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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Vaccine. Author manuscript; available in PMC 2011 February 23.
Published in final edited form as:
PMCID: PMC2830906

Engineering superior DNA vaccines: MHC class I single chain trimers bypass antigen processing and enhance the immune response to low affinity antigens


It is commonly believed that delivery of antigen into the class I antigen presentation pathway is a limiting factor in the clinical translation of DNA vaccines. This is of particular concern in the context of cancer vaccine development as many immunodominant peptides derived from self tumor antigens are not processed and presented efficiently. To address this limitation, we have engineered completely assembled peptide/MHC class I complexes whereby all three components (class I heavy chain, β2m, and peptide) are attached by flexible linkers and expressed as a single polypeptide (single chain trimers or SCT). In this study, we tested the efficacy of progressive generations of SCT DNA vaccines engineered to (1) enhance peptide binding, (2) enhance interaction with the CD8 coreceptor, and/or (3) activate CD4+ helper T cells. Disulfide trap SCT (dtSCT) have been engineered to improve peptide binding, with mutations designed to create a disulfide bond between the class I heavy chain and the peptide linker. dtSCT DNA vaccines dramatically enhance the immune response to model low affinity antigens as measured by ELISPOT analysis and tumor challenge. SCT engineered to enhance interaction with the CD8 coreceptor have a higher affinity for the TCR/CD8 complex, and are associated with more robust CD8+ T cell responses following vaccination. Finally, SCT constructs that coexpress a universal helper epitope PADRE, dramatically enhance CD8+ T cell responses. Taken together, our data demonstrate that dtSCT DNA vaccines coexpressing a universal CD4 epitope are highly effective in generating immune responses to poorly processed and presented cancer antigens.


The observation that direct administration of recombinant DNA can generate potent immune responses in rodents established the field of DNA vaccines in the early 1990s (Tang et al., 1992; Cox et al., 1993; Davis et al., 1993; Fynan et al., 1993; Ulmer et al., 1993; Wang et al., 1993). Since then, DNA vaccines have remained an area of intense research interest, and vaccines targeting infectious disease and cancer have progressed into clinical trials. Advantages of the DNA vaccine platform include the remarkable safety profile of DNA vaccines, and ease of manufacture relative to proteins and other biologics (Donnelly et al., 1997; Gurunathan et al., 2000). Perhaps most important, however, is the flexibility and molecular precision of the platform, with the ability to genetically manipulate the encoded antigens, and/or incorporate other genes to amplify the immune response (Kutzler and Weiner, 2008). For instance DNA vaccines have been engineered to improve antigen expression (Lee et al., 1997; Andre et al., 1998), target dendritic cells (Trumpfheller et al., 2006; Nchinda et al., 2008), and/or coexpress molecular adjuvants capable of enhancing immune responses such as costimulatory molecules (Chan et al., 2001), cytokines (Boyer et al., 2005; Schadeck et al., 2006; Chong et al., 2007; Hirao et al., 2008), or chemokines (Sumida et al., 2004). Unfortunately, despite the dramatic preclinical success of DNA vaccines, and greater than 200 ongoing or completed human clinical trials, immune responses in non-human primates and humans have been disappointing (Calarota et al., 1998; MacGregor et al., 1998; Wang et al., 1998), and no DNA vaccines have been approved for human use. One of the limitations of DNA vaccination is the requirement for intracellular processing and presentation of encoded antigens by MHC class I molecules through cross-presentation, a very inefficient process (Yewdell and Del Val, 2004). This is of particular concern in the context of cancer vaccine development as many immunodominant peptides derived from (self) tumor antigens are not presented efficiently at the plasma membrane (Chapatte et al., 2006). This inefficiency results from the fact that surface expression of peptide-MHC complexes is influenced by a variety of factors including efficiency of antigen processing (Del Val et al., 1991; Eisenlohr et al., 1992), specificity of peptide translocation into the ER (Heemels and Ploegh, 1995), and the biogenesis and kinetic stability of the peptide-MHC complex itself (Deng et al., 1997).

To address these limitations, we have engineered completely assembled peptide/MHC class I complexes, termed single chain trimers (SCT), whereby all three components of the complex (class I heavy chain, β2m, and peptide) are attached by flexible linkers and expressed as a single polypeptide (Yu et al., 2002). To date, over 30 different peptide-MHC allele combinations have been successfully constructed and validated, confirming the general applicability of this approach (Jaramillo et al., 2004; Crew et al., 2005; Huang et al., 2005; Primeau et al., 2005; Hung et al., 2007a; Lilienfeld et al., 2007; Zhang et al., 2007). Of particular note, we and others have demonstrated that first generation SCT DNA vaccines are capable of generating antigen-specific T cell responses in a number of model systems (Yu et al., 2002; Jaramillo et al., 2004; Huang et al., 2005; Hung et al., 2007a). In addition to the obvious clinical potential of SCT as DNA vaccines, these reagents have begun to provide unique and significant insights into our understanding of the complex role of MHC class I molecules in lymphocyte development and activation (Choudhuri et al., 2005; Hudrisier et al., 2005; Kim et al., 2005).

