Diverse CD4 Response to Vaccinia Virus in Human is Revealed by Proteome-Wide T cell Profiling

Authors: Lichen Jing,1 D. Huw Davies,2 Tiana M. Chong,3 Sookhee Chun,2 Christopher L. McClurkan,3 Jay Huang,3 Brian T. Story,3 Douglas M. Molina,4 Siddiqua Hirst,2 Philip L. Felgner,2 and David M. Koelle1,2,5,6,7*
Department of Medicine, University of Washington, Seattle, Washington981021
Department of Medicine, University of California Irvine, Irvine, California 926972
Department of Laboratory Medicine, University of Washington, Seattle, Washington3
ImmPORT Therapeutics Inc., Irvine, California 926184
Benaroya Research Institute, Seattle, Washington5;
Fred Hutchinson Cancer Research Center, Seattle, Washington6; and 
Program in Pathobiology, University of Washington, Seattle, Washington7

Preventative infection with replication-competent vaccinia virus has been successfully used to eradiate smallpox transmission; however, zoonotic orthopoxviruses can still periodically emerge.1 Moreover, there remains a potential for use of smallpox as a bioweapon and vaccination with live vaccinia virus has complications.2 These issues, along with the use of vaccinia virus as a vector for vaccines and immunotherapy, have raised interest in further understanding of the immune response to wild type and replication-incompetent vaccinia virus.3

CD4 T cells have been shown to play an important role in the maintenance and recall of antiviral CD8 T cells and for antibody responses.1-3 Recently, the breadth of CD8 T cell responses to vaccine virus has been studied using various approaches in human and mice;4 however, much less is known about the overall architecture of the CD4 response to complex microbial pathogens. The goal of the current study was to define the breadth and populationdominant antigens of the vaccinia virusspecific CD4 response using a nonpredictive approach by expressing recombinant antigens covering the entire predicted vaccinia virus proteome.

Totally 180 predicted open reading frames (ORFs) in the vaccinia virus genome were expressed with Escherichia coli-based rapid translation system (RTS) and are therefore termed RTS proteins. These RTS proteins were tested using responder cells from 20 blood samples from 11 vaccinees. Validation assays confirmed that proteins expressed by in vitro transcription/translation allowed sensitive and specific detection of CD4 responses. For proliferative responses, the positivity cutoff was separately calculated for each assay data set containing the complete set of responses for each polyclonal lymphocyte line. Three most frequently recognized RTS proteins were further characterized at the peptide level.

Epitope mapping experiments were conducted using Sigma’s PEPscreen® custom peptide library sets covering a variety of ORFs or sub-ORF regions. For example, in Figure 1, peptides from two distinct regions of vaccinia ORF L4R were evaluated by ELISPOT. As expected, either a single peptide, or two adjacent and partially overlapping peptides, were antigenic in each region of L4R evaluated. Peptides were directly used as provided by the manufacturer to make a 10 mg/mL stock DMSO solution. Singlepeptide ELISPOT and ICC assays used a final concentration of 1 μg/ml. ELISPOT assays were performed as described previously.5 For ICC assays, negative and positive controls were UV-treated mock virus, medium, DMSO, and phorbol myristate acetate/ionomycin, respectively.6 Each example pursued to the peptide epitope level showed the CD4+ phenotype. Compared to CD8 T cells, CD4 T cells had far more efficient in vitro proliferative response by memory T cells to exogenous protein antigens. Furthermore, in similar epitope mapping work currently being prepared for submission to a peer-reviewed journal, the Koelle lab has used Sigma peptide sets covering additional vaccinia ORFs. These peptide sets have performed well, typically yielding one T cell reactive peptide within each ORF that had previously been identified as driving CD4 T cell responses.

Figure 1. Synthetic peptides confirm reactivities assigned using RTS reactions. Single peptides correspond to predicted antigenic regions of L4R. Data are means standard deviations of duplicate ELISPOT assay results.


Among all the samples tested, CD4 responses were detected for 122 ORFs (68%). A mean of 39 ORFs were recognized per person (range, 13 to 63). The most frequently recognized ORFS were present in virions, including A3L and A10L (core proteins), WR148 (a fragmented homolog of an orthopoxvirus protein that forms inclusions in cells), H3L (a membrane protein), D13L (a membrane scaffold protein), and L4R (a nucleic acid binding protein).

RTS proteins containing each vaccinia virus ORF were also printed onto nitrocellulose slides to prepare protein microarrays. Serum immunoglobulin G profiling by these microarrays detected responses to 45 ORFs among 180 ORFs expressed. These widespread responses confirmed recent studies showing a diverse response directed to membrane and nonmembrane antigens.

In summary, this study provides the first empirical whole-proteome data set regarding the global CD4 response to full-length proteins in a complex virus. Highly diverse responses to vaccinia virus, with a mean of 39 ORFs being recognized per subject, are consistent with the theory that abundant structural proteins are immunodominant.

The 70% homology of the entire gp160 sequence for the same strains. These data strongly support the high amount of crossclade reactivity previously observed in the recombinant vaccinia assays. The observation that a diverse protein sequence can nevertheless generate cross-reactive T cell responses is an important consideration in developing vaccines targeted towards a diverse strain of viruses.

Adapted by Wu Yao, Ph.D. (Sigma R&D Scientist) from J Virol. Jul; 82(14):7120-34. (2008).


  1. Resch, W., K. K. Hixson, R. J. Moore, et al., 2007. Virology 358:233-247.
  2. Halsell, J. S., J. R. Riddle, J. E. Atwood, et al., 2003. JAMA 289:3283-3289.
  3. Amanna, I. J., M. K. Slifka, and S. Crotty. 2006. Immunol. Rev. 211:320-337.
  4. Dong, Y., and T. N. Denny. 2006. J. Infect. Dis. 194:168-175.
  5. Koelle, D. M., Z. Liu, C. L. McClurkan, et al., 2003. PNAS 100:12899-12904.
  6. Jing, L., T. M. Chong, B. Byrd, et al., 2007. J. Immunol. 178:6374-6386.

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