Abstract
The report by Trentin and associates (1) on the oncogenic properties of Adenovirus Type 12 has been confirmed by Huebner et al.(2) who have further demonstrated the production of tumors in hamsters with Adenovirus Type 18. These studies suggest a need for an in vitro test to differentiate strains of a particular Adenovirus type. The following pilot study on antibody production to Adenovirus in rabbits was initiated with the ultimate goal of demonstrating immunological differences among Adenovirus Type 12 strains. Previous reports(3–7) involved hyperimmunization with multiple doses of large amounts of infectious virus. As here shown, satisfactory antibody titers have been obtained following a single injection of relatively small amounts of viral antigen.
Methods. New Zealand rabbits, 3.5 to 4.5 kg, were bled immediately prior to inoculation, and at 1, 7, 15, 21, 28, 30, 35 and 42 days thereafter. Sera were clarified by light centrifugation and stored at −20°C until tested.
Adenovirus Type 12 antigen was prepared by making 3 serial passages in KB tissue culture of the American Type Culture Collection prototype strain (Huie). Infected cells and fluid from the third passage harvest were freeze-thawed 3 times and lightly centrifuged to remove cellular debris. The supernatant virus fluid represented the antigen used in these studies. This pool† has since been demonstrated to be oncogenic by Huebner et al. (2). Infectivity titrations in HeLa cells have shown the virus pool to titer 103.5/0.1 ml after 7 days. Considerably higher titers up to 106.3/0.1 ml were obtained when the virus was titrated for 14 days in primary human embryonic kidney (HEK) tissue culture. All virus titers listed in the following discussion and tables are based on titrations in HEK.
Three groups of 2 rabbits each were inoculated intravenously with 1 1/2 ml of Adenovirus Type 12 with 30,000,000 tissue culture infective doses—50% (TCID50); 300,000 TCID50; and 3,000 TCID50. Rabbits #3012, #3014 and # 3016 were given a booster inoculation of 300,000 TCID50 intravenously on day 28.
Serum neutralization tests were carried out in 24-hour-old HeLa tube cultures. Cells were maintained for the duration of the test on Eagle's Basal Medium(8) in Earle's Balanced Salt Solution (BSS) containing 5% Agamma Calf Serum.§
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