New p35 (H3L) Epitope Involved in Vaccinia Virus Neutralization and Its Deimmunization

Abstract

Vaccinia virus (VACV) is a promising oncolytic agent because it exhibits many characteristic features of an oncolytic virus. However, its effectiveness is limited by the strong antiviral immune response induced by this virus. One possible approach to overcome this limitation is to develop deimmunized recombinant VACV. It is known that VACV p35 is a major protein for B- and T-cell immune response. Despite the relevance of p35, its epitope structure remains insufficiently studied. To determine neutralizing epitopes, a panel of recombinant p35 variants was designed, expressed, and used for mice immunization. Plaque-reduction neutralization tests demonstrated that VACV was only neutralized by sera from mice that were immunized with variants containing both N- and C- terminal regions of p35. This result was confirmed by the depletion of anti-p35 mice sera with recombinant p35 variants. At least nine amino acid residues affecting the immunogenic profile of p35 were identified. Substitutions of seven residues led to disruption of B-cell epitopes, whereas substitutions of two residues resulted in the recognition of the mutant p35 solely by non-neutralizing antibodies.

Keywords: H3L; deimmunization; epitope; immunogenicity; neutralizing antibodies; oncolytic vaccinia virus; oncolytic virus; orthopoxvirus; p35; vaccinia virus.

Figure 1

Characterization of truncated variants of VACV p35: (a) Ribbon and surface representations of rp35Δ, rp35Δ12, rp35Δ6, rp35Δ3, rp35Δfuse, predicted using a homology-based online modeling service I-TASSER (http://zhanglab.ccmb.med.umich.edu/I-TASSER/, access date 14 April 2022) and molecular coordinates for the VACV p35 protein (PDB 5EJ0); (b) Western blot analysis of VACV proteins fractionated by SDS-PAGE (12%) and developed with serum samples obtained from immunized (lanes1–6) and a nonimmunized (lane 7) volunteers, M, protein molecular weight markers; (c) Western blot analysis of purified recombinant variants of VACV p35, which were fractionated by SDS-PAGE (12.5%) and developed with anti-VACV pooled human sera diluted to 1:200.

Figure 2

Reactivities of obtained sera samples: (a) Indirect ELISA evaluating binding of pooled anti-rp35Δ, anti-rp35Δ3, anti-rp35Δ6, anti-rp35Δ12, and anti-rp35Δfuse sera with the corresponding immunogen; (b) indirect ELISA evaluating binding of pooled anti-rp35Δ sera with rp35Δ, rp35Δ3, rp35Δ6, rp35Δ12, and rp35Δfuse; (c) indirect ELISA evaluating binding of pooled anti-rp35Δ3, anti-rp35Δ6, anti-rp35Δ12, and anti-rp35Δfuse with rp35Δ. Tick-borne encephalitis trx-NS1 was used as a control of nonspecific binding. Absorbance was measured at 450 nm.

Figure 3

In vitro neutralization of VACV with serial dilutions of pooled anti-rp35Δ, anti-rp35Δ3, anti-rp35Δ6, anti-rp35Δ12, anti-rp35Δfuse, and anti-rp35Δfuse4 sera samples. The initial dilution of sera was 1:20. VACV was diluted to 320 pfu/mL. The data from three or more independent experiments are shown.

Figure 4

Characterization of pooled anti-rp35Δ sera after depletion. (a) Indirect ELISA evaluating binding of depleted anti-rp35Δ sera with the corresponding depletion antigen; (b) in vitro neutralization of VACV with serial dilutions of pooled anti-rp35Δ sera depleted with rp35Δ, rp35Δ3, rp35Δ6, rp35Δ12, and rp35Δfuse. The initial dilution of sera was 1:20. VACV was diluted to 320 pfu/mL. The data from three or more independent experiments are shown.

Figure 5

Alignment of rp35Δfuse mutant variants.

Figure 6

Characterization of mutated variants of rp35Δfuse. Western blot analysis of purified mutated variants of rp35Δfuse, which were fractionated by SDS-PAGE (12.5%) and developed with neutralizing pooled human sera of volunteers vaccinated with VACV (diluted to 1:200).