In this study, we tested the efficacy of various generations of SCT engineered to (1) enhance peptide binding, (2) enhance interaction with CD8 coreceptor, and (3) activate CD4+ helper T cells. Initial proof-of-principle experiments were performed using the ovalbumin antigen SIINFEKL (OVAp), and the known altered peptide ligand, SIINYEKL (p5Y), which binds poorly to H-2Kb (Howarth et al., 2004). Additional experiments were performed using SCT encoding a peptide from the human breast cancer antigen, mammaglobin-A (Watson and Fleming, 1996). Our data suggest that disulfide trap SCT DNA vaccines coexpressing a universal CD4 epitope are highly effective in generating immune responses to poorly processed cancer antigens.

Materials and Methods

DNA Constructs

SCT DNA constructs were generated using standard techniques as previously described (Yu et al., 2002; Lybarger et al., 2003; Mitaksov et al., 2007; Truscott et al., 2007) and were confirmed by DNA sequencing analysis. Mutations were introduced using the QuikChange II Site-Directed Mutagenesis System (Stratagene) following manufacturer’s instructions. Table 1 summarizes the progressive engineering of SCT DNA constructs used in this study. Plasmid DNA encoding SIINFEKL (mini-OVA), SIINYEKL (mini-p5Y), and AKFVAAWTLKAAA (mini-PADRE) peptides, as well as full-length chicken ovalbumin cDNA (OVA cDNA and mutated OVAp5Y cDNA) were also used as DNA vaccines.

Table 1
Progressive engineering of SCT constructs


All animals were used between the age of eight and twelve weeks. C57BL/6 (H-2b) mice were purchased from NCI-Frederick. OT-1 mice (CD45.1 congenic, JAX) were routinely screened for TCR Vα2/Vβ5 expression. hCD8+/HLA-A2+ mice were generated by crossing human CD8 transgenic mice to HLA-A*0201 transgenic mice (both from JAX) and used as F1 generation. All protocols followed the guidelines approved by the Animal Studies Committee at Washington University School of Medicine.

Cell Lines and Antibodies

The E.G7 cell line, which expresses full-length OVA cDNA, and its parental thymoma cell line EL4 were provided by Dr. M Salem (Medical University of South Carolina). The CHO cell line was provided by Dr. T Murphy (Washington University). Cells were cultured in RPMI1640 or DMEM complete media (Invitrogen) supplemented with L-glutamine, 10% fetal bovine serum (AtlantaBiologicals), sodium pyruvate, non-essential amino acids, and penicillin-streptomycin. CHO cells were transfected with SCT using jetPEI transfection reagent (PolyPlus Transfection) according to the manufacturer’s recommendations. Transfectants were selected by the addition of 0.6 mg/mL of Zeocin (InVivogen). B8-24-3 is a monoclonal antibody that recognizes folded H-2Kb molecules. Monoclonal antibody 25-D1.16 (a gift from Dr. J. Yewdell, NIH, Bethesda, MD) recognizes Kb plus SIINFEKL peptide (Porgador et al., 1997).

Flow Cytometry

Data from viable cells, gated by forward and side scatter, were acquired on a FACSCalibur (BD Biosciences) and analyzed using Flowjo software (TreeStar). Staining with 25-D1.16 culture supernatant was visualized using FITC-conjugated goat anti-mouse IgG (BD). The binding affinity of SCT to OT-1 transgenic T cells was determined by tetramer staining. SCT tetramers were prepared as described previously (Lybarger et al., 2003). After the staining procedure, aliquots of the stained OT-1 cells were allowed to sit on ice and analyzed at different time points. The mean fluorescence was compared to that of t = 0 min and the half-life of the tetramer binding was determined using a nonlinear regression model.

CTL Assay

CHO cells transfected with the indicated class I constructs were labeled with Na51CrO4 for 1 h. Activated OT-1 T cells were plated at various concentrations into 96-well microtiter plates and incubated with target cells for 4 h at 37°C in 5% CO2. Radioactivity in supernatants was measured in an Isomedic gamma counter (ICN Biomedicals). The mean of triplicate samples was calculated, and specific lysis was determined as follows: specific lysis = 100 × ((experimental 51Cr release – control 51Cr release)/(maximum 51Cr release – control 51Cr release)), where experimental 51Cr release represents counts from target cells mixed with effector cells, control 51Cr release represents counts from target cells in medium alone, and maximum 51Cr release represents counts from target cells lysed with 5% (v/v) Triton X-100 (Sigma-Aldrich).

