Accuri C6 Flow Cytometer System
Originally published online as doi:10.1189/jlb.1204722 on April 27, 2005

Published online before print April 27, 2005
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(Journal of Leukocyte Biology. 2005;78:401-411.)
© 2005 by Society for Leukocyte Biology

Enhancement of antigen acquisition by dendritic cells and MHC class II-restricted epitope presentation to CD4+ T cells using VP22 DNA vaccine vectors that promote intercellular spreading following initial transfection

Waithaka Mwangi*,1, Wendy C. Brown*, Gary A. Splitter{dagger}, Yan Zhuang*, Kimberly Kegerreis* and Guy H. Palmer*

* Department of Veterinary Microbiology and Pathology, Washington State University, Pullman; and
{dagger} Department of Animal Health & Biomedical Sciences, University of Wisconsin-Madison

1 Correspondence: Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040. E-mail: waitham{at}vetmed.wsu.edu


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ABSTRACT
 
Induction of immune responses against microbial antigens using DNA is an attractive strategy to mimic the immunity induced by live vaccines. Although DNA vaccines are efficacious in murine models, the requirement for multiple immunizations using high doses in outbred animals and humans has hindered deployment. This requirement is, in part, a result of poor vaccine spreading and suboptimal DC transfection efficiency. Incorporation of a signal that directs intercellular spreading of a DNA-encoded antigen is proposed to mimic live vaccine spreading and increase dendritic cell (DC) presentation. Bovine herpes virus 1 tegument protein, BVP22, is capable of trafficking to surrounding cells. To test the hypothesis that BVP22 enhances spreading and antigen presentation to CD4+ T cells, a DNA construct containing BVP22, fused in-frame to a sequence encoding a T cell epitope of Anaplasma marginale, was generated. A construct with reversed BVP22 sequence served as a negative control. Immunocytometric analysis of transfected primary keratinocytes, human embryonic kidney 293, COS-7, and Chinese hamster ovary cells showed that BVP22 enhanced intercellular spreading by ≥150-fold. Flow cytometric analysis of antigen-presenting cells (APCs) positively selected from cocultures of transfected cells and APCs showed that 5% of test APCs were antigen-positive, compared with 0.6% of control APCs. Antigen-specific CD4+ T cell proliferation demonstrated that BVP22 enhanced DC antigen presentation by ≥20-fold. This first report of the ability of BVP22 to increase DNA-encoded antigen acquisition by DCs and macrophages, with subsequent enhancement of major histocompatibility complex class II-restricted CD4+ T cell responses, supports incorporating a spreading motif in a DNA vaccine to target CD4+ T cell-dependent immunity in outbred animals.

Key Words: primary keratinocytes • tegument protein • trafficking • bovine herpes virus


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INTRODUCTION
 
Induction of antigen-specific effector and memory immune responses against microbial antigens using DNA vaccines is a contemporary strategy that combines vaccine stability, cost effectiveness, and safety [1 , 2 ]. Host cell transfection by a DNA vaccine results in de novo expression of the encoded antigen [3 ], which subsequently induces major histocompatibility complex (MHC) class I [4 ]- and class II [5 ]-restricted lymphocyte as well as antibody responses [6 ]. Additional signals can be incorporated into DNA vaccines to direct expressed antigen to a specific processing and presentation pathway [7 ] or target antigen to a specific receptor [8 ] and thus, induce a desired immune response [9 ]. In addition, DNA vaccine vector modification such as inclusion of stimulatory CpG motifs [10 ] or cytokine gene coexpression [11 , 12 ] with the vaccine antigen enhances early stages of antigen processing, presentation, and priming of antigen-specific B and T lymphocytes. However, despite promising results achieved in inbred murine models [13 ], widespread deployment of DNA vaccines for induction of protective immunity in humans and other outbred animal species, the actual populations to be protected, has not been achieved [4 ]. Effective induction of immunity has required multiple immunizations using high vaccine doses, negating the attractive features of DNA vaccination [4 , 14 ]. This requirement is, in part, a result of insufficient antigen expression [15 ], poor vaccine spreading [16 ], and suboptimal dendritic cell (DC) transfection efficiency [17 , 18 ]. Cellular uptake and intracellular expression of DNA vaccine vectors allow prolonged antigen expression [3 ] and thus, mimic a live vaccine. However, unlike live vectors, DNA vaccines lack the ability to amplify and spread intercellularly at the inoculation site. Recombinant bacterial and viral vaccine vectors such as Listeria [19 ] and poxviruses [20 ] are more efficacious antigen-delivery vehicles than DNA vaccines, at least in part, as they replicate and spread in vivo and thus, increase the availability of antigens to DCs [21 ]. Transfection efficiency of DCs in vivo by DNA vaccines is poor, and it is estimated that only 0.4% of DCs is transfected [16 17 18 , 22 ]. As somatic cells are the main transfectants following DNA vaccination [16 , 18 , 22 23 24 ], incorporation of a motif that directs active intercellular spreading of DNA-encoded antigen would mimic live vaccine in vivo spreading and may significantly increase the number of DCs processing and presenting antigen [25 , 26 ].

