Peripheral tolerance to human papillomavirus E7 oncoprotein occurs by cross-tolerization, is largely Th-2-independent, and is broken by dendritic cell immunization.

The E7 oncoprotein of human papillomavirus 16 functions as a tumor-specific antigen in transformed epithelial cells of the uterine cervix to which immunotherapeutic strategies aimed at CTL induction may be directed. We previously have shown in mice transgenic for the E7 gene driven off an epithelial specific (keratin-14) promoter, that expression of E7 protein in peripheral epithelium is sufficient to tolerize E7-directed CTL precursors (pCTL; Doan et al, J. Virol., 73: 6166-1670, 1999). Here we show that E7 is presented to T cells for tolerization by cells of bone marrow origin ("cross-tolerization"). We demonstrate that tolerization of E7-directed pCTLs occurs within 2 weeks of exposure to E7 in epithelium. It is maintained in the near absence of CD4+ cells and in the absence of the thymus, and is independent of a coexisting E7-directed Th2-type antibody response. Tolerance was broken by immunization with E7 CTL epitope-pulsed dendritic cells. These findings have implications for immunotherapy of patients with human papillomavirus 16-associated cervical carcinoma, whose immune systems may have experienced long-term exposure to E7-expressing epithelial cells.

Vaccines for human papillomavirus-associated anogenital disease and cervical cancer: practical and theoretical approaches.

The association of genital warts, cervical dysplasia and cervical cancer with certain human papillomavirus (HPV) types indicates that vaccine strategies that target the virus could be effective in controlling disease onset and progression. Three vaccine strategies are available. Firstly, a prophylactic approach of immunisation with HPV virus-like particles to elicit neutralising antibody would prevent infection. Secondly, vaccination targeting replicating virus in suprabasal cells of infected anogenital epithelium would be an effective therapy for infection and early dysplasias. Thirdly, immunotherapy directed to the oncoprotein products of the HPV E6 and E7 open reading frames would be effective in the control of cervical carcinoma. We examine how these strategies may be augmented by contemporary vaccine technologies, in particular through the use of live recombinant vaccine vectors, specific targeting of antigen processing pathways, dendritic cell and 'polytope' approaches, to produce 'designer' vaccines of maximum specificity and efficacy. How these approaches are being exploited by vaccine manufacturers and in clinical trials is discussed.

Expression of jumper ant (Myrmecia pilosula) venom allergens: post-translational processing of allergen gene products.

N-terminal analyses of electrophoretically-separated allergenic polypeptides of the venom of the jumper ant M. pilosula showed that five out of the six allergenic polypeptides identified are homologous with the cloned major allergen Myr p I and may be derived from a single precursor polypeptide. The sixth polypeptide is homologous with a second cloned major allergen, Myr p II which is expressed as a single precursor polypeptide but exists in its native form as a disulphide bond-linked complex.

Molecular cloning and characterization of the major allergen Myr p II from the venom of the jumper ant Myrmecia pilosula: Myr p I and Myr p II share a common protein leader sequence.

A major allergen Myr p II of the Australian jumper ant Myrmecia pilosula has been cloned, immunocharacterized and nucleotide sequenced. An open reading frame of 225 bases was identified and found to encode a deduced amino acid sequence of 75 residues which contained a typical hydrophobic peptide leader sequence. Expressed fusion proteins of Myr p II in both phage and plasmid vectors bind high levels of ant venom-specific IgE and the expressed clones are recognised by 35% of ant venom-allergic individuals. IgE antibodies that recognise the expressed clone have been shown to recognise IgE-binding bands in blots of native venom after separation by SDS-PAGE. The amino acid sequence of Myr p II shares close structural homology with the other major jumper ant allergen Myr p I, differing by only three amino acids in the first 47 residues of both sequences. However, N-terminal analysis of IgE-binding bands derived from Tricine-SDS-PAGE gel blots indicates that both Myr p I and Myr p II undergo extensive post-translational proteolytic processing to unique peptides of 45 and 27 residues, respectively.

Separation of jumper ant (Myrmecia pilosula) venom allergens: a novel group of highly basic proteins.

The sting of the jumper ant (Myrmecia pilosula) causes severe allergic reactions, including anaphylaxis in sensitized individuals. Two of the major allergens, Myr p I and Myr p II, have been cloned, immunocharacterized and nucleotide-sequenced and they encode 112 and 75 residue polypeptides, respectively. Both allergens are highly basic proteins having isoelectric point values greater than 10. However, electrophoretic analysis has generated conflicting results as to the actual sizes of the allergens in the native venom. Electrophoretic, immunological and N-terminal analyses suggested that these allergens undergo extensive post-translational processing to final forms of 45 and 27 residues, respectively. The results highlight the difficulties in the study of small, basic proteins and polypeptides by electrophoretic techniques.

Immediate allergic reactions to Myrmecia ant stings: immunochemical analysis of Myrmecia venoms.

IgE antibody reactivities to the venoms of Myrmecia pilosula, Myrmecia nigrocincta, Myrmecia tarsata, Myrmecia pyriformis, Myrmecia simillima and Myrmecia gulosa have been identified in sera from subjects allergic to ant venom. Sera with IgE reactivity to only a single Myrmecia venom most often recognize M. pilosula venom although all six venoms appear capable of inducing IgE antibodies. The six different Myrmecia venoms were electrophoretically separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotted to nitrocellulose and probed with sera from ant venom-allergic subjects. Four to six IgE-binding components ranging in size from 2 to 25 kDa were identified in each of the venoms. Similarities in molecular weights of the IgE-binding components exist and close taxonomic relationships between the species suggest that common or similar peptides may be present in the different venoms.

Identification of an IgE-binding determinant of the major allergen Myr p I from the venom of the Australian jumper ant Myrmecia pilosula.

The structure of the single allergenic determinant of the major ant venom allergen, Myr p I from the Australian jumper ant Myrmecia pilosula has been determined by inhibition studies with synthetic peptides. A 14 amino-acid C-terminal peptide sequence has been shown to constitute this determinant. Half-maximal inhibition of binding of ant venom-specific IgE antibodies to the native venom was obtained with this peptide at a concentration of 5 x 10(-8) M. This allergenic determinant was invariant for all ant venom-allergic subjects tested whose IgE antibodies recognized this allergen.