Characterization of murine antibody responses to vaccinia virus envelope protein A14 reveals an immunodominant antigen lacking of effective neutralization targets

Abstract

Vaccinia virus (VACV) A14 is a major envelope protein and a dominant antibody target in the smallpox vaccine. However, the role of anti-A14 antibodies in immunity against orthopoxviruses is unclear. Here, we characterized 22 A14 monoclonal antibodies (mAb) from two mice immunized with VACV. Epitope mapping showed that 21 mAbs targeted the C-terminal hydrophilic region, while one mAb recognized the middle region predicted to be across the viral envelope from the C-terminus. However, none of the mAbs bound to virions in studies with electron microscopy. Interestingly, some mAbs showed low VACV neutralization activities in the presence of complement and provided protection to SCID mice challenged with VACV ACAM2000. Our data showed that, although A14 is an immunodominant antigen in smallpox vaccine, its B cell epitopes are either enclosed within the virions or are inaccessible on virion surface. Anti-A14 antibodies, however, could contribute to protection against VACV through a complement-dependent pathway.

Keywords: A14; Antibody; Epitope; Immunization; Neutralization; Smallpox; Vaccinia.

Figure 1. Mapping the epitopes of A14 mAbs. A and B)

Mapping the epitopes by Western blot of GST-A14 proteins. E. coli strains were either not induced (−) or induced with IPTG (+) to express GST fusion protein with the indicated A14 fragments. Proteins from the whole cell lysates were resolved by SDS-PAGE and analyzed by either Coomassie staining or by Western blot with the indicated antibodies. Prominent protein bands that are only present in induced samples are marked with *. Only Western blots with representative antibodies are shown. C). Predicted topology of A14 on MV with two possible orientations. The two grey lines represent the viral envelope, and the dark lines represent an A14 dimer. The amino acid residue numbers are indicated. The “-S-” denotes the disulfide bond via Cys71. The internal and external side of the virion are indicated in the two models. D). Further define the 8C6 epitope by ELISA of A14 mutants. 293T cells were transfected with plasmids encoding A14 alleles under the control of a VACV promoter and subsequently infected with an IPTG-inducible A14 mutant VACV (VACV-iA14) either in the absence of IPTG. The cell lysates were used to coat a microtiter plate, and ELISA was performed with either 8C6 or HE6. The A14 plasmids are named after the A14 residues expressed (12-75, 12-90) or A14 residues substituted with alanines (26–30->A, 32–35->A, 39–44->A).

Figure 2. Immunogold staining of purified VACV MV with anti-VACV antibodies

Sucrose-gradient purified virions were incubated with the indicated antibodies at 2 μg/ml followed by incubation with secondary gold-conjugated antibodies. The virions were stained with uranyl-acetate and visualized with an electron microscope.

Figure 3. Neutralization of VACV MV by A14 mAbs

Sucrose-gradient purified VACV mature virions were incubated with the indicated concentration of purified antibodies in the presence or absence of rabbit complement for 1 hr at 4°C. The mixture was then added to monolayers of BS-C-1 cells, and the inoculum was removed after one hour. The number of plaques that appeared after 2 days was enumerated. The number of plaques obtained under the indicated condition as the percentage to the number of plaques from untreated inoculums is shown. The average and standard deviation are from three independent inoculums.

Figure 4. Protection of SCID mice from intravenous VACV challenge by anti-A14 mAbs

Groups of six SCID mice were treated with either 400 μL PBS or 400 μL of the indicated antibodies at a concentration of 0.25 mg/mL (=100 μg) on day −1. PBS/antibodies were injected at 200 μL intraperitoneal and 200 μL retro-orbitally. Then, mice were challenged with 1 x 10⁵ PFU (200 μL) of VACV ACAM2000 by the retro-orbital route on day 0. (A) Body weights. (B) Body weight loss at day 58. (C) Survival. (D) Clinical scores over time.