Chimpanzee/human mAbs to vaccinia virus B5 protein neutralize vaccinia and smallpox viruses and protect mice against vaccinia virus

Abstract

Chimpanzee Fabs against the B5 envelope glycoprotein of vaccinia virus were isolated and converted into complete mAbs with human gamma 1 heavy chain constant regions. The two mAbs (8AH8AL and 8AH7AL) displayed high binding affinities to B5 (Kd of 0.2 and 0.7 nM). The mAb 8AH8AL inhibited the spread of vaccinia virus as well as variola virus (the causative agent of smallpox) in vitro, protected mice from subsequent intranasal challenge with virulent vaccinia virus, protected mice when administered 2 days after challenge, and provided significantly greater protection than that afforded by a previously isolated rat anti-B5 mAb (19C2) or by vaccinia immune globulin. The mAb bound to a conformational epitope between amino acids 20 and 130 of B5. These chimpanzee/human anti-B5 mAbs may be useful in the prevention and treatment of vaccinia virus-induced complications of vaccination against smallpox and may also be effective in the immunoprophylaxis and immunotherapy of smallpox.

Fig. 1.

Amino acid sequences of variable domains of heavy (a) and light (b) chains of chimpanzee/human anti-B5 mAbs and ELISA titration of anti-B5 8AH8AL (c). Complementarity-determining regions (CDR1, CDR2, and CDR3) and framework regions (FWR1, FWR2, and FWR3) are indicated above the sequence or sequence alignment. Dashes indicate an identical residue. For the ELISA binding assay, the wells of ELISA plates were coated with recombinant B5 (275t) or unrelated proteins (BSA, thyroglobulin, lysozyme, and phosphorylase-b) and then incubated with 8AH8AL at various concentrations. Bound IgG was detected by the addition of peroxidase-conjugated anti-human (Fab)₂ followed by tetramethylbenzidine substrate.

Fig. 2.

Epitope mapping by Western blotting. (a) Similar amounts of different-sized fragments of B5 expressed in bacteria were blotted onto the membrane, and anti-B5 mAb was added. The bound anti-B5 was detected by HRP-conjugated anti-human IgG (Fab`)_2. The positive bands were visualized with addition of LumiGLO chemiluminescent peroxidase substrate and exposing the membrane to x-ray film. The result is summarized in b, where the peptides that reacted with antibody are scored as positive (+). Faint intensity of the bands is scored as +/−. The numbers denote the starting and ending amino acids.

Fig. 3.

In vitro neutralizing activity of anti-B5 mAbs measured by a comet-reduction assay. (a) BS-C-1 cells were infected with ≈50 pfu of VACV, strain IHD-J. After 2 h at 37°C, the monolayer was washed, and fresh medium containing indicated amounts of chimpanzee anti-B5 8AH7AL or 8AH8AL was added. PBS and rabbit hyperimmune serum served as negative and positive controls, respectively. After 48 h, the monolayers were stained with crystal violet. For the smallpox assay (b), monolayers of BS-C-40 cells in six-well cell culture plates were infected with the Solaimen strain of variola virus at 50 pfu per well in RPMI medium containing 2% FBS. After 1 h, the medium was aspirated; cells were washed twice and overlaid with RPMI medium containing 25, 2.5, or 0 μg of anti-B5 IgG. The plates were then incubated in a CO₂ incubator for 4 days at 35.5°C. Cells were fixed and reacted with polyclonal rabbit anti-variola virus antibody. After incubation with goat anti-rabbit–HRP conjugate, comets were visualized by addition of TrueBlue peroxidase substrate.

Fig. 4.

Prophylactic and therapeutic protection in mice by anti-B5 mAbs. Groups of five BALB/c mice were inoculated i.p. with 90 μg of purified IgG (a) or different amounts of IgG (b). Twenty-four hours later, mice were challenged intranasally with 10⁵ pfu of the WR strain of VACV. Ninety micrograms of rat anti-B5 19C2 IgG (a) or 5 mg of human VIG (b) were used for comparison. (c) Groups of five BALB/c mice were inoculated intranasally with 10⁵ pfu of VACV, strain WR. After 48 h, the mice were injected i.p. with 90 μg of purified IgG or 5 mg of human VIG. Mice were weighed individually, and mean percentages of starting weight ± standard error were plotted. Controls were unimmunized (No antibody) or unchallenged (No virus). †, died naturally or were killed because of 30% weight loss.

Fig. 5.

Antibody responses elicited by challenge with the WR strain of VACV. Mice were bled 22 days after challenge with WR. Individual sera were assayed for binding to EV-associated proteins B5 (a) and A33 (b) and MV-associated proteins L1 (c) and A27 (d). The sera were also assayed for neutralizing antibodies to MV (e). IC₅₀, the reciprocal serum dilution that can neutralize 50% of virus. Reciprocal endpoint binding titers were determined by ELISA by using anti-mouse-HRP. Filled and open bars represent animals immunized with 8AH8AL and VIG, respectively. Those groups that received postexposure immunization are indicated by “Post.”