Herpes virus contains a tegument protein, designated VP22, which has a remarkable property of intercellular trafficking and is capable of distributing protein to surrounding cells [27 ]. Intercellular transport of a VP22 fusion protein has been demonstrated after endogenous synthesis and may thus have potential in enhancing DNA vaccine efficacy in outbred animals [27 , 28 ]. As DNA vaccine-encoded antigens are synthesized de novo within the transfected cell [3 ], the antigens are preferentially processed by proteosomes and presented mainly by MHC class I molecules to CD8+ T cells [4 , 11 , 12 , 17 , 18 , 25 , 29 30 31 ]. However, somatic cells, the main transfectants following DNA vaccination [22 ], lack expression of MHC class II molecules [32 ], and the DNA-encoded antigen is poorly released from the transfected somatic cells [29 ] for uptake by DCs. Thus, DNA vaccines tend to induce suboptimal CD4+ lymphocyte responses [14 , 18 ]. Priming and expansion of MHC class II-restricted CD4+ T cells and their recall upon re-exposure to antigen are required for vaccine efficacy. CD4+ T cells are necessary for generation of high-affinity neutralizing antibody, immunoglobulin (Ig) class switching, stimulation of macrophage (M{phi}) killing of intracellular pathogens, and optimal expansion of CD8+ cytotoxic T lymphocytes [33 34 35 36 ]. Consequently, identifying DNA vaccine modifications that enhance MHC class II presentation and CD4+ T cell responses is fundamental in optimizing vaccine-induced immunity against a broad spectrum of microbial pathogens.

The goal of this study was to test whether incorporation of VP22 intercellular spreading motif in a DNA vaccine would enhance DNA-encoded antigen acquisition by antigen-presenting cells (APCs) and MHC class II-restricted antigen presentation to CD4+ T cells. The VP22 ortholog in bovine herpes virus 1, BVP22, was selected, as it is the most efficient intercellular spreading motif identified among the related herpes viruses [27 ]. This study was conducted in two parts. First, chimeric constructs containing the full-length bvp22 gene and bvp22 truncations were generated and tested to determine if specific domains were required for optimal intercellular spreading. Second, the ability of BVP22-mediated intercellular spreading to enhance DNA vaccine-encoded antigen acquisition by APCs and MHC class II-restricted presentation to T cells was evaluated using a defined CD4+ T cell epitope of Anaplasma marginale major surface protein (MSP)1a [37 38 39 40 ]. This conserved CD4+ T cell epitope, designated F2–5, is MHC class II DR-restricted, and responses to F2–5 are induced by immunization with native MSP1 protein antigen or DNA vaccine-encoded MSP1a in calves with diverse class II haplotypes [39 , 40 ]. In this manuscript, we report testing the hypothesis that BVP22-mediated intercellular spreading enhances APC acquisition of a DNA-encoded T cell epitope and MHC class II-restricted presentation to specific CD4+ T cells.


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MATERIALS AND METHODS
 
Plasmid construction and purification
To identify a construct that would mediate optimal intercellular spreading, chimeric DNA constructs, containing bvp22, linked in-frame at the 3' end to the gene encoding the MSP1a F2–5 T cell epitope, were generated in VR-1055 (Vical, San Diego, CA) eukaryotic expression vector [40 ]. The full-length BVP22 open-reading frame (encoding 258 amino acids) was polymerase chain reaction (PCR)-amplified from the cloned cDNA [27 ] using a forward primer that corresponds to the 5' sequence (bases 1–18) and a reverse primer that corresponds to the complementary sequence of the 3' end (bases 756–774) [27 ]. The forward primer incorporated an EcoRV restriction site at the 5' end, and the reverse primer incorporated a XbaI site at the 3' end of the PCR product, which was designated BVP22. The sequence encoding the CD4+ T cell epitope (MSP1a peptide F2–5, GGVSYNDGNASAARSVLETLAGHVDALGIS) [39 ] was PCR-amplified from the pVRMSP1aEDm construct [40 ] using a forward primer that corresponds to the DNA sequence encoding the first six residues of F2–5 (GGVSYN). The reverse primer corresponds to the complementary sequences of the codons encoding the last six residues of F2–5 (DALGIS) and FLAG-tag (DYKDDDDK) [41 ]. The forward primer introduced a XbaI site at the 5' end, and the reverse primer introduced a BglII site at the 3' end of the PCR product, which was designated msp1aF2–5flag. Following restriction enzyme digestion, bvp22 (EcoRV and XbaI) and msp1aF2–5flag (XbaI and BglII) fragments were subcloned into an EcoRV-BglII-cut pGCVII vector (5 Prime->3 Prime, Boulder, CO) to generate a bvp22msp1aF2–5flag chimera. This EcoRV-BglII fragment was then subcloned into an EcoRV-BamHI-cut VR-1055 eukaryotic expression vector to generate the Vp-BVP22MSP1F2–5FLAG construct, designated pBVP22. Similarly, a chimeric construct Vp-BVP22RMSP1F2–5FLAG, designated pBVP22R, in which the full-length bvp22 gene is reversed, was also generated to serve as a negative control for the intercellular spreading analysis. A second negative-control construct was generated by replacing the bvp22r gene with a gene encoding the N-terminal region (258 amino acids) of Babesia bovis rhoptry-associated protein 1 (RAP1) [42 ], and the resultant construct Vp-RAP1MSP1F2–5FLAG was designated pRAP1.

