ADENOVIRUS VECTORS: ADAPTIVE IMMUNITY AND VACCINES angiogenesis and arteriogenesis, but also exerts a remarkable antiapoptotic and pro-regenerative activity during severe ischemic injury. Based on these observations, the aim of the present study was to test whether rAAV-VEGF165 delivery into cardiac tissue, during the acute phase of myocardial infarction, exerts a protective effect on the injured myocardium and promotes long-term functional recovery. Acute infarction of the anterior LV wall was induced in twelve chronically instrumented dogs by permanent occlusion of the LAD coronary artery. Four hours after occlusion, using an echoguided needle, we injected rAAV-VEGF (n= 6; 4x1012 viral particles per animal) or rAAV-LacZ (n=6) directly into the paradoxical segment. Hemodynamics, echocardiographic data and segmental shortening of the infarcted region were measured at baseline (pre-occlusion) and monitored repeatedly during the subsequent four weeks. Histological analysis was performed at the end of the study. LV and arterial systolic and mean pressure, LV dP/dtmax and ejection fraction were not significantly different between the two groups over time. In contrast, the indexes of contraction of the infarcted area displayed marked differences after the second week post-infarction: at four weeks, the fractional shortening was 75 ±18% and -3 ±15% of baseline and regional shortening work was 54 ±15% and 0.8 ±15% of baseline in rAAV-VEGF165 vs. LacZ group, respectively (P<0.05). Histological analysis of the border regions between the infarcted and viable cardiac tissue showed a marked increase in the number of α-SMA-positive arterioles (50-120 µm diameter; 68±2.8 vs. 100±3.8 vessels per 100x microscopic field in the LacZ and VEGF-treated animals respectively; p<0.05). In the same areas, few c-kit-positive cells (1-3 per field) were observed and these only in the VEGF-treated group. The histological analysis of transmural sections showed a significant improvement in myocardial viability in the VEGF group; in accordance, several troponin T-expressing cardiomyocytes displayed a clear nuclear positivity for the proliferation marker PCNA suggesting an ongoing process of active myocardial regeneration. Interestingly, in both groups of animals, the expression of the three main VEGF receptors (VEGFR-1, VEGFR-2 and NP-1) was clearly up-regulated in the border of the infarct. In particular, VEGFR-2 was found to be expressed diffusely on the surviving cardiomyocytes, reinforcing the notion of a possible direct role of VEGF in myocardial protection and/or regeneration.
hypothesis, we used the major antigenic site HA from the hemagglutinin protein of influenza virus type A as model epitope. Replication deficient Ad vectors were engineered expressing the HA sequence YPYDVPDYAGG on the surface of the adenoviral virion as part of following 4 capsid proteins: in HVR5 loop of hexon protein (Ad.Hexon-HA), in HI loop of fiber protein (Ad.FiberHA), in RGD loop of penton protein (Ad.Penton-HA), and attached to the C-terminus of Protein IX (Ad.pIX-HA). The HA epitope is present at various copy numbers in each virion: hexon, 720; protein IX 240; penton base 60; and fiber 36. Using an anti-HA antibody, in vitro studies demonstrated that the modified capsid proteins were incorporated into virions and express the HA-sequences in the expected capsid proteins of each recombinant vector. CBA mice were immunized by the intramuscular route with the same dose of adenoviral vectors (5x109 particle units) and serum anti-HA titer was evaluated at 4 wk by ELISA against purified recombinant HIStagged HA fusion protein. Titers against HA in Ad.Fiber-HA immunized mice were three times higher compared to Ad.HexonHA immunized mice (555±40 vs 162±36, p<0.05) in spite of the lower HA copy number. Very low titers were observed with Ad.Penton-HA and Ad.pIX-HA. Similarly, when the 4 vectors were dosed in the basis of HA equivalents, the titer with Ad.Fiber-HA was up to 10-fold greater than with the Ad.Hexon-HA vector ( 486±13 vs 42±6, p<0.05). The predominant isotype contribution was IgG2a > IgG2b for Ad.Fiber-HA and Ad-Hexon-HA. In contrast to the variable response to a specific antigen inserted into different locations of the capsid, anti-Ad capsid titer were the same for all 4 HA modified vectors and also for an AdNull vector with the wild type capsid delivered by the same dose and route. We conclude that incorporation of an epitope into the fiber gene is the most potent strategy to elucidate an anti-epitope response, at least for this model system . The fact that immune response was independent of the dose of the epitope suggested that antigen position in the capsid and its recognition by B cells is the critical component of the response to the epitope incorporated into the Ad capsid.
ADENOVIRUS VECTORS: ADAPTIVE IMMUNITY AND VACCINES
Yan Zhi,1 Di Wu,1 Heather Jordan,1 Guangping Gao,1 James M. Wilson.1 1 Gene Therpy Program, University of Pennsylvania, Philadelphia, PA.
1088. Antigen Epitopes Expressed in Different Adenovirus Capsid Proteins Induce Different Levels of Epitope-Specific Immunity
Replication-defective adenoviral vectors based on human serotype 5 (H5) has emerged as very effective vaccine carriers. However, the neutralizing antibodies to AdH5 vector by previous natural infections will very likely impair its vaccine efficacy. Therefore, our lab developed replication-defective simian adenoviral C7 vectors to circumvent interference by pre-existing neutralizing antibodies to human AdH5. Nevertheless, cross-reactivity between virus-specifc T-cell has been identified in a variety of different forms in both human and murine models. Therefore, it is of interest to directly examine the cross-reactivity of vector-specific T cells. C57BL/6 mice were i.m. immunized with either AdH5Null or AdC7Null vector at the dose of 1011particles/mouse. Splenocytes were harvested after immunization. A small portion of splenocytes were stimulated with UV-inactivated AdH5 or AdC7 viral particles in vitro and supernatants were assayed for production of IFN-g by ELISA. The results suggested that there was cross reactivity of vector-specific Th1 subsets of T helper cells between AdH5 and AdC7 vector, since a significant level of IFN g was secreted when immunized splenocytes were stimulated with heterologous UV-inactivated viral particles in vitro. The majority of splenocytes were adoptively transferred into AdH5LacZ vector-i.v. injected Rag I mice. Two weeks after adoptive transfer, livers of Rag I mice were isolated and
Anja Krause,1 Stefan Worgall,1 Ju H. Joh,1 Neil R. Hackett,1 Peter W. Roelvink,1 Joseph T. Bruder,1 Thomas J. Wickham,1 Imre Kovesdi,1 Ronlad G. Crystal.1 1 Genetic Med, Weill Medical College of Cornell Univ, NY, NY. The immune response against adenovirus gene transfer vectors (Ad) is directed against the capsid proteins and transgene expressed by the vector. Incorporation of pathogen-derived epitopes into capsid proteins may be a useful strategy to evoke an anti-pathogen immunity and may complement the anti-transgene immune response. Our previous data (Worgall et al Mol Ther 2004; 9:S261) demonstrate that incorporating an epitope of the outer membrane protein of Pseudomonas aeruginosa into the hexon of the adenovirus capsid provides protection against pulmonary challenge by P. aeruginosa in experimental animals. In this study, we compare the immune response against an epitope incorporated into different capsid proteins (hexon, fiber, penton base and protein IX) and determine if the immune response against the epitopes will be proportional to the number of copies of that protein in the capsid. To test this Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
1089. Examine the Cross-Reactivity of VectorSpecific T Cells between Human Adenoviral H5 and Simian Adenoviral C7 Vector in Mice
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