Western blot analysis of transiently expressed A30, F8 and B7 proteins from the cell lysate (L) and culture supernatant (S) of 293T cells transfected with their respective variola major antigen-expressing DNA vaccine (A30L, F8L or B7R). Recombinant variola major proteins rA30, rB7 and rF8 produced in E.coli are also included in the analysis. Vaccinia WR strain, grown in Vero cells, is included as a positive control. The empty DNA vaccine vector is included as a negative control. The B7 proteins, which were glycosylated when expressed in 293T or Vero cells, were subjected to PNGase treatment.

Neutralizing antibody activities against the vaccinia intracellular mature virus (IMV) as measured by the plaque reduction test. Sera were collected from Balb/C mice after 3 monthly immunizations with DNA vaccines expressing either individual vaccinia antigens (A27 and D8L) or individual variola antigens (A30 and F8). Sera from mice immunized with VACV served as a positive control. Sera from the group that received an empty DNA vaccine vector were used as the negative control. Data are shown as average titers from 10 mice per group.

Immunogenicity and protection efficacy of polyvalent recombinant variola protein vaccines and polyvalent DNA vaccines expressing variola antigens. (A) ELISA analysis of end titration IgG titers against A30, B7 or F8 antigens in immunized mice sera. Data are shown as the geometric mean titers of each group (5 per group) after two bi-weekly immunizations. Animals were immunized with either a combination of recombinant A30, B7 and F8 proteins (Protein), or a combination of three DNA vaccines expressing A30, B7 and F8 antigens (DNA). Control group animals received a one-time vaccinia (WR) immunization (VACV). The protein immunization elicited significantly higher antibody when compared to DNA immunization (* indicates p<0.01). (B) Protection against a lethal intranasal challenge of VACV (WR) in mice that received two bi-weekly immunizations of either the combination of three recombinant variola proteins (rA30, rB7 and rF8) or the combination of three DNA vaccines expressing A30, B7 and F8. Positive control animals received a one-time vaccinia (WR) immunization and negative control animals received three immunizations of an empty DNA vaccine vector. Each curve shows the daily percentage of survivals for each group (5 mice per group) after challenge.

Sequence analysis of the selected protective variola major and vaccinia antigens. Amino acid sequences of the proteins encoded by the WR and COP strains of the vaccinia virus and those encoded by variola India1967 and Bangladesh1975 were compared The numbers at the top are amino acid positions. Amino acids that were different between these strains are shown in bold, and amino acids that are not shown but identical in all viruses are presented as dots.

Recognition of variola major and vaccinia antigens by ELISA using the same polyvalent mouse serum induced with the combination of three DNA vaccines expressing variola major antigens A30, B7 and F8. Each curve is the average of sera assayed from 10 mice that have received three monthly immunizations. Each panel shows one pair of ortholog antigens from both variola and vaccinia.

Variola antigen-specific antibody responses and protection against lethal intraperitoneal (i.p.) VACV challenge in Balb/C mice immunized with DNA vaccines expressing variola antigens. (A) ELISA analysis of end titration IgG titers of mouse sera immunized with monovalent DNA vaccines expressing either A30, B7 or F8 against the autologous antigens expressed in 293T cells. Sera were collected after one, two or three monthly DNA immunizations. The data are shown as the geometric mean titers of 5 animals. (B) Protection against i.p. VACV challenge in mice immunized with either the monovalent or the combination of A30L, B7R and F8L DNA vaccines. Mice immunized with either the positive control vaccinia (WR) or the negative control empty DNA vaccine vector are also included. Body weight loss, shown as the percentage of pre-challenge weight, was measured daily. Each curve shows the group average weight loss (5 mice per group) after challenge. (C) Protection against i.p. VACV challenge in mice immunized with either combination of vaccinia DNA vaccines (A27, B5R and D8L) or combination of variola DNA vaccines (A30, B7R and F8L) or an empty DNA vaccine vector as the negative control. Body weight loss, shown as the percentage of pre-challenge weight, was measured daily. Each curve shows the group average weight loss ± standard deviation (5 mice per group) after challenge.