The sequence variability of protective antigens is a major challenge to the development of vaccines. For Neisseria meningitidis, the bacterial pathogen that causes meningitis, the amino acid sequence of the protective antigen factor H binding protein (fHBP) has more than 300 variations. These sequence differences can be classified into three distinct groups of antigenic variants that do not induce cross-protective immunity. Our goal was to generate a single antigen that would induce immunity against all known sequence variants of N. meningitidis. To achieve this, we rationally designed, expressed, and purified 54 different mutants of fHBP and tested them in mice for the induction of protective immunity. We identified and determined the crystal structure of a lead chimeric antigen that was able to induce high levels of cross-protective antibodies in mice against all variant strains tested. The new fHBP antigen had a conserved backbone that carried an engineered surface containing specificities for all three variant groups. We demonstrate that the structure-based design of multiple immunodominant antigenic surfaces on a single protein scaffold is possible and represents an effective way to create broadly protective vaccines.
Helper-dependent adenoviral vectors deleted of all viral coding sequences have shown an excellent gene expression profile in a variety of animal models, as well as a reduced toxicity after systemic delivery. What is still unclear is whether long-term expression and therapeutic dosages of these vectors can be obtained also in the presence of a preexisting immunity to adenovirus, a condition found in a high proportion of the adult human population. In this study we performed intramuscular delivery of helper-dependent vectors carrying mouse erythropoietin as a marker transgene. We found that low doses of helper-dependent adenoviral vectors can direct long-lasting gene expression in the muscles of fully immunocompetent mice. The best performance-i.e., 100% of treated animals showing sustained expression after 4 months-was achieved with the latest generation helper-dependent backbones, which replicate and package at high efficiency during vector propagation. Moreover, efficient and prolonged transgene expression after intramuscular injection was observed with limited vector load also in animals previously immunized against the same adenovirus serotype. These data suggest that human gene therapy by intramuscular delivery of helper-dependent adenoviral vectors is feasible. R eplication-deficient adenoviral (Ad) vectors deleted of one or more early genes (first-and second-generation Ad vectors) are among the most efficient vehicles for in vivo gene delivery, but their utilization for therapeutic purposes is limited by the transient nature of transgene expression and the systemic toxicity, which are both due to inflammatory and immune responses triggered by the residual expression of viral proteins (1-5). The development of Ad vectors deleted of all viral coding sequences offers the prospect of a safer and more efficient way to deliver genes (6). Production of fully deleted Ad vectors, also called helper-dependent (HD) Ad vectors, is possible thanks to the supply in trans of the viral proteins required for replication and packaging by a helper first-generation virus (7).Previous studies by several laboratories have shown improved performances of HD vs. first-generation vectors after intravenous (i.v.) injection as to efficacy of transduction, longevity of transgene expression, and systemic toxicity (8-11). These studies strongly suggested that latest generation adenoviruses may be useful for human applications. However, delivery of Ad vectors (including HD vectors) to human recipients may be rendered particularly difficult when a preexisting immunity against an adenovirus serotype identical to, or cross-reactive with, the one used for gene transfer has previously developed as a consequence of a natural infection. This interfering effect is expected to be particularly pronounced in the case of i.v. delivery, because transducing vector particles will be exposed to circulating neutralizing antibodies before reaching the main target organ, the liver. It is therefore important to evaluate performance, therapeutic efficacy, an...
Scaling up experimental protocols from rodents to humans is often not a straightforward procedure, and this particularly applies to cancer vaccines, where vaccination technology must be especially effective to overcome a variety of immune suppressive mechanisms. DNA electroporation (DNA-EP) and adenoviral vectors (Ad) have shown high potency and therapeutic efficacy for different antigens in several pre-clinical models. To evaluate the ability of DNA-EP and Ad to break tolerance to a self-antigen in large animals, we have cloned the CEA homologue (rhCEA) from rhesus monkeys (Macaca mulatta) colon tissue samples. rhCEA is a 705 aa protein and shares 78.9% homology to human CEA protein. Immunogenicity of rhCEA expressing vectors was tested in mice and subsequently in rhesus monkeys. To further increase the immunogenic potency of these vectors, a synthetic codon optimized rhCEA cDNA (rhCEAopt) was constructed. Genetic vaccination of rhesus monkeys was effective in breaking immune tolerance to rhCEA in all immunized animals, maintaining over time the elicited immune response, and most importantly, neither autoimmunity nor other side-effects were observed upon treatment. Our data confirm the efficacy of genetic cancer vaccines in large animals such as nonhuman primates and show that development of modified expression cassettes that result in increased potency of plasmid DNA and adenovirus may have a significant impact on vaccine development against malignancies expressing tumor associated antigens in patients. ' 2007 Wiley-Liss, Inc.
Delivery of the tet transactivators using as vehicle a HD-Ad vector induced an immune response directed against the transactivators themselves, causing short-term therapeutic transgene expression. Regulated, long-term therapeutic transgene expression was, however, obtained by reducing the vector dose or expressing the transactivators under the control of a muscle-specific promoter.
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