DNA Vaccination and Tumor Challenge

DNA plasmids were amplified in E. coli DH5α (Invitrogen) and purified using NucleoBond Maxi Plasmid DNA Purification kits (Macherey-Nagel). DNA concentration was determined by the optical density measured at 260nm. DNA vaccination was performed using a Helios gene gun (Bio-Rad) according to the protocol provided by the manufacturer. Each cartridge carries 0.5 mg of gold (1 μm diameter) coated with 1 μg of plasmid DNA. A total of 4 μg of DNA was delivered to non-overlapping shaved and depilated mice abdominal areas. The discharge helium pressure was set to 400 p.s.i. Vaccination was administered at 3-day intervals for a total of three doses (Bins et al., 2005).

The tumor protection experiment was carried out as following. E.G7 cells were transplanted (s.c.) in the right flank of C57BL/6 mice four days before adoptive transfer of 2.5 × 106 splenocytes from OT-1 mice. Mice were vaccinated twice by a gene gun with 4 μg DNA at one day and one week after the OT-1 transfer. Tumors were measured using a caliper every other day after tumors became palpable. Tumor volumes were calculated by multiplying the two perpendicular diameters. 1 × 106 EL-4 cells were injected in the left flank as control.


To measure primary CTL immune responses, IFN-γ ELISPOT assays were performed at five days after the final DNA vaccination. Erythrocyte-free single cell suspensions were prepared from spleens, inguinal lymph nodes, or peripheral blood. A MultiScreen 96-well PVDF filtration plate (Millipore) was coated overnight with 15 μg/mL of capture antibody (clone AN18, Mabtech). After washing, the membrane was blocked with complete RPMI supplemented with 10% FBS for 1 hour at room temperature. Appropriate amounts of responder cells (2 – 4 × 105) were added into wells in triplicate. Cells were incubated for 20 hr with or without the presence of 1 μM of OVAp or 40 μg/mL of the mammaglobin-A-derived peptide, MamA2.1 (LIYDSSLCDL) (Jaramillo et al., 2004) (both synthesized by AnaSpec). After extensive washes, 1 μg/mL of biotinylated detection antibody (clone R4-6A2, Mabtech) was added and incubated at room temperature for 2 hr. Streptavidin-ALP (Mabtech) and BCIP/NBT (Moss Substrates) were then used for color development. The reaction was stopped by rinsing the plate extensively under tap water. The plates were left to dry before scanning and analysis on an ImmunoSpot ELISPOT Reader (C.T.L.).


Data were analyzed using GraphPad Prism 4 software (GraphPad). The Mann-Whitney test was used to compare between vaccination groups. A P value equal or less than 0.05 is considered statistically significant.


Disulfide trap SCT DNA vaccines enhance the immune response to low affinity peptides

In an effort to enhance the immunogenicity of first generation SCT, we have engineered SCT constructs to improve peptide binding (Figure 1A). The mutation (Y84C) creating a disulfide bond between Kb and an appropriately spaced cyteine residue in the peptide linker (disulfide trap SCT or dtSCT) was reported previously and showed enhanced peptide binding in vitro (Truscott et al., 2007; Truscott et al., 2008). It has been reported that improvements in the affinity of peptide/MHC interaction can trigger activation of naïve T cells against self antigens (Dyall et al., 1998; Gold et al., 2003; Yu et al., 2004). We hypothesized that the disulfide trap could increase the immunogenicity of SCT incorporating tumor antigen-derived peptides, most of which have low binding affinity to MHC class I molecules (Houghton and Guevara-Patino, 2004). We tested this hypothesis using the well-characterized OVA/Kb model system, in which the binding affinities of the immunodominant epitope SIINFEKL and its altered peptide ligands have been studied extensively (Howarth et al., 2004). In vitro validation studies demonstrated that the disulfide trap does not affect the expression or assembly of the MHC complex, even when the low affinity peptide p5Y is incorporated, as determined by flow cytometry and 51Cr-release assays (Figure 1, B and C).