To evaluate the contribution to intercellular spreading by domains conserved among VP22 orthologs, BVP22 domains that play a role in intercellular spreading were mapped by generating bvp22msp1aF2–5flag chimeras containing 5'-end bvp22 truncations. The 5'-truncated bvp22msp1aF2–5flag chimeras were PCR-amplified from the pBVP22 chimeric construct using six forward primers corresponding to BVP22 amino acid: 61–67, 114–120, 141–147, 164–170, 197–202, and 229–235 [28 ]. Each forward primer introduced an EcoRV restriction site and incorporated a transcription initiation methionine codon. The F2–5FLAG reverse primer described above was used for PCR amplification of the six truncated chimeras. The resultant chimeric genes, designated bvp22.1–bvp22.6, were then subcloned into VR-1055. Similarly, controls were generated by reversing each truncated bvp22 gene, and the resultant chimeric genes, designated bvp22.1r–bvp22.6r, were also subcloned into VR-1055. After sequencing the chimeric constructs, positive clones as well as the empty VR-1055 vector were amplified in TOP 10 Escherichia coli cells (Invitrogen, Carlsbad, CA), and endotoxin-free plasmid DNA was purified using the Endo-Free Plasmid Maxi-Prep kit (Qiagen, Valencia, CA).

Protein expression
Protein expression by the chimeric constructs described above was tested by immunocytometric analysis of transfected human embryonic kidney (HEK) 293-F cells (Invitrogen), as described previously [40 ]. The HEK 293-F cells transfected with the VR-1055 empty vector served as a negative control. Briefly, the transfected HEK 293-F monolayers were incubated with a 1/1000 dilution of a mouse anti-FLAG M2-alkaline phosphatase conjugate (Sigma-Aldrich, St. Louis, MO) in blocking buffer [1x phosphate-buffered saline (PBS) with 5% fetal bovine serum (FBS)]. Duplicate, transfected monolayers were reacted with an isotype control monoclonal antibody (mAb), and following washes in blocking buffer, the monolayers were incubated with a 1/1000 dilution of alkaline phosphatase-conjugated goat anti-mouse mAb (Tropix, Bedford, MA) in blocking buffer. Following washes in blocking buffer, the alkaline phosphatase activity was detected using Fast Red TR/Naphthol AS-MX substrate (Sigma-Aldrich). Stained cells were visualized and photographed using an inverted phase contrast microscope model CK-2 (Olympus Optical, Tokyo, Japan).

Intercellular spreading
The BVP22-mediated intercellular spreading was tested in 100-mm petri dishes of HEK 293-F, COS-7L, Chinese hamster ovary (CHO)-S, and primary human keratinocyte (all from Invitrogen) cell monolayers transfected with the DNA constructs described above. Cell monolayers were transfected with the DNA constructs for 6 h using Lipofectamine 2000 (Invitrogen) and washed three times with Opti-MEM (Invitrogen), and fresh growth medium was added. Spreading was evaluated at a single-cell transfectant level in cells transfected with 0.05 µg DNA followed by immunocytometric analysis using the mouse anti-FLAG M2-alkaline phosphatase conjugate (Sigma-Aldrich), and spreading was quantified by flow cytometry using a mouse anti-FLAG M2-fluorescein isothiocyanate (FITC) conjugate (Sigma-Aldrich) following intracellular staining of cells transfected with 0.2 µg DNA and permeabilized with 2% saponin. Prior to intracellular staining, Trypan blue analysis was used to determine cell viability and cell counts. The pBVP22 construct containing the full-length bvp22 gene was the positive control, whereas the pBVP22R construct, in which the full-length bvp22 gene is reversed, the pRAP1 construct, and the VR-1055 empty vector served as negative controls. Similarly, intercellular spreading by the truncated chimeric constructs and the respective controls was evaluated and quantified. The results of the intercellular spreading assay are presented as a percentage of FLAG-positive cells. To determine the efficiency of BVP22-mediated intercellular spreading, the percentage of FLAG-positive HEK 293-F cells transfected with 0.2 µg of the test chimeric construct was divided by the percentage of FLAG-positive HEK 293-F cells transfected with an equal amount of the respective control construct containing reversed bvp22. The intercellular spreading efficiency is presented as intercellular spreading index (ISI).

Temporal characteristics of BVP22-mediated intercellular spreading were analyzed at a single-cell transfectant level in 100-mm petri dishes of HEK 293-F cell monolayers transfected with 0.05 µg of the pBVP22 construct, and intercellular spreading was evaluated by immunocytochemistry at 24 h intervals for 6 days using the mouse anti-FLAG M2-alkaline phosphatase conjugate (Sigma-Aldrich). The pBVP22R and the pRAP1 constructs served as controls for the intercellular spreading, whereas the VR-1055 empty vector served as a negative control for protein expression.

Intercellular spreading from transfectants to APCs
Generation of bovine M{phi} and monocyte-derived DCs
Bovine M{phi} were generated from peripheral blood mononuclear cells (PBMCs) by plastic adhesion followed by culturing the cells for 6 days in complete medium consisting of RPMI 1640 (Invitrogen) supplemented with 25 mM HEPES (Invitrogen), 10% heat-inactivated FBS (Hyclone, Logan, UT), 2 mM GlutaMax (Invitrogen), 50 ug/mL gentamicin sulfate (Invitrogen), and 5 x 10–5 M 2-mercaptoethanol (Sigma-Aldrich). To generate bovine monocyte-derived DCs, PBMC-derived plastic adherent cells were cultured for 6 days in complete RPMI-1640 medium (Invitrogen) containing 100 ng/ml recombinant bovine granulocyte macrophage-colony stimulating factor (GM-CSF) [40 ], 200 ng/ml recombinant bovine interleukin-4 (IL-4; the gene was a gift from Dr. Volker T. Heussler) [43 ], and 200 ng/ml recombinant bovine fetal liver tyrosine kinase 3 ligand (FLT3L) [44 ]. After 3 days, fresh, complete RPMI-1640 medium and cytokines were added to the cells. The recombinant cytokines used in this protocol were expressed as FLAG-tagged proteins in HEK 293 Free-Style cells (Invitrogen) and affinity-purified using anti-FLAG M2-agarose beads (Sigma-Aldrich). Live DCs were visualized using a Nikon microscope model Eclipse E400, and images were captured using a Nikon Digital Sight DS-L1 apparatus (Nikon Corp., Tokyo, Japan). The phenotype of the DCs was determined by two-color flow cytometry using mAb specific for bovine DEC-205 (CC98) [45 ], MHC class II (TH14B), CD14 (CAM36A), CD21 (BAQ44), and CD172A (DH59B), purchased from the Washington State University Monoclonal Antibody Center (Pullman). Surface expression of CD80/CD86 was detected using a bovine cytotoxic T-lymphocyte antigen-4FLAG fusion protein (expressed and purified as above).

