Despite the eradication of naturally occurring smallpox, the licensed smallpox vaccine is still administered to military personnel and first responders due to the threat of bioterrorism
, as well as to individuals with potential exposure to monkeypox. In February 2008, the Centers for Diseases Control and Prevention (CDC) disposed of the last of its 12 million doses of Dryvax, the licensed first generation smallpox vaccine grown on the skin of calves. A new vaccine, ACAM2000, was licensed by the Food and Drug Administration in 2007 as a replacement. ACAM2000 is a replication-competent vaccinia virus clone derived from Dryvax and manufactured in large scale mammalian cell cultures. Efficacy was determined in a number of animal models and found to be non-inferior to Dryvax in eliciting an immunological response; however ACAM2000 has a similar safety profile when compared to Dryvax and introduces a level of risk for a small subset of individuals
. These complications may be severe and life-threatening. Severe adverse events following vaccination may include eczema vaccinatum (EV) in patients with atopic dermatitis and certain other skin conditions, and progressive vaccinia (PV) in immunocompromised patients
Vaccinia Immune Globulin Intravenous (Human) (VIGIV), a polyclonal antibody preparation manufactured from plasma of vaccinia-immunized donors, is the only licensed therapy for smallpox vaccine complications. While no placebo-controlled clinical trials were performed with the currently available VIGIV product, the use of similar products has historically decreased mortality, from 100% to 50% for PV, and from 30-40% to 3-4% for EV (reviewed in
). In severe cases very high repeated doses of VIGIV have been used and in the context of widespread vaccination, VIGIV supply could be limiting
[6, 8]. Enhancing the potency of licensed VIGIV is challenging in part because virus neutralizing assays for screening donor plasma are laborious, require live virus, and are subject to the variability typically encountered in biological assays. Binding assays to quantitate antibody levels are problematic in the absence of specific epitope binding information or in the context of polyclonal preparations that may contain a mixture of neutralizing and non-neutralizing antibodies, and are therefore typically supported by use of a plaque reduction neutralization assay
. Since immunogenicity is a critical consideration in vaccine development, structural understanding of critical viral protein epitopes would aid development of feasible assays capable of measuring important antibody specificities in donor plasma and VIGIV.
During the poxvirus infectious life cycle, approximately 1% of intracellular mature virions (IMV) are wrapped with additional membrane and exocytosed as extracellular enveloped virus (EEV) (reviewed in
). While IMV may mediate host-to-host transmission
[11, 12], EEV are thought to be uniquely responsible for rapid spread of virus in vivo and present an important antibody target. Antibody-mediated inhibition of EEV release from infected cells and blockade of EEV entry have been demonstrated
[13–15]. Passive immunization is more effective in polyclonal antibody preparations containing higher EEV antibody titers
, and anti-EEV monoclonals provide protection in a mouse vaccinia intranasal challenge model
. Vaccination with EEV proteins can also elicit a protective immune response
. Unfortunately, in immunized individuals anti-EEV titers vary considerably and may decline over time post-vaccination
[19, 20]. Anti-EEV antibody levels are also variable among different VIG products (M. Kennedy and R. Fisher, unpublished data) suggesting that potency gains might be realized by selecting plasma of donors with more robust responses to EEV neutralizing surface determinants. However, identification and characterization of EEV neutralizing determinants is still incomplete and assays to measure EEV neutralizing activity are subject to a high degree of variability.
The EEV envelope contains several viral proteins, including A56R
[21, 22], F13L
[23, 24], B5R
[13, 25], A36R
[27, 28], and A33R
. Among those, B5
 and A33
 proteins are known neutralization or viral spread inhibition targets associated with the EEV membrane and/or infected cells. The A33 protein appears to regulate EEV egress from cells and interacts with A36 to antagonize superinfection of neighboring cells, promoting more rapid long-distance dissemination
[32–34]. Antibodies such as MAb-1G10 directed against A33 block comet formation in vitro and can protect against poxvirus challenge in vivo in passive transfer models
MAb-1G10 was initially characterized as an A33-binding monoclonal antibody that could provide partial protection in vivo against an intranasal VACV-WR challenge in a mouse model, as well as block EV spread in cell culture
. Although a disconnect between protective efficacy and antibody affinity has been demonstrated for antibodies raised against A33
, A33 has been evaluated as part of an effort to identify epitopes which might be cross-protective against multiple pathogenic poxviruses
. This analysis showed that the β-mercaptoethanol sensitive MAb-1G10 epitope on vaccinia A33 was not present in the monkeypox A33 ortholog A35; the interpretation was that the MAb-1G10 binding epitope was conformational in nature. Binding of MAb-1G10 to the monkeypox A35 protein could be restored by single-residue exchanges at positions 117, 118, and 120 changing the monkeypox sequence to the vaccinia sequence. Based on this information, residues 117–120 were implicated as core residues forming the MAb-1G10 epitope. The importance of this region was reinforced by crystallographic data from a fragment of the ectodomain of A33 (residues 98–185)
. A dimeric, β-strand rich structural model of vaccinia A33 with structural similarity with C-type lectins was proposed. The described structure featured 5 β-strands and 2 α-helices stabilized by 2 intramolecular disulfide bonds (C100-C109 and C126-C180). Residues 117–120 were mapped to a surface-exposed edge on the proposed monomer structure, well removed from the dimer and proposed ligand-binding interfaces.
To provide additional characterization of the epitope involved in cell to cell spread of vaccinia, we considered whether additional residues might influence MAb-1G10 binding in the context of the vaccinia A33 protein. In this study, we screened a random peptide phage display library to find peptides specifically bound by MAb-1G10. A conformationally constrained consensus motif of seven residues was analyzed against available A33 sequence and structural information to generate an epitope model, which was tested and confirmed by an alanine site directed mutagenesis approach. The results demonstrated that the negatively charged D115 is required for MAb-1G10 binding, and helps establish the minimum epitope core for MAb-1G10 binding in the intact vaccinia A33 protein. Our data also confirm that residue L118 contributes to epitope formation, in agreement with previous observations. Our study shows that an unbiased mapping strategy utilizing random peptide display technology can effectively map linear and conformational epitopes involved in facilitating cell to cell spread of vaccinia. This work also expands understanding of an important orthopoxvirus epitope, which may be exploited to improve and inform therapies for vaccinia and potentially smallpox.