Immunization with Non-Infectious Formalin Derivative of Purified Equine Encephalomyelitis Virus Protein. ∗

Abstract

Previous ultracentrifugal studies 1 of crude equine encephalomyelitis chick vaccines 2 have failed to yield definite evidence of the character of the immunizing principle. Information concerning its probable nature has been sought in the present work by study of the purified virus protein treated with formaldehyde.

Purified protein (Eastern strain), 3 dissolved in Ringer's solution, pH 8-9, 2.0 mg per cc, was treated with various concentrations of CH₂O. Before exposure to CH₂O, one m.i.u. 4 of protein was 10^-13.5 g. When the concentration of CH₂O was less than 0.01 M, inactivation was not always complete in 2 weeks, and tests for immunizing capacity were not made. With other concentrations of CH₂O used, inactivation was complete in the time shown in Table I, as judged by the failure of 10^-5.5 g of protein to infect mice.

The effect of CH₂O on the protein molecules was somewhat similar to its action on tobacco mosaic virus protein. 5 With CH₂O 0.01 M or less, the protein remained in solution but lost slightly in homogeneity, showing a slightly diffuse boundary with a sedimentation pattern similar to that of the untreated protein 3 persisting with little change for more than 3 weeks. In 0.02 M and higher concentrations of CH₂O, the protein rapidly became quite inhomogeneous and increasingly insoluble, losing the definite boundary in 2-4 days. In no instance was there evidence of molecular disruption to small soluble protein fragments of uniform size, nor was there evidence of the component S₂₀° = ca 60 × 10^-13 cm sec^-1 dynes^-1 previously described. 6

The non-infectious, soluble though slightly inhomogeneous protein derivative with 0.01 M CH₂O has immunized all guinea pigs receiving it, as shown in the typical experiment in Table I.