Interference with virus and bacteria replication by the tissue specific expression of antibodies and interfering molecules

Historically, protection against virus infections has relied on the use of vaccines, but the induction of an immune response requires several days and in certain situations, like in newborn animals that may be infected at bir th and die in a few days, there is not sufficient time to elicit a protecti ve immune response. Immediate protection in new born could be provided eith er by vectors that express virus-interfering molecules in a tissue specific form, or by the production of animals expressing resistance to virus repli cation. The mucosal surface is the largest body surface susceptible to viru s infection that can serve for virus entry. Then, it is of high interest to develop strategies to prevent infections of these areas. Virus growth can be interfered intracellularly, extracellularly or both. The antibodies neut ralize virus intra- and extracellularly and their molecular biology is well known. In addition, antibodies efficiently neutralize viruses in the mucos al areas. The autonomy of antibody molecules in virus neutralization makes them functional in cells different from those that produce the antibodies a nd in the extracellular medium. These properties have identified antibodies as very useful molecules to be expressed by vectors or in transgenic anima ls to provide resistance to virus infection. A similar role could be played by antimicrobial peptides in the case of bacteria. Intracellular interfere nce with virus growth (intracellular immunity) can be mediated by molecules of very different nature: (i) full length or single chain antibodies: (ii) mutant viral proteins that strongly interfere with the replication of the wild type virus (dominant-negative mutants); (iii) antisense RNA and ribozy me sequences; and (iv) the product of antiviral genes such as the Mr protei ns. All these molecules inhibiting virus replication may be used to obtain transgenic animals with resistance to viral infection built in their genome s. We have developed two strategies to target into mucosal areas either antibo dies to provide immediate protection, or antigens to elicit immune response s in the enteric or respiratory surfaces in order to prevent virus infectio n. One strategy is based on the development of expression vectors using cor onavirus derived defective RNA minigenomes, and the other relies on the dev elopment of transgenic animals providing virus neutralizing antibodies in t he milk during lactation. Two types of expression vectors are being enginee red based on transmissible gastroenteritis coronavirus (TGEV) defective min igenomes. The first one is a helper virus dependent expression system and t he second is based on self-replicating RNAs including the information requi red to encode the TGEV replicase. The minigenomes expressing the heterologo us gene have been improved by using a two-step amplification system based o n cytomegalovirus (CMV) and viral promoters. Expression levels around 5 mu g per 10(6) cells were obtained. The engineered minigenomes will be useful to understand the mechanism of coronavirus replication and for the tissue s pecific expression of antigen, antibody or virus interfering molecules. To protect from viral infections of the enteric tract, transgenic animals s ecreting virus neutralizing recombinant antibodies in the milk during lacta tion have been developed. Neutralizing antibodies with isotypes IgG1 or IgA were produced in the milk with titers of 10(6) in RIA that reduced virus i nfectivity by one million-fold. The recombinant antibodies recognized a con served epitope apparently essential for virus replication. Antibody express ion levels were transgene copy number independent and were related to the t ransgene integration site. This strategy may be of general use since it cou ld be applied to protect newborn animals against infections of the enteric tract by viruses or bacteria for which a protective MAb has been identified . Alternatively the same strategy could be used to target the expression of antibiotic peptides to the enteric tract in order to protect against bacte rial or virus infections.