Intercellular spreading to APCs
To test whether BVP22 directs intercellular spreading from transfected cells to nontransfected APCs, HEK 293-F cells were transfected with 0.2 µg of the pBVP22 construct or the pBVP22R control construct, and the empty VR-1055 vector served as a negative control. One day post-transfection, the HEK 293-F cell transfectants were harvested, washed with PBS, and then mixed, at a ratio of 3:1, with M{phi} or monocyte-derived DCs at the fifth day of differentiation. The cell mixture was plated on poly-D-lysine (Sigma-Aldrich)-coated, 100-mm petri dishes and left overnight to establish a mixed cell monolayer with maximum cell-to-cell contact. The M{phi} in the cell mixture were labeled with the mouse anti-bovine CD14 mAb CAM36A, whereas the monocyte-derived DCs were labeled with the mouse anti-bovine DEC-205 mAb CC98, followed by positive selection using goat anti-mouse IgG microbeads (Miltenyi Biotec, Auburn, CA). Intercellular spreading from HEK 293-F cell transfectants to nontransfected APCs was quantified by two-color flow cytometry using the mouse anti-FLAG M2-FITC conjugate (Sigma-Aldrich) following intracellular staining for the FLAG epitope and surface staining for the CD14 or the DEC-205 marker. The surface-stained CD14 and DEC-205 markers were detected using phycoerythrin-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA). The results are presented as a percentage of FLAG-positive APCs.

Enhanced M{phi} and DC MHC class II presentation
The influence of BVP22-mediated intercellular spreading on MHC class II-restricted MSP1a F2–5 epitope presentation was tested by proliferation assays using M{phi} or monocyte-derived DCs, positively selected from the HEK 293-F cell transfectant-APC cocultures described above. Irradiated (3000 rad) M{phi} (5x102, 5x103, or 104 cells per well) were cultured in triplicate wells with 2 x 104 MSP1a F2–5 epitope-specific, short-term CD4+ T cells generated from MSP1-immunized cow number 87 [39 ]. Monocyte-derived DCs were similarly cultured but with a MSP1a F2–5 epitope-specific CD4+ T cell clone 87.2.4E8, as described previously [39 ]. The positive control was APCs (5x102, 5x103, or 104 cells per well) pulsed with an optimal dose of MSP1a F2–5 peptide (0.1 ug/ml) [39 ], whereas APCs similarly pulsed but with an irrelevant peptide, MSP2-P1 [46 ], served as a negative control for the 3-day assay. Cells were radiolabeled for the last 18 h of culture with 0.25 µCi 3H-thymidine, harvested using an automated cell harvester (Tomtec, Orange, CT), and counted with a liquid scintillation counter. Results are presented as the stimulation index (SI), which represents the mean counts per minute (cpm) of triplicate cultures of CD4+ T cells plus antigen-containing APCs, divided by the mean cpm of triplicate cultures of cells plus medium. The significance of the differences in proliferation of the CD4+ T cells from the antigen-treated and control APCs was analyzed by Student’s t-test using cpm values. A value of P < 0.05 was considered significant.


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RESULTS
 
Plasmid constructs
A chimeric construct, designated pBVP22, containing full-length bvp22 and the sequences encoding the MSP1a F2–5 CD4+ T cell epitope [39 ] and a FLAG-tag [41 ], was generated in VR-1055 eukaryotic expression vector (Fig. 1A ). Similarly, a chimeric construct, designated pBVP22R, in which the bvp22 gene is reversed, was also generated to serve as a negative control for the intercellular spreading analysis (Fig. 1B) . The reversed bvp22 sequence did not contain transcription-termination codons. To control for the possible existence of a cryptic intercellular spreading signal in the reversed bvp22 sequence, a second negative-control construct was generated by replacing bvp22r with a sequence encoding B. bovis RAP1 [42 ]. This construct was designated pRAP1 (Fig. 1C) . To evaluate the contribution of domains conserved among VP22 orthologs to intercellular spreading, six chimeric constructs, containing 5'-end bvp22 truncations were generated. Selection of truncation sites was based on existence of potential conserved domains among VP22 orthologs (Fig. 2 ), and the resultant constructs were designated pBVP22.1–pBVP22.6 (Fig. 3 ). Similarly, controls were generated by reversing each truncated bvp22 sequence, and the resultant chimeric constructs were designated pBVP22.1R–pBVP22.6R.



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  		Figure 1. Generation of MSP1TF2–5FLAG chimeric constructs. (A) A chimeric DNA construct (pBVP22), containing a full-length bvp22 sequence fused in-frame to a sequence encoding a defined CD4+ T cell epitope (F2–5) of A. marginale MSP1a and a FLAG-tag, was generated. (B) A construct (pBVP22R), generated similarly but with reversed bvp22 sequence, and (C) a construct (pRAP1), containing a gene encoding an irrelevant sequence, B. bovis rap1, served as negative controls for intercellular spreading. Empty vector served as a negative control for protein expression. hCMVP, Human cytomegalovirus promoter.



