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Mouse monoclonal antibodies (mAbs) were raised against the major capsid protein, L1, of human papillomavirus type 16 (HPV16), produced in Escherichia coli with the expression plasmid pTrcL1. Epitope specificity could be assigned to 11 of these 12 antibodies using a series of linear peptides and fusion proteins from HPV16. One mAb (MC53) recognized a novel linear epitope that appears to be unique to the HPV16 genotype. A further 11 mAbs were characterized as recognizing novel and previously defined linear and conformational epitopes shared among more than one HPV genotype. The apparently genotype specific mAb could be useful for the development of diagnostic tests for vegetative virus infection in clinical specimens.

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Melanomas tend to become less pigmented in the course of malignant progression. Thus, as proliferation increases, the tumors are decreasingly characterized by the tissue-specific phenotype of normally differentiated melanocytes. To learn whether the decline in melanization is associated with a shift from constitutive to alternative splicing of some pigment gene pre-mRNAs, melanomas were collected from Tyr-SV40E transgenic mice of the standard C57BL͞6 strain. The mRNAs of the tyrosinase gene, which has a key role in melanogenesis, were analyzed by quantitative reverse transcriptase-PCR in 34 samples from 16 cutaneous tumors and 9 metastases. The cutaneous tumors included some cases with distinct melanotic and amelanotic zones, which were separately analyzed. All tyrosinase transcripts found in the melanomas were also found in normal skin melanocytes. However, the ⌬1b and ⌬1d alternatively spliced transcripts, due to deletions within the first exon, were specifically augmented in most of the tumors over their very low levels in skin; the exceptions were some all-amelanotic tumors in which no tyrosinase transcripts were detected. The level of ⌬1b rose as high as 11.3% of total tyrosinase mRNAs as compared with 0.6% in skin; ⌬1d reached 4.0% as compared with 0.8% in skin. Expression of these splice variants was highest in the melanotic components of zonal primary tumors, relatively lower in their amelanotic components, and still lower in all-amelanotic primary tumors and amelanotic metastases. The increase in ⌬1b and ⌬1d transcripts may be predicted to increase the levels of unusual peptides, which could have antigenic potential in the tumors, especially in the relatively early phases of malignancy. Analyses of the alternative transcripts of other pigment genes may identify additional candidate antigens, ultimately enabling melanoma cells in all phases of the disease to be represented as a basis for immune intervention.

The expression of cell-specialization genes is likely to be changing in tumor cells as their differentiation declines. Functional changes in these genes might yield unusual peptide epitopes with anti-tumor potential and could occur without modification in the DNA sequence of the gene. Melanomas undergo a characteristic decline in melanization that may ref lect altered contributions of key melanocytic genes such as tyrosinase. Quantitative reverse transcriptase-PCR of the wild-type (C) tyrosinase gene in transgenic (C57BL͞6 strain) mouse melanomas has revealed a shift toward alternative splicing of the pre-mRNA that generated increased levels of the ⌬1b and ⌬1d mRNA splice variants. The spontaneous c 2j albino mutation of tyrosinase (in the C57BL͞6 strain) changes the pre-mRNA splicing pattern. In c 2j ͞c 2j melanomas, alternative splicing was again increased. However, while some mRNAs (notably ⌬1b) present in C͞C were obligatorily absent, others (⌬3 and ⌬1d) were elevated. In c 2j ͞c 2j melanomas, the percentage of total tyrosinase transcripts attributable to ⌬3 reached approximately 2-fold the incidence in c 2j ͞c 2j or C͞C skin melanocytes. The percentage attributable to ⌬1d rose to approximately 2-fold the incidence in c 2j ͞c 2j skin, and to 10-fold that in C͞C skin. These differences provide a basis for unique mouse models in which the melanoma arises in skin grafted from a C͞C or c 2j ͞c 2j transgenic donor to a transgenic host of the same or opposite tyrosinase genotype. Immunotherapy designs then could be based on augmenting those antigenic peptides that are novel or overrepresented in a tumor relative to the syngeneic host.

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