Infectious intracellular and extracellular forms of vaccinia virus have different outer membrane proteins,
presenting multiple targets to the immune system. We investigated the immunogenicity of soluble forms of L1,
an outer membrane protein of the intracellular mature virus, and of A33 and B5, outer membrane proteins of
the extracellular enveloped virus. The recombinant proteins, in 10-g amounts mixed with a Ribi- or saponintype
adjuvant, were administered subcutaneously to mice. Antibody titers to each protein rose sharply after the
first and second boosts, reaching levels that surpassed those induced by percutaneous immunization with live
vaccinia virus. Immunoglobulin G1 (IgG1) antibody predominated after the protein immunizations, indicative
of a T-helper cell type 2 response, whereas live vaccinia virus induced mainly IgG2a, indicative of a T-helper
cell type 1 response. Mice immunized with any one of the recombinant proteins survived an intranasal
challenge with 5 times the 50% lethal dose of the pathogenic WR strain of vaccinia virus. Measurements of
weight loss indicated that the A33 immunization most effectively prevented disease. The superiority of protein
combinations was demonstrated when the challenge virus dose was increased 20-fold. The best protection was
obtained with a vaccine made by combining recombinant proteins of the outer membranes of intracellular and
extracellular virus. Indeed, mice immunized with A33 plus B5 plus L1 or with A33 plus L1 were better protected
than mice immunized with live vaccinia virus. Three immunizations with the three-protein combination were
necessary and sufficient for complete protection. These studies suggest the feasibility of a multiprotein
smallpox vaccine.