato leaf protoplasts and cultured human cells. The results showed very low but measurable activity of 35S promoter in human 293T-cells (0.01% of that revealed when using pCMV) and in 293 cells that do not produce SV40 T antigen this activity was even lower. On the other hand, in potato protoplasts, pCMV displayed nearly 1% activity seen with p35S.

Enhancement of DNA vaccine potency against legumain.

The asparaginyl endopeptidase legumain that is overexpressed in M2-polarized tumor-associated macrophages has been identified as a suitable target for elimination of these cells supporting tumor progression. To enhance the efficacy of DNA immunization against legumain, we performed several modifications in this protein that could improve induction of immune responses. First, we mutated the RGD motif into GGD or RGG sequences. This alteration resulted in diminished maturation of legumain and impaired cellular localization. Then, as tolerance to self-antigens can be broken by the activation of CD4+ T-cell help, we tried to enhance the immunogenicity of legumain by the insertion of a foreign helper epitope, namely the p30 epitope from the tetanus toxin. Finally, the 2 modifications were combined. After gene gun DNA immunization of C57BL/6 mice with these constructs, we identified the Lgmn111-119 CD8+ T-cell epitope that binds to H-2Db molecules. Furthermore, we showed that mutagenesis in the RGD motif significantly enhanced the immune response against legumain. The addition of the p30 helper epitope induced the specific production of IFN-&ggr; by T cells, but did not significantly increase legumain-specific immunity activated after mutagenesis in the RGD motif which might be caused by simultaneous activation of a Th2 response demonstrated by the production of IL-4. However, the beneficial effect of the helper epitope on legumain-specific response was proved after the depletion of regulatory T cells by antibody against CD25 that preferentially stimulated Th1 immunity. The antitumor effect of the modified legumain gene was shown in the immunization against tumors induced by MK16 cells.