sepharose and Adenoviridae-Infections

Recombinant adeno-associated viral (rAAV) vectors based on serotype 2 are currently being evaluated most extensively in animals and human clinical trials. rAAV vectors constructed from other AAV serotypes (serotypes 1, 3, 4, 5, and 6) can transduce certain tissues more efficiently and with different specificity than rAAV2 vectors in animal models. Here, we describe reagents and methods for the production and purification of AAV2 inverted terminal repeat-containing vectors pseudotyped with AAV1 or AAV5 capsids. To facilitate pseudotyping, AAV2rep/AAV1cap and AAV2rep/AAV5cap helper plasmids were constructed in an adenoviral plasmid backbone. The resultant plasmids, pXYZ1 and pXYZ5, were used to produce rAAV1 and rAAV5 vectors, respectively, by transient transfection. Since neither AAV5 nor AAV1 binds to the heparin affinity chromatography resin used to purify rAAV2 vectors, purification protocols were developed based on anion-exchange chromatography. The purified vector stocks are 99% pure with titers of 1 x 10(12) to 1 x 10(13)vector genomes/ml.

Topics: Adenoviridae; Adenoviridae Infections; Animals; Anion Exchange Resins; Cell Line; Chromatography; DNA, Recombinant; Gene Expression Regulation, Viral; Genetic Therapy; Genetic Vectors; Humans; Male; Plasmids; Rats; Rats, Inbred F344; Sepharose; Virion; Virus Replication

The interaction between antibodies and surface antigens of intact immobilized adenovirus particles was studied by single radial immunodiffusion tests (SRDT) in agarose gels. Purified virus particles and antisera against virus particles of selected serotypes representing different subgroups and against structural components of certain serotypes were employed. Antibodies against hexons, pentons and fibres all gave visible zones. Antisera against the latter components were more efficient in forming zones and this was though to be due to the occurrence of relatively smaller amounts of vertex capsomers and fibre antigen at the virus particle surface. The capacity of antibodies against vertex capsomers to form zones was demonstrated in tests with virus particles of intermediate types. The SRDT with immobilized virus particles was found to be highly sensitive for the demonstration of virus particle surface antigens shared between serotypes. Results obtained in previous studies using virus particles haemagglutination-inhibition (HI) tests including anti-antiserum were confirmed. Cross-reactions mainly occurred within subgroups and were ascribed to a sharing of vertex capsomere antigens. In preliminary studies a rapid typing was achieved by examination of paired human sera from five cases of adenovirus infections.

Topics: Adenoviridae; Adenoviridae Infections; Antibodies, Viral; Antigen-Antibody Reactions; Antigens, Viral; Cross Reactions; HeLa Cells; Humans; Immunodiffusion; Sepharose; Serotyping; Viral Proteins