Figure 1
dtSCT constructs encoding low affinity peptides generate functional MHC I complexes

To test the ability of dtSCT to enhance the CD8 T cell response to low affinity peptide antigens in vivo, we vaccinated C57BL/6 mice with full-length cDNA or SCT constructs following adoptive transfer of OT-1 T cells. T cell responses were measured using IFN-γ ELISPOT assays. Conventional DNA vaccines (OVA cDNA) and disulfide-trap SCT DNA vaccines (OVAp/Kb dtSCT) generated comparable antigen-specific T cell responses to Kb/SIINFEKL, a high affinity peptide that is processed efficiently. However, for the low affinity peptide p5Y, the p5Y/Kb dtSCT vaccine was clearly superior to the p5Y cDNA vaccine (Figure 2A). The immune response generated through dtSCT DNA vaccination was able to suppress E.G7 tumor growth in vivo (Figure 2B). This antitumor response was antigen-specific as growth of the parental EL4 tumor was comparable among vaccination groups. The data strongly suggest that dtSCT are capable of inducing CD8+ T cell responses against weak binding epitopes and therefore are valuable for the development of tumor-specific DNA vaccines.

Figure 2
dtSCT DNA vaccines dramatically enhance the immune response to low affinity peptide antigens in vivo

Q115E mutation enhances SCT interaction with CD8 co-receptor

SCT are recognized as intact molecules by the immune system, providing the ability to further engineer these constructs to enhance T cell activation. A critical component of the T cell receptor complex is the CD8 coreceptor. We have engineered an SCT to enhance interaction with the CD8 coreceptor by mutating a single amino acid residue (Q115E) in the α2 domain of the MHC class I heavy chain. This mutation has been shown to functionally enhance the interaction between the TCR and the pMHC complex (Wooldridge et al., 2005; Wooldridge et al., 2007). To test the affinity of the engineered SCT constructs, we created tetramers derived from wildtype and Q115E Kb molecules. The dissociation rates of the tetramers from OT-1 T cells were measured in a “tetramer decay” experiment (Holmberg et al., 2003). The half-life of the wildtype and Q115E Kb SCT tetramers were 6.6 and 19.7 minutes, respectively (Figure 3A). This indicates that the Q115E mutation results in a higher affinity interaction between the SCT and TCR/CD8 complex. We then vaccinated C57BL/6 mice with wildtype and Q115E Kb SCT DNA vaccines. The data in Figure 3B demonstrate that the Q115E SCT DNA vaccine is associated with an enhanced CD8 T cell response compared to its wildtype counterpart.

Figure 3
Q115E mutation increases SCT affinity for the TCR/CD8 complex

Coexpression of the PADRE helper epitope increases immunogenicity of SCT

Concurrent activation of CD4+ T cells is critical for the induction and maintenance of cellular anti-tumor immune responses (Shedlock and Shen, 2003; Sun and Bevan, 2003). SCT vaccines have been optimized to enhance CD8 T cell responses, and were not initially engineered to elicit a CD4 T cell response. To address this potential deficiency, we have engineered a construct that drives simultaneous expression of the OVAp/Kb dtSCT and the universal pan DR epitope peptide (PADRE) (Figure 1A). PADRE is an artificial peptide sequence (AKFVAAWTLKAAA) engineered to achieve high binding affinity to most of all HLA-DR alleles and certain murine class II molecules (Alexander et al., 1994). Previous studies have shown that PADRE is immunogenic in vivo and is able to elicit robust helper effect when delivered as either peptide (Alexander et al., 1994) or DNA construct (Hung et al., 2007b).

In vitro expression studies demonstrated that the expression levels of SCT with or without PADRE coexpression were comparable (data not shown). We tested the construct in vivo by gene gun immunization of mice followed by in vitro analysis of splenocytes. The frequency of IFN-γ producing cells in mice vaccinated with OVAp/Kb dtSCT PADRE were three- to five-fold higher than OVA cDNA or OVAp/Kb dtSCT vaccination, suggesting that PADRE coexpression dramatically increases the efficacy of the SCT DNA vaccine. The ELISPOT results from a representative experiment are shown in Figure 4.

Figure 4
Coexpression of the universal CD4 helper epitope PADRE in the dtSCT construct dramatically enhances the efficacy of the SCT vaccine

SCT constructs targeting mammaglobin-A

Mammaglobin-A is a human breast cancer-specific antigen (Watson and Fleming, 1996; Watson et al., 1999; Fleming and Watson, 2000) whose immunodominant epitopes have been identified (Jaramillo et al., 2002; Manna et al., 2003; Jaramillo et al., 2004; Narayanan et al., 2004). Generation of CD8 T cell responses to mammaglobin-A appears to be associated with anti-tumor activity, and there is evidence that mamA2.1 epitope is immunodominant in this response (Bharat et al., 2008). In preclinical studies, we assessed the efficacy of an SCT construct in mice expressing both HLA-A2 and human CD8 transgenes (Figure 5A). This construct is identical to what would be used in human breast cancer patients. The SCT encode human β2-microglobin, HLA-A2 heavy chain, and the MamA2.1 peptide, and coexpress PADRE (MamA2.1 dtSCT PADRE). As shown in Figure 5B, vaccination with the MamA2.1 dtSCT PADRE induced stronger mammaglobin-A specific T cell responses than vaccination with MamA2.1 peptide or mammaglobin-A cDNA.

