Session 51Poster Presentations Novel Vaccine Approaches Session Day and Time: Thursday 1:30 - 3:30 pm Room: Hall D
422 Tat Protein Production in Plants; A New Approach to Mucosal Delivery of an HIV-1 Vaccine Candidate A. V. Karasev*, K. J. Shon, B. Aiamkitsumrit, I. Zwierzynski, H. Koprowski Thomas Jefferson Univ, Doylestown, PA
Background: The HIV-1 Tat protein has been recently explored as a prospective vaccine candidate with broad, subtype non-specific action. We approached the problem of delivery of Tat through the mucosal route, e.g., mimicking the normal route of HIV-1 entry into the body, by expressing Tat in an edible plant, like spinach. As a first step, we focused our research on optimization of the Tat production in spinach and initial toxicity testing of plant-produced Tat in mice.
Methods: Tat gene, HIV-1 strain MN, was assembled from synthetic overlapping oligonucleotides and was subsequently cloned into a plant virus-based vector for expression in spinach. A table of codon usage for spinach was extracted from the database and utilized for the optimized Tat gene design. Several constructs expressing Tat alone and fused to different stabilizing entities were tested. Spinach plants inoculated with the Tat-producing constructs were observed 7-14 days post-inoculation, collected, and fed to mice.
Results: Codon optimization of the Tat gene expressed in spinach plants resulted in several-fold yield increase as detected in immunoblots, and did not cause severe symptoms in inoculated plants. Some plant virus proteins used as stabilizing tags, apparently caused host-specific hypersensitive response in Nicotiana benthamiana after inoculation with these constructs. However, spinach was relatively tolerant to high levels of Tat or Tat fusions, showing relatively mild, if any, symptoms of infection. Groups of 5 mice were fed with the Tat-producing or control, vector-inoculated spinach, as well as with uninoculated spinach. After 3 voluntary feedings, 1 wk apart, 1 g per mice, no differences were detected in the growth rate or behavior of the animals fed with these 3 types of spinach.
Conclusions: Codon optimization allowed to produce up to 300-500 micrograms of Tat antigen per 1 g of leaf tissue in spinach using plant virus-based expression system. The plant-produced Tat did not seem to have any apparent adverse effect on mice growth or behavior when fed with spinach for 3 wks.