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  		Figure 2. Determination of potentially conserved domains in VP22 orthologs. Amino acid alignment of VP22 orthologs, bovine (BVP22), human (HVP22), Turkey (TVP22), and Marek’s (MVP22) herpes viruses revealed conserved domains, designated 1–5. Each domain is overlined with a dotted line with an arrowhead as the domain end. Definition of these potentially conserved domains allowed selection of truncation sites (22.1–22.6) for evaluating the contribution of each domain to intercellular spreading.



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  		Figure 3. Evaluation of the contribution of domains conserved among VP22 orthologs to intercellular spreading. The contribution of each domain conserved among VP22 orthologs to intercellular spreading was determined using chimeric constructs containing 5'-end bvp22 truncations (bvp22.1–bvp22.6) generated by PCR. A control for each chimera was generated by reversing the truncated bvp22 fragment (bvp22.1r–bvp22.6r), and the empty vector served as a negative control for antigen expression. Efficiency of intercellular spreading, mediated by the truncated bvp22 chimeras, presented as ISIa, was determined by dividing the percentageb of FLAG-positive HEK 293-F cells transfected with 0.2 µg of each truncated bvp22 chimeric construct (bvp22.1–bvp22.6) with the percentagec of FLAG-positive cells transfected with an equal amount of the respective control construct containing the truncated sequence in reverse orientation (bvp22.1r–bvp22.6r). The pBVP22 chimeric construct containing the full-length bvp22 gene served as a positive control, whereas the pBVP22R construct containing the full-length bvp22 gene in reverse orientation served as a negative control.

Plasmid DNA expression of BVP22 chimeras
Expression of BVP22 chimeric proteins by the constructs described above was tested by immunocytometric analysis of transfected HEK 293-F cells, and cells transfected with the VR-1055 empty vector served as a negative control. Expression was detected in the pBVP22, pBVP22R, and pRAP1 HEK 293 cell transfectants but not the empty vector transfectants (Fig. 4A and 4B ). Expression was also detected in HEK 293 cells transfected with the pBVP22.1–pBVP22.6 and the pBVP22.1R–pBVP22.6R constructs (data not shown).



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  		Figure 4. Evaluation of BVP22-mediated intercellular spreading. Monolayers of HEK 293-F cell transfectants were evaluated for BVP22-mediated intercellular spreading by in situ immunocytochemistry using a mouse anti-FLAG alkaline phosphatase conjugate. (i) Cells transfected with the pBVP22 construct; (ii) cells transfected with the pBVP22R construct; (iii) cells transfected with the pRAP1 construct; and (iv) cells transfected with the empty vector. (A) Intercellular spreading was evaluated at a single, transfected cell level in cells transfected with 0.05 ug DNA. (B) Intercellular spreading efficiency was evaluated in cells transfected with 0.2 ug DNA. (C) Intercellular spreading in cells transfected with 0.2 ug DNA was quantified by flow cytometry following intracellular staining for the FLAG-tag using mouse anti-FLAG-FITC conjugate and presented as a percentage of FLAG-positive cells. (D) Intercellular spreading in cells transfected with 0.2 ug pBVP22 was compared with spreading in cells transfected with 30 ug pBVP22R, pRAP1, and empty vector, respectively (ii–iv). (E) Intercellular spreading in D was quantified by flow cytometry, as described in C. aNB: To evaluate intercellular spreading at a single-cell level, 100 mm petri dishes of HEK 293 cells were transfected with 0.05 ug DNA to achieve areas of a monolayer with single-cell transfectants as shown.

Intercellular spreading
At a single-cell transfectant level, immunocytochemical detection using a mouse anti-FLAG M2 mAb revealed that BVP22-mediated intercellular antigen spreading in HEK 293-F cells (Fig. 4Ai) , primary human keratinocytes (see Fig. 5A ), COS-7L, and CHO-S cells (data not shown) transfected with 0.05 µg of the pBVP22 construct but not the control constructs (Figs. 4Aii and 4Aiii and 5B ). In a preliminary DNA titration study, 0.05 µg pBVP22 construct had been found to generate single-cell transfectants in a 100-mm petri dish of HEK 293-F cell monolayer. No protein expression was detected in duplicate pBVP22 transfectants reacted with an isotype control mAb (data not shown). In addition, there was no expression in nontransfected cells or empty VR-1055 transfectants when reacted identically with the anti-FLAG M2 mAb [Figs. 4A and 4Biv and 5C ]. Intense FLAG staining was detected in pBVP22 transfectants as compared with neighboring cells that acquired antigen through intercellular spreading (Fig. 4Ai) . This pattern was evident by flow cytometric analysis, whereby a small fraction of FLAG-positive cells stained intensely, but the majority of FLAG-positive cells stained less intensely (Fig. 4Ci) . Trypan blue analysis revealed that cells transfected with the VP22 constructs did not have significantly higher numbers of dead cells as compared with controls. Intracellular staining for the FLAG epitope in 0.2 ug DNA transfectants using a mouse anti-FLAG M2-FITC conjugate followed by flow cytometric analysis revealed >98% FLAG-positive cells in pBVP22 HEK 293-F cell transfectants (Fig. 4Ci) as compared with 1.5% and 2.9% in pBVP22R and pRAP1 HEK 293-F cell transfectants, respectively (Fig. 4Cii and 4Ciii) . Increasing the amount of pBVP22R or pRAP1 DNA used to transfect HEK 293-F cells incrementally increased the number of FLAG-positive cells; however, even with a 150-fold increase in DNA (0.2–30 ug), the pBVP22R or pRAP1 DNA could only generate 30% FLAG-positive cells (Fig. 4D and 4E) . Transfection with >30 ug pBVP22R or pRAP1 DNA was toxic (data not shown). Thus, compared with the 0.2 ug pBVP22 construct that yielded >98% FLAG-positive cells (Fig. 4Ci) , results from the HEK 293-F cells, transfected identically but with 30 ug pBVP22R or the pRAP1 DNA, indicate that BVP22 enhanced antigen spreading to neighboring cells by >150-fold.