Figure 5
MamA2.1 dtSCT PADRE DNA vaccine is more robust than comparable peptide or conventional mammaglobin-A cDNA vaccines


Delivery of sufficient antigen into the class I antigen presentation pathway to elicit T cell activation is a major limitation in the clinical translation of DNA vaccines. To address this limitation, we have developed innovative SCT constructs for use as DNA vaccines. SCT encode completely assembled peptide/MHC class I complexes as single polypeptide chain, therefore bypassing the requirement for antigen presentation and/or cross-presentation. First generation SCT have proven to be highly effective DNA vaccines in selected preclinical models (Jaramillo et al., 2004; Huang et al., 2005; Hung et al., 2007a; Zhang et al., 2007). Unfortunately, immunodominant tumor-derived peptides usually have low binding affinity to MHC class I molecules, contributing to the difficulty in trying to induce immune responses against cancer cells (Houghton and Guevara-Patino, 2004; Chapatte et al., 2006). We report in this study that engineering a disulfide trap into SCT constructs has a significant impact on the level of vaccine efficacy, particularly when a low affinity peptide is encoded. For proof-of-concept studies we chose the OVA/Kb model system because there are a number of reagents available that facilitate mechanistic studies in this system. These include the OT-1 TCR transgenic mouse, the Kb/OVA-specific mAb 25D1.16, and detailed experimental information available on altered peptide ligands. Our results provide critical insights into the development of SCT vaccines against tumor-associated antigens, which are commonly “self antigens” with relatively low binding affinity for MHC molecules.

The specificity of T cell activation depends upon TCR interaction with the peptide/MHC class I complex. However, the CD8 coreceptor is considered to be a key component of this interaction, by stabilizing the complex at the cell surface and facilitating TCR-mediated antigen recognition (Luescher et al., 1995; Gao et al., 1997; Arcaro et al., 2001; Wooldridge et al., 2005). We have previously reported that this interaction is particularly important for the generation of primary CD8 T cell responses to low affinity or poorly processed peptides, or by low avidity TCR (Alexander et al., 1991). To directly enhance this interaction we engineered SCT to incorporate the Q115E mutation, previously shown to enhance CD8+ T cell responses in vitro (Wooldridge et al., 2007). Our data demonstrate that this mutation leads to increased half-life of the TCR-pMHC complex, which is associated with enhanced SCT vaccine efficacy in vivo.

CD4+ T cells play a critical role in the generation and maintenance of antigen-specific CD8+ T cell immune responses. It has been well documented that CD4+ T cells are required for the establishment of CD8+ T cell effector and memory responses (Shedlock and Shen, 2003; Sun and Bevan, 2003). Several models have been proposed to illustrate how CD4+ and CD8+ T cell are collaborating, although the exact mechanism is still unclear. In one model, antigen presenting cells (APC) deliver co-stimulatory signals to the CD4+ T cells. CD4+ T cells produce IL-2, which in turn activate CD8+ T cells (Mitchison and O’Malley, 1987; Bennett et al., 1997; Xiang et al., 2005). In another model, the engagement of CD4+ T cells with APC leads to APC maturation, which subsequently activate CD8+ T cells (Bevan, 2004; Xiang et al., 2005). A recent article suggested an important role of IL-15 in promoting longevity of antigen-specific CD8+ T cells (Oh et al., 2008). MHC class I SCT vaccines have been optimized to enhance CD8 T cell responses, and were not initially engineered to elicit a CD4 T cell response. One strategy explored by Dr T-C Wu’s group involves the engineering of invariant chain (Ii), in which the CLIP region was replaced by antigen-specific or the universal helper epitope PADRE (Hung et al., 2007b). Co-administration of the Ii-PADRE DNA and SCT vaccines encoding HPV E6 or E7 antigens resulted in more robust E6- or E7-specific CTL responses (Hung et al., 2007b). The main drawback of this regimen is that the helper epitope and SCT are encoded in two separate plasmids. In the current study we employed a novel approach in which both SCT and the PADRE helper epitope are encoded in a single plasmid. Our data demonstrate that coexpression of PADRE in the same plasmid is able to enhance the efficacy of SCT DNA vaccines. This approach has the potential to facilitate the clinical translation of SCT DNA vaccines. With a single plasmid, vaccine manufacture, product release tests, and regulatory approval are streamlined.

