Infection of cells with adeno-associated virus (AAV) type 2 (AAV-2) is mediated by binding to heparan sulfate proteoglycan and can be competed by heparin. Mutational analysis of AAV-2 capsid proteins showed that a group of basic amino acids (arginines 484, 487, 585, and 588 and lysine 532) contribute to heparin and HeLa cell binding. These amino acids are positioned in three clusters at the threefold spike region of the AAV-2 capsid. According to the recently resolved atomic structure for AAV-2, arginines 484 and 487 and lysine 532 on one site and arginines 585 and 588 on the other site belong to different capsid protein subunits. These data suggest that the formation of the heparin-binding motifs depends on the correct assembly of VP trimers or even of capsids. In contrast, arginine 475, which also strongly reduces heparin binding as well as viral infectivity upon mutation to alanine, is located inside the capsid structure at the border of adjacent VP subunits and most likely influences heparin binding indirectly by disturbing correct subunit assembly. Computer simulation of heparin docking to the AAV-2 capsid suggests that heparin associates with the three basic clusters along a channel-like cavity flanked by the basic amino acids. With few exceptions, mutant infectivities correlated with their heparin-and cell-binding properties. The tissue distribution in mice of recombinant AAV-2 mutated in R484 and R585 indicated markedly reduced infection of the liver, compared to infection with wild-type recombinant AAV, but continued infection of the heart. These results suggest that although heparin binding influences the infectivity of AAV-2, it seems not to be necessary.

Overexpression of the major myelin protein of the CNS, proteolipid protein (PLP), leads to late-onset degeneration of myelin and pathological changes in axons. Based on the observation that in white matter tracts of these mutants both CD8ϩ T-lymphocytes and CD11bϩ macrophage-like cells are numerically elevated, we tested the hypothesis that these cells are pathologically involved in the primarily genetically caused neuropathy. Using flow cytometry of mutant brains, CD8ϩ cells could be identified as activated effector cells, and confocal microscopy revealed a close association of the T-cells with MHC-Iϩ (major histocompatibility complex class I positive) oligodendrocytes. Crossbreeding the myelin mutants with mice deficient in the recombination activating gene-1 (RAG-1) lacking mature T-and B-lymphocytes led to a reduction of the number of CD11bϩ cells and to a substantial alleviation of pathological changes. In accordance with these findings, magnetic resonance imaging revealed less ventricular enlargement in the double mutants, partially because of more preserved corpora callosa. To investigate the role of CD8ϩ versus CD4ϩ T-lymphocytes, we reconstituted the myelin-RAG-1 double mutants with bone marrow from either CD8-negative (CD4ϩ) or CD4-negative (CD8ϩ) mice. The severe ventricular enlargement was only found when the double mutants were reconstituted with bone marrow from CD8ϩ mice, suggesting that the CD8ϩ lymphocytes play a critical role in the immune-related component of myelin degeneration in the mutants. These findings provide strong evidence that a primary glial damage can cause secondary immune reactions of pathological significance as it has been suggested for some forms of multiple sclerosis and other leukodystrophies.

Expression of the structural proteins L1 and L2 of the human papillomaviruses (HPV) is tightly regulated. As a consequence, attempts to express these prime-candidate genes for prophylactic vaccination against papillomavirus-associated diseases in mammalian cells by means of simple DNA transfections result in insufficient production of the viral antigens. Similarly, in vivo DNA vaccination using HPV L1 or L2 expression constructs produces only weak immune responses. In this study we demonstrate that transient expression of the HPV type 16 L1 and L2 proteins can be highly improved by changing the RNA coding sequence, resulting in the accumulation of significant amounts of virus-like particles in the nuclei of transfected cells. Data presented indicate that, in the case of L1, adaptation for codon usage accounts for the vast majority of the improvement in protein expression, whereas translation-independent posttranscriptional events contribute only to a minor degree. Finally, the adapted L1 genes demonstrate strongly increased immunogenicity in vivo compared to that of unmodified L1 genes.The human papillomaviruses (HPV) comprise a heterogeneous group of epitheliotropic DNA viruses. It is assumed that each of the more than 80 described HPV genotypes represents also a separate serotype (23, 24). The papillomavirus life cycle requires the infection of differentiating epithelia. In this environment, expression of the viral genes is controlled by the cell differentiation program (5, 17). Infections by human papillomaviruses are the major cause of uterine cancer in humans (22,43,44). It is estimated that worldwide half a million new cases of cervical cancer are caused by these viruses every year. The most important HPV type in this respect is HPV type 16 (HPV-16), accounting for approximately 50% of all cases of cervical cancer. Since the recognition of HPV infection as a major health burden, efforts have been undertaken to interrupt the cycle of papillomavirus infections in order to prevent virusinduced disease. Most promising for the prevention of papillomavirus-associated cancer seems to be the development of subviral vaccines that evoke protective immunity by the induction of neutralizing, capsid-directed antibodies. In fact, viruslike particles (VLP) based on the viral capsid protein L1 or L1 plus L2 are currently being developed for prophylactic and therapeutic vaccination against papillomavirus infections (19,26,27). Because they require costly production and purification protocols, it is predictable that it will require a long time for VLP-based vaccines to become affordable in the lessdeveloped countries, which suffer most from papillomaviruscaused cancer. For the same reasons, production and purification of VLP-based vaccines likely have to be restricted to a very limited number of HPV serotypes. As an alternative approach, capsid-specific neutralizing antibodies could be induced by simple DNA vaccination strategies. Since production of DNA vaccines are standardized, it is feasible to produce vaccines against ...

Recently it has become clear that more potent methods for DNA vaccine delivery need to be developed to enhance the efficacy of DNA vaccines. In vivo electroporation has emerged as a potent method for DNA vaccine delivery. In a mouse model, we evaluated the CD8(+) T lymphocyte response to a prostate cancer DNA vaccine encoding prostate-specific antigen (PSA) after intradermal electroporation. A significantly increased gene expression (100- to 1000-fold) and higher levels of PSA-specific T cells, compared to DNA delivery without electroporation, was demonstrated. Interestingly, investigation of a panel of different electroporation conditions showed that only some conditions that induce high levels of gene expression additionally induced cellular immunity. This suggests that electroporation parameters should be carefully optimized, not only to enhance transfection efficiency, but also to enhance the immune response to the vaccine. This study demonstrates the applicability of intradermal electroporation as a delivery method for genetic cancer vaccines and other DNA vaccines relying on antigen-specific T cell induction.