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  		Figure 5. Evaluation of BVP22-mediated intercellular spreading in primary keratinocytes. Monolayers of primary human keratinocyte transfectants were evaluated for BVP22-mediated intercellular spreading using the mouse anti-FLAG alkaline phosphatase conjugate by in situ immunocytochemistry. (A) Primary human keratinocytes transfected with the pBVP22 construct; (B) primary human keratinocytes transfected with the pBVP22R construct; and (C) primary human keratinocytes transfected with the empty vector.

Transfection using 0.2 ug truncated constructs pBVP22.1 and pBVP22.2 revealed 47.5% and 50.5% FLAG-positive cells in HEK 293-F cells, respectively, as compared with 1.4% and 2.1% using the reversed sequence controls, pBVP22.1R and pBVP22.2R (Fig. 3) . However, minimal or no intercellular spreading was detected in HEK 293-F cells transfected with the pBVP22.3-to-pBVP22.6 chimeric constructs as compared with each reversed sequence control (Fig. 3) . These data demonstrated that deletion of the first domain conserved among BVP22 orthologs reduced intercellular spreading by ~50% and remained at this level upon deletion of the second conserved domain (Fig. 2) . However, deletion of the third conserved domain diminished intercellular spreading to near-background levels (Fig. 3) . Exclusion of the fifth domain completely abrogated intercellular spreading (data not shown), indicating that the C-terminal region is required for BVP22 to mediate intercellular spreading.

The efficiency of BVP22-mediated intercellular spreading was determined using an ISI: The percentage of FLAG-positive HEK 293-F cells transfected with the test chimeric construct divided by the percentage of FLAG-positive HEK 293-F cells identically transfected with an equal amount of the respective control construct containing BVP22 in reverse orientation. The pBVP22 construct gave an ISI = 65.5 as compared with an ISI = 33.9 for the pBVP22.1 construct, and an ISI = 24.1 for the pBVP22.2 construct, respectively (Fig. 3) . However, all of the other bvp22-truncated constructs gave an ISI < 5 (Fig. 3) . These data showed that maximal intercellular spreading requires the N-terminal region as well as the C terminus. Consequently, the full-length bvp22 sequence in the pBVP22 (Vp-BVP22MSP1F2–5FLAG) construct was used in all subsequent experiments.

The kinetics of BVP22-mediated intercellular spreading was analyzed at a single-cell transfectant level in HEK 293-F cell monolayers transfected with the pBVP22 construct, whereas the pBVP22R and the pRAP1 constructs served as controls. Determination of when intercellular spreading starts and peaks was important for testing BVP22-mediated intercellular spreading from transfectants to nontransfected APCs. At a single-cell transfectant level, immunocytometric analysis revealed that intercellular spreading was detectable 24 h post-transfection and peaked at 48–72 h post-transfection in pBVP22 but not pBVP22R transfectants (Fig. 6 ). The peak intercellular spreading remained steady, and no significant differences were observed at 96, 120, and 144 h post-transfection (data not shown).



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  		Figure 6. Kinetics of BVP22-mediated intercellular spreading. BVP22-mediated intercellular spreading was determined at 24 h intervals by immunocytochemistry, as described in Materials and Methods. (A) Empty vector HEK 293-F cell transfectants after 24 h; (B–D) pBVP22 HEK 293-F cell transfectants after 24 h, 48 h, or 72 h post-transfection, respectively. (E) pBVP22 HEK 293-F cell transfectants probed with an isotype control mAb 24 h post-transfection. (F–H) pBVP22R HEK 293-F cell transfectants after 24 h, 48 h, or 72 h post-transfection, respectively.

Intercellular spreading from transfectants to nontransfected APCs
Bovine monocyte-derived DCs and M{phi}
Bovine monocyte-derived DCs were generated using PBMC-derived plastic adherent cells cultured for 6 days in complete RPMI medium. The purity of the DCs, generated using recombinant bovine GM-CSF, IL-4, and FLT3L, was shown to be >90% by flow cytometry and by morphological analysis (Fig. 7 ). The DCs grow in suspension and are MHC class II+, DEC-205+, CD80+/CD86+, CD172Alow, CD14–, and CD21– (Fig. 7) . The M{phi} grow as adherent cells and are CD14+, DEC-205–, and CD172Ahigh (data not shown). Intercellular spreading from transfected cells to nontransfected DCs or M{phi} was quantified by two-color flow cytometry following surface staining for an APC-specific marker and intracellular staining for the FLAG epitope in nontransfected APCs, positively selected from pBVP22- or pBVP22R-transfected cell-APC monolayer cocultures. Flow cytometric analysis showed that >5% of APCs from the pBVP22-transfected HEK 293 cell-APC cocultures were FLAG epitope-positive (Fig. 8A ) compared with 0.61% of APCs from the pBVP22R-transfected HEK 293 cell-APC cocultures (Fig. 8B) . Background staining was 0.47% (Fig. 8C) .