We tested the engineered SCT in preclinical studies targeting the human breast caner antigen mammaglobin-A. Mammaglobin-A is a novel breast cancer-associated antigen that is expressed exclusively in normal breast epithelium and breast cancer (Watson and Fleming, 1994). It is consistently overexpressed in breast cancer, and may ultimately prove to be a candidate for breast cancer prevention. Our data demonstrated that the MamA2.1 dtSCT PADRE DNA vaccine is superior to conventional cDNA and peptide vaccines. After thorough investigations of the basic biology/immunology of mammaglobin-A (Watson and Fleming, 1994,Watson and Fleming, 1996; Watson et al., 1998; Watson et al., 1999; Fleming and Watson, 2000; Manna et al., 2003; Tanaka et al., 2003; Goedegebuure et al., 2004; Jaramillo et al., 2004; Narayanan et al., 2004; Viehl et al., 2005; Viehl et al., 2007; Bharat et al., 2008) and the functional immunology of SCT DNA vaccines (Yu et al., 2002; Lybarger et al., 2003; Jaramillo et al., 2004; Huang et al., 2005; Mitaksov et al., 2007; Truscott et al., 2007), we are in a unique position to take the first step in the clinical translation of a rationally designed, innovative, and highly effective breast cancer DNA vaccine.


This research was supported by grants from Susan G. Komen for the Cure, KG080476 (W.E.G. and L.L.); Department of Defense, W81XWH-06-1-0677 (W.E.G.); National Institute of Health, AI055849 (T.H.H.); and Frank Cancer Research Fund, awarded by the Barnes-Jewish Hospital Foundation (P.G.).


Author Disclosure Statement

S.M. Truscott and T.H. Hansen are listed on a pending patent based on single chain format and disulfide traps.

