Class switch DNA recombinations change the constant (C) region of the antibody heavy (H) chain expressed by a B cell and thereby change the antibody effector function. Unusual tandemly repeated sequence elements located upstream of H chain gene exons have long been thought to be important in the targeting and/or mechanism of the switch recombination process. We have deleted the entire switch tandem repeat element (Sμ) from the murine μ H chain gene. We find that the Sμ tandem repeats are not required for class switching in the mouse immunoglobulin H-chain locus, although the efficiency of switching is clearly reduced. Our data demonstrate that sequences outside of the Sμ tandem repeats must be capable of directing the class switch mechanism. The maintenance of the highly repeated Sμ element during evolution appears to reflect selection for a highly efficient switching process rather than selection for a required sequence element.

Deficiencies of the Msh2 protein or the Smu tandem repeat (SmuTR) sequences each reduce isotype switching in mice by about 2- to 3-fold. We find that switching in mice deficient for both Msh2 and SmuTR is nearly ablated. We propose that the SmuTR provides closely spaced cleavage sites that can undergo switch recombination independent of Msh2, whereas cleavages in sequences flanking the SmuTR require Msh2 processing to allow recombinational joining. We also find that changes in Smu sequences alter the focus of switch junctions within Sgamma sequences, indicating that sequences of switch regions act together in the choice of switch recombination junctions. These findings help to explain the conservation of tandemly repeated switch regions associated with heavy chain constant genes in species capable of switching.

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a Melanocyte-stimulating hormone (aMSH) is a 13 amino acid peptide with potent anti-inflammatory effects. We created two DNA expression constructs (miniPOMC and pACTH1-17) that encode bioactive versions of the aMSH peptide, and tested these constructs for therapeutic effects in experimental autoimmune encephalomyelitis (EAE). Each construct contained the sequences for aMSH, as well as the sequences that are involved in the secretion and processing of the POMC gene with the assumption that these sequences would promote processing and release of the encoded aMSH peptide. The differences between the two constructs lie at the C-terminal end where amino acids necessary for amidation of aMSH were included in only the pACTH1-17 construct. These two constructs were tested in vitro in bioassays, and in vivo in a mouse model of EAE. The results show that although bioactive peptides are secreted from cells transfected with either construct, there appears to be a significant therapeutic effect only with the pACTH1-17 construct which contains the extra C-terminal amino acids. The data suggest that it is possible to engineer DNA expression vectors encoding small secreted peptides such as aMSH, and that similar type constructs may be useful as therapeutics for the treatment of inflammatory diseases.

A phase II trial was conducted in subjects with human papillomavirus (HPV) associated high-grade cervical dysplasia testing the safety and efficacy of a microparticle encapsulated pDNA vaccine. Amolimogene expresses T cell epitopes from E6 and E7 proteins of HPV types 16 and 18. An analysis was performed on a subset of HLA-A2+ subjects to test whether CD8+ T cells specific to HPV 16, 18, 6 and 11 were increased in response to amolimogene immunization. Of the 21 subjects receiving amolimogene, 11 had elevated CD8+ T cell responses to HPV 16 and/or 18 peptides and seven of these also had increases to corresponding HPV 6 and/or 11 peptides. In addition, T cells primed and expanded in vitro with an HPV 18 peptide demonstrated cross-reactivity to the corresponding HPV 11 peptide. These data demonstrate that treatment with amolimogene elicits T cell responses to HPV 16, 18, 6 and 11.

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