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  		Figure 7. Morphology and phenotype of monocyte-derived DCs, which were generated using bovine GM-CSF, bovine IL-4, and bovine FLT3L, as described in Materials and Methods. Live images of 6-day-cultured DCs were captured using a Nikon Digital Sight DS-L1 apparatus. The DCs were stained for bovine MHC class II, DEC-205, CD14, CD21, CD172A, CD80, and CD86, as described in Materials and Methods. The stained cells were analyzed by two-color flow cytometry.



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  		Figure 8. Evaluation of BVP22-mediated intercellular spreading from transfected cells to nontransfected APCs. Intercellular spreading from transfected cells to nontransfected APCs was quantified by two-color flow cytometry. MHC class II+ cells were gated and analyzed for the FLAG-epitope intracellular staining. M{phi} from (A) pBVP22-, (B) pBVP22R-, or (C) vector-transfected cell-M{phi} monolayer cocultures.

Enhanced M{phi} and DC MHC class II presentation of antigen
The ability of BVP22-mediated intercellular spreading to enhance MHC class II-restricted, DNA-encoded MSP1a F2–5 epitope presentation to specific CD4+ T cells by APCs was tested by proliferation assays using M{phi} or monocyte-derived DCs positively selected from the HEK 293-F cell transfectant-APC cocultures described above. APCs pulsed with the MSP1a F2–5 peptide were the positive control [39 ], whereas APCs pulsed with an irrelevant peptide, MSP2-P1 [46 ], served as a negative control. The M{phi} positively selected from pBVP22 HEK 293-F cell transfectants-M{phi} monolayer cocultures induced statistically significant (P<0.001), MSP1a F2–5-specific, CD4+ T cell-proliferative responses compared with MSP1a F2–5-specific, CD4+ T cell-proliferative responses induced by M{phi} from pBVP22R HEK 293-F cell transfectants-M{phi} monolayer cocultures (Fig. 9A ). Relevant to priming using DNA vaccines, DCs positively selected from pBVP22 HEK 293-F cell transfectants-DC monolayer cocultures also induced statistically significant (P<0.001), MSP1a F2–5-specific, CD4+ T cell-proliferative responses compared with MSP1a F2–5-specific, CD4+ T cell-proliferative responses induced by DCs from pBVP22R HEK 293-F cell transfectants-DC monolayer cocultures (Fig. 9B) . The MSP1a F2–5-specific, CD4+ T cell proliferation assays using 5 x 103 APCs showed that BVP22 enhanced M{phi} and DC MHC class II-restricted antigen presentation by 14- and 20-fold, respectively, as compared with the pBVP22R construct (Fig. 9A and 9B) . As compared with M{phi} pulsed with an optimal concentration of the MSP1a F2–5 peptide, the same number of M{phi} positively selected from pBVP22 HEK 293-F cell transfectant-APC monolayer cocultures induced statistically significant (P<0.01), MSP1a F2–5-specific, CD4+ T cell-proliferative responses (Fig. 10 ). Similar results were obtained using DCs as APCs (data not shown).



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  		Figure 9. BVP22-mediated intercellular spreading enhances antigen presentation to MHC class II-restricted CD4+ T cells. The effect of BVP22-mediated intercellular spreading on MHC class II-restricted MSP1a F2–5 epitope presentation to an F2–5-specific, short-term CD4+ T cell line from cow 87 or CD4+ T cell clone 87.2.4E8 was tested by proliferation assays using M{phi} (A) or DCs (B), respectively, positively selected from overnight monolayer cocultures of transfected cells and nontransfected APCs. Results are presented as the SI. The significance of the differences in proliferation (cpm) of the CD4+ T cells from the antigen-treated and control APCs was analyzed by Student’s t-test. Compared with the nonspreading control, the BVP22-mediated intercellular spreading induced statistically significant (P<0.001), MSP1a F2–5-specific, CD4+ T cell-proliferative responses by the M{phi} and DCs. Background cpm of cells cultured with medium ranged from 48.76 to 50.



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  		Figure 10. Intercellular spreading mediates more efficient MHC class II-restricted antigen presentation to CD4+ T cells than peptide pulsing. MHC class II-restricted MSP1a F2–5 epitope presentation to F2–5-specific CD4+ T cells by 5 x 103 APCs positively selected from overnight monolayer cocultures of pBVP22-transfected cells and nontransfected APCs was compared with MHC class II-restricted presentation of the same epitope to the same CD4+ T cells by an equivalent number of APCs pulsed with 0.1 ug/ml F2–5 peptide by proliferation assay. Results are presented as mean cpm of [3H] thymidine incorporation ± 1 SD. The significance of the differences in proliferation of the CD4+ T cells was analyzed by Student’s t-test. Compared with the APCs pulsed with the F2–5 peptide, the BVP22-mediated intercellular spreading induced statistically significant (P<0.01), MSP1a F2–5-specific, CD4+ T cell-proliferative responses.


