We initiated the current study with the simple goal of generating some monoclonal antibodies (mAbs) against VACV as reagents for studying VACV biology. In the end, 66 mAbs against 11 different VACV antigens were developed from a single immunized mouse. To our knowledge, it is the first time since 1980s that such a large number of mAbs against such a diverse group of VACV proteins have been reported. Several reports published decades ago had described a large number of mAbs against VACV (Ichihashi and Oie, 1988
; Wilton et al., 1986
). However, the antigenic targets of these mAbs were largely unknown, and the mAbs are no longer available. Ten years ago, a panel of VACV-specific mAbs were generated by Alan Schmaljohn’s group (Hooper et al., 2000
), but, unfortunately, they have never been fully described in a publication. Nevertheless, some of the mAbs, including a neutralizing antibody against L1, were made available to a few groups and have been very useful in a number of studies (Lustig et al., 2005
). More recently, multiple mAbs against recombinant L1 and B5 proteins were generated from recombinant proteins by Cohen, Eisenberg and co-workers (Aldaz-Carroll et al., 2005a
; Aldaz-Carroll et al., 2005b
), and these mAbs have been very usefully in studies of MV and EV neutralization. Here, we report for the first time mAbs against A14, A10 and D13, which are all proteins playing critical role in VACV morphogenesis. The mAbs we generated in this study target nonstructural proteins (WR148, D13L, C3) as well as structural proteins in the virion core (A10) or on the membranes of MV (D8, H3, A14) or EV (B5, A33, F13, A56). Collectively, they make up an excellent molecular “toolkit” for studying the life cycle of VACV.
We are pleasantly surprised with the number and the diversity of the antibodies that we obtained from a single immunized mouse. This success could perhaps be attributed to our immunization and screening methodology. The mouse was initially infected with a live attenuated VACV, which elicited an immune response that was strong enough to protect against a subsequent high dose VACV challenge. The mouse was finally given an intravenous dose of UV-inactivated VACV three days before the harvest of the spleen. This immunization scheme resulted in high titer of serum antibody against VACV and ultimately led to a large number of hybridomas that are specific for VACV. For screening the hybridomas, we performed immunofluorescence assay of cells infected with VACV at low MOI. This method was more labor-intensive than other commonly used methods such as ELISA, but we found it to be highly specific and very sensitive. In our hands, ELISA with infected cell lysate has a high percentage of false positive, while ELISA with purified virus appears to only detect antibody against major virion membrane proteins. In contrast, immunofluorescence of infected and uninfected cells in the same slide unambiguously identified hybridomas that are specific for VACV. In addition, cellular localization of the target proteins showed by immunofluorescence aided subsequent target identification.
Although the immunization protocol that we used here differed from the practice of smallpox vaccination, the spectrum of mAbs that we generated matches nicely with the profile of polyclonal antibody response to smallpox vaccination (Davies et al., 2005a
; Davies et al., 2007
). A proteome array of recombinant VACV proteins consistently detected polyclonal antibodies to around 25 VACV proteins (Davies et al., 2007
), against 10 of which we have found hybridomas in this study. For some VACV proteins that elicited strong polyclonal antibody responses, including D8, A14, WR148 and D13, we found multiple clones of hybridomas against the same protein. Therefore, the spectrum of the hybridomas, which was generated and detected with native VACV proteins, largely supports the polyclonal antibody profiling done with recombinant proteins produced with a prokaryotic system. There are some major targets of polyclonal antibodies, against which we did not find any hybridoma in this study. This may be due partly to inherent limits in hybridoma generation and partly to differences in antibody response to VACV in individual hosts. It was shown that smallpox vaccination in humans could result in different repertoire of antibody responses (Benhnia et al., 2008
). On the other hand, we found 2 hybridomas against C3, which was not detected by the proteomic array as a polyclonal antibody target. C3 is the VACV complement control protein (VCP), which is secreted by infected cells and inhibits both the classical and the alternative pathways of complement activation (Kotwal and Moss, 1988
). Recently, two studies showed that C3 is a target of vaccinia immune globulin (VIG) (Adamo et al., 2009
; Liszewski et al., 2009
), which was harvested from individuals vaccinated with smallpox vaccine. It is possible that the proteome array failed to detect antibody response to C3 because the C3 antibodies elicited by VACV only recognize native C3, which forms intramolecular disulfide bonds in eukaryotic cells (Liszewski et al., 2006
). In addition to the advantage of detecting additional antibody response to native VACV proteins, our generation of hybridomas resulted in monoclonal antibodies against a variety of VACV antigens, which could be used to map B cell epitopes in smallpox vaccine that contribute to immune protection.