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  • ALEXANDER J, SIDNEY J, SOUTHWOOD S, RUPPERT J, OSEROFF C, MAEWAL A, SNOKE K, SERRA HM, KUBO RT, SETTE A, et al. Development of high potency universal DR-restricted helper epitopes by modification of high affinity DR-blocking peptides. Immunity. 1994;1:751–761. [PubMed]
  • ALEXANDER MA, DAMICO CA, WIETIES KM, HANSEN TH, CONNOLLY JM. Correlation between CD8 dependency and determinant density using peptide-induced, Ld-restricted cytotoxic T lymphocytes. J Exp Med. 1991;173:849–858. [PMC free article] [PubMed]
  • ANDRE S, SEED B, EBERLE J, SCHRAUT W, BULTMANN A, HAAS J. Increased immune response elicited by DNA vaccination with a synthetic gp120 sequence with optimized codon usage. J Virol. 1998;72:1497–1503. [PMC free article] [PubMed]
  • ARCARO A, GREGOIRE C, BAKKER TR, BALDI L, JORDAN M, GOFFIN L, BOUCHERON N, WURM F, VAN DER MERWE PA, MALISSEN B, LUESCHER IF. CD8beta endows CD8 with efficient coreceptor function by coupling T cell receptor/CD3 to raft-associated CD8/p56(lck) complexes. J Exp Med. 2001;194:1485–1495. [PMC free article] [PubMed]
  • BENNETT SR, CARBONE FR, KARAMALIS F, MILLER JF, HEATH WR. Induction of a CD8+ cytotoxic T lymphocyte response by cross-priming requires cognate CD4+ T cell help. J Exp Med. 1997;186:65–70. [PMC free article] [PubMed]
  • BEVAN MJ. Helping the CD8(+) T-cell response. Nat Rev Immunol. 2004;4:595–602. [PubMed]
  • BHARAT A, BENSHOFF N, FLEMING TP, DIETZ JR, GILLANDERS WE, MOHANAKUMAR T. Characterization of the role of CD8+T cells in breast cancer immunity following mammaglobin-A DNA vaccination using HLA-class-I tetramers. Breast Cancer Res Treat. 2008;110:453–463. [PubMed]
  • BINS AD, JORRITSMA A, WOLKERS MC, HUNG CF, WUTC, SCHUMACHER TN, HAANEN JB. A rapid and potent DNA vaccination strategy defined by in vivo monitoring of antigen expression. Nat Med. 2005;11:899–904. [PubMed]
  • BOURGEOIS C, VEIGA-FERNANDES H, JORET AM, ROCHA B, TANCHOT C. CD8 lethargy in the absence of CD4 help. Eur J Immunol. 2002;32:2199–2207. [PubMed]
  • BOYER JD, ROBINSON TM, KUTZLER MA, PARKINSON R, CALAROTA SA, SIDHU MK, MUTHUMANI K, LEWIS M, PAVLAKIS G, FELBER B, WEINER D. SIV DNA vaccine co-administered with IL-12 expression plasmid enhances CD8 SIV cellular immune responses in cynomolgus macaques. Journal of medical primatology. 2005;34:262–270. [PubMed]
  • CALAROTA S, BRATT G, NORDLUND S, HINKULA J, LEANDERSSON AC, SANDSTROM E, WAHREN B. Cellular cytotoxic response induced by DNA vaccination in HIV-1-infected patients. Lancet. 1998;351:1320–1325. [PubMed]
  • CHAN K, LEE DJ, SCHUBERT A, TANG CM, CRAIN B, SCHOENBERGER SP, CORR M. The roles of MHC class II, CD40, and B7 costimulation in CTL induction by plasmid DNA. J Immunol. 2001;166:3061–3066. [PubMed]
  • CHAPATTE L, AYYOUB M, MOREL S, PEITREQUIN AL, LEVY N, SERVIS C, VAN DEN EYNDE BJ, VALMORI D, LEVY F. Processing of tumor-associated antigen by the proteasomes of dendritic cells controls in vivo T-cell responses. Cancer Res. 2006;66:5461–5468. [PubMed]
  • CHONG SY, EGAN MA, KUTZLER MA, MEGATI S, MASOOD A, ROOPCHARD V, GARCIA-HAND D, MONTEFIORI DC, QUIROZ J, ROSATI M, SCHADECK EB, BOYER JD, PAVLAKIS GN, WEINER DB, SIDHU M, ELDRIDGE JH, ISRAEL ZR. Comparative ability of plasmid IL-12 and IL-15 to enhance cellular and humoral immune responses elicited by a SIVgag plasmid DNA vaccine and alter disease progression following SHIV(89.6P) challenge in rhesus macaques. Vaccine. 2007;25:4967–4982. [PubMed]
  • CHOUDHURI K, WISEMAN D, BROWN MH, GOULD K, VAN DER MERWE PA. T-cell receptor triggering is critically dependent on the dimensions of its peptide-MHC ligand. Nature. 2005;436:578–582. [PubMed]
  • COX GJ, ZAMB TJ, BABIUK LA. Bovine herpesvirus 1: immune responses in mice and cattle injected with plasmid DNA. J Virol. 1993;67:5664–5667. [PMC free article] [PubMed]
  • CREW MD, CANNON MJ, PHANAVANH B, GARCIA-BORGES CN. An HLA-E single chain trimer inhibits human NK cell reactivity towards porcine cells. Mol Immunol. 2005;42:1205–1214. [PubMed]
  • DAVIS HL, MICHEL ML, WHALEN RG. DNA-based immunization induces continuous secretion of hepatitis B surface antigen and high levels of circulating antibody. Hum Mol Genet. 1993;2:1847–1851. [PubMed]
  • DEL VAL M, SCHLICHT HJ, RUPPERT T, REDDEHASE MJ, KOSZINOWSKI UH. Efficient processing of an antigenic sequence for presentation by MHC class I molecules depends on its neighboring residues in the protein. Cell. 1991;66:1145–1153. [PubMed]
  • DENG Y, YEWDELL JW, EISENLOHR LC, BENNINK JR. MHC affinity, peptide liberation, T cell repertoire, and immunodominance all contribute to the paucity of MHC class I-restricted peptides recognized by antiviral CTL. J Immunol. 1997;158:1507–1515. [PubMed]
  • DONNELLY JJ, ULMER JB, SHIVER JW, LIU MA. DNA vaccines. Annu Rev Immunol. 1997;15:617–648. [PubMed]
  • DYALL R, BOWNE WB, WEBER LW, LEMAOULT J, SZABO P, MOROI Y, PISKUN G, LEWIS JJ, HOUGHTON AN, NIKOLIC-ZUGIC J. Heteroclitic immunization induces tumor immunity. J Exp Med. 1998;188:1553–1561. [PMC free article] [PubMed]
  • EISENLOHR LC, YEWDELL JW, BENNINK JR. Flanking sequences influence the presentation of an endogenously synthesized peptide to cytotoxic T lymphocytes. J Exp Med. 1992;175:481–487. [PMC free article] [PubMed]
  • FLEMING TP, WATSON MA. Mammaglobin, a breast-specific gene, and its utility as a marker for breast cancer. Ann N Y Acad Sci. 2000;923:78–89. [PubMed]
  • FYNAN EF, WEBSTER RG, FULLER DH, HAYNES JR, SANTORO JC, ROBINSON HL. DNA vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations. Proc Natl Acad Sci U S A. 1993;90:11478–11482. [PubMed]
  • GAO GF, TORMO J, GERTH UC, WYER JR, MCMICHAEL AJ, STUART DI, BELL JI, JONES EY, JAKOBSEN BK. Crystal structure of the complex between human CD8alpha(alpha) and HLA-A2. Nature. 1997;387:630–634. [PubMed]
  • GOEDEGEBUURE PS, WATSON MA, VIEHL CT, FLEMING TP. Mammaglobin-based strategies for treatment of breast cancer. Curr Cancer Drug Targets. 2004;4:531–542. [PubMed]
  • GOLD JS, FERRONE CR, GUEVARA-PATINO JA, HAWKINS WG, DYALL R, ENGELHORN ME, WOLCHOK JD, LEWIS JJ, HOUGHTON AN. A single heteroclitic epitope determines cancer immunity after xenogeneic DNA immunization against a tumor differentiation antigen. J Immunol. 2003;170:5188–5194. [PubMed]
  • GURUNATHAN S, KLINMAN DM, SEDER RA. DNA vaccines: immunology, application, and optimization*. Annu Rev Immunol. 2000;18:927–974. [PubMed]
  • HEEMELS MT, PLOEGH H. Generation, translocation, and presentation of MHC class I-restricted peptides. Annual review of biochemistry. 1995;64:463–491. [PubMed]
  • HIRAO LA, WUL, KHAN AS, HOKEY DA, YAN J, DAI A, BETTS MR, DRAGHIA-AKLI R, WEINER DB. Combined effects of IL-12 and electroporation enhances the potency of DNA vaccination in macaques. Vaccine. 2008;26:3112–3120. [PMC free article] [PubMed]
  • HOLMBERG K, MARIATHASAN S, OHTEKI T, OHASHI PS, GASCOIGNE NR. TCR binding kinetics measured with MHC class I tetramers reveal a positive selecting peptide with relatively high affinity for TCR. J Immunol. 2003;171:2427–2434. [PubMed]
  • HOUGHTON AN, GUEVARA-PATINO JA. Immune recognition of self in immunity against cancer. J Clin Invest. 2004;114:468–471. [PMC free article] [PubMed]
  • HOWARTH M, WILLIAMS A, TOLSTRUP AB, ELLIOTT T. Tapasin enhances MHC class I peptide presentation according to peptide half-life. Proc Natl Acad Sci U S A. 2004;101:11737–11742. [PubMed]
  • HUANG CH, PENG S, HE L, TSAI YC, BOYD DA, HANSEN TH, WUTC, HUNG CF. Cancer immunotherapy using a DNA vaccine encoding a single-chain trimer of MHC class I linked to an HPV-16 E6 immunodominant CTL epitope. Gene Ther. 2005;12:1180–1186. [PMC free article] [PubMed]
  • HUDRISIER D, RIOND J, GARIDOU L, DUTHOIT C, JOLY E. T cell activation correlates with an increased proportion of antigen among the materials acquired from target cells. Eur J Immunol. 2005;35:2284–2294. [PubMed]
  • HUNG CF, CALIZO R, TSAI YC, HE L, WUTC A DNA vaccine encoding a single-chain trimer of HLA-A2 linked to human mesothelin peptide generates anti-tumor effects against human mesothelin-expressing tumors. Vaccine. 2007a;25:127–135. [PubMed]
  • HUNG CF, TSAI YC, HEL, WUTC DNA vaccines encoding Ii-PADRE generates potent PADRE-specific CD4+ T-cell immune responses and enhances vaccine potency. Mol Ther. 2007b;15:1211–1219. [PMC free article] [PubMed]
  • JANSSEN EM, LEMMENS EE, WOLFE T, CHRISTEN U, VON HERRATH MG, SCHOENBERGER SP. CD4+ T cells are required for secondary expansion and memory in CD8+ T lymphocytes. Nature. 2003;421:852–856. [PubMed]
  • JARAMILLO A, MAJUMDER K, MANNA PP, FLEMING TP, DOHERTY G, DIPERSIO JF, MOHANAKUMAR T. Identification of HLA-A3-restricted CD8+ T cell epitopes derived from mammaglobin-A, a tumor-associated antigen of human breast cancer. Int J Cancer. 2002;102:499–506. [PubMed]
  • JARAMILLO A, NARAYANAN K, CAMPBELL LG, BENSHOFF ND, LYBARGER L, HANSEN TH, FLEMING TP, DIETZ JR, MOHANAKUMAR T. Recognition of HLA-A2-restricted mammaglobin-A-derived epitopes by CD8+ cytotoxic T lymphocytes from breast cancer patients. Breast Cancer Res Treat. 2004;88:29