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DISCUSSION
 
This study has shown for the first time that BVP22 mediated significant intercellular spreading of a defined CD4+ T cell epitope from transfected cells to cocultured, but nontransfected, APCs. This observation is consistent with another report demonstrating that immunofluorescence analysis of COS-1 cells microinjected with a plasmid encoding VP22 revealed intercellular spreading of VP22 [28 ]. BVP22-mediated intercellular spreading significantly enhanced acquisition and presentation of a MHC class II-restricted T cell epitope by M{phi} and DCs to epitope-specific CD4+ T cells. The MSP1a F2–5-specific CD4+ T cell proliferation assays using positively selected APCs showed that BVP22 enhanced DC and M{phi} MHC class II-restricted antigen presentation by 20- and 14-fold, respectively. Furthermore, APCs positively selected from pBVP22 HEK 293-F cell transfectant-APC monolayer cocultures induced statistically significant proliferation of the MSP1a F2–5-specific CD4+ T cells than APCs pulsed with an optimal concentration of the MSP1a F2–5 peptide. This strongly supports accepting the hypothesis that BVP22-mediated intercellular spreading enhances DNA-encoded antigen acquisition by APCs and MHC class II-restricted presentation to CD4+ T cells, as exogenous antigens, represented by the F2–5 peptide, are presented by MHC class II molecules with high efficiency [47 ].

DNA-encoded BVP22 significantly enhanced intercellular spreading of the MSP1a F2–5 CD4+ T cell epitope in primary keratinocyte monolayers, and cytoplasmic localization of the spread antigen was clearly observable. Following intradermal (i.d.) DNA vaccine inoculation, keratinocytes are the main transfected cell type that persistently expresses the encoded antigen, and the expressed antigen has to be released for cross-priming by DCs to occur [22 ]. Thus, our demonstration that BVP22 significantly enhanced intercellular spreading of a defined CD4+ T cell epitope in keratinocytes suggests that BVP22-mediated intercellular spreading could significantly increase the number of dermal DCs and Langerhan’s cells acquiring antigen following i.d. DNA vaccine immunization. BVP22 also enhanced intercellular spreading by ≥150-fold in HEK 293-F, COS-7L, and CHO-S cells, indicating that BVP22-mediated spreading is not tightly restricted by cell type. These observations are consistent with other reports demonstrating that BVP22 orthologs, human herpes virus VP22 and Marek’s disease virus VP22, can direct intercellular spreading of chimeric proteins in many different cells and tissues [25 , 28 , 48 , 49 ]. This suggests that DNA-encoded VP22 can mediate significant intercellular spreading of chimeric antigens following immunization using diverse routes and in different species.

Intercellular spreading is conserved among herpes virus VP22 orthologs, and experiments were conducted to determine the contribution of domains conserved among VP22 orthologs to intercellular spreading. Deletion of the first and second conserved domains located at the N terminus of BVP22 reduced intercellular spreading by 50%, whereas deletion of the third or fourth domain located in the C-terminal half of the BVP22 completely abrogated intercellular spreading. Although this is consistent with a prior report that deletion of the C-terminal 34 residues in VP22 abrogated intercellular spreading [28 ], our results demonstrate that the BVP22-spreading motif is not solely contained within the C terminus. Instead, the spreading phenotype requires contributions from or interactions among domains extending throughout the protein. Thus, the full-length BVP22 protein mediates optimal intercellular spreading with the requirement of conserved domains throughout the protein.

VP22 is a cytoplasmic and nuclear protein that lacks a signal sequence [28 , 50 ]. In mixed cultures, untreated Vero cells acquire mRNA encoding green fluorescent protein (GFP) from HEp-2 cells doubly transduced with the genes encoding VP22 and GFP [51 ]. Thus, within a VP22-expressing cell, the protein binds mRNA and efficiently transports it [51 ], via actin cytoskeleton but in a Golgi-independent mechanism [28 ], to adjacent, uninfected (or nontransfected) cells. In addition to the spread of VP22 protein, the transported mRNA is expressed endogenously in the cytoplasm [51 ]. This is consistent with favored proteosome processing for MHC class I presentation and is supported by the enhanced class I presentation and increase in CD8+ cytotoxic lymphocytes in mice immunized with a DNA vector linking VP22 to the E7 papillomavirus antigen [25 , 26 ]. The significant presentation by the MHC class II molecules in our study indicates that antigen fused to BVP22 traffics to the endocytic pathway. Whether this cross-presentation reflects direct or indirect targeting to the endocytic pathway is unknown.

BVP22-mediated intercellular trafficking was detectable as early as 24 h post-transfection, and significant spreading peaked at 72 h post-transfection with sustained expression for more than 6 days. This suggests that BVP22 is capable of mediating sustained and significant antigen spreading to APCs within or newly recruited into the transfected tissue. Thus, incorporation of the BVP22 intercellular spreading motif in a DNA vaccine is a strategy that may mimic live vaccine spreading and persistence at the immunization site. We have previously shown that i.d. inoculation of calves representing diverse MHC class II haplotypes with a DNA vector encoding bovine FLT3L and GM-CSF significantly increases DC recruitment to the i.d. immunization site [40 ]. Combining DC recruitment and BVP22-mediated intercellular trafficking is hypothesized to increase the number of DC that process and present DNA-encoded antigen and thus, enhance the ability of DNA vaccines to strongly prime and amplify CD4+ T cell-dependent, immune responses in outbred animals.


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ACKNOWLEDGEMENTS
 
This work was supported by U.S. Department of Agriculture-National Research Initiative Competitive Grants Program Grant 2004-35204-14206, BARD US-3315-02C, and National Institutes of Health Grant R01 GM060986. W. M. is supported by Immunology Training Program T32-AI07025. We thank Dr. Chris J. Howard (Institute of Animal Health, Compton, UK) for providing the bovine GM-CSF gene and anti-bovine DEC-205 mAb (CC98), Dr. Volker T. Heussler (University of Berne, Switzerland) for providing the bovine IL-4 gene, and Dr. Travis C. McGuire for critically reviewing the manuscript. We also thank Beverly Hunter for excellent technical assistance.

Received December 13, 2004; revised March 22, 2005; accepted April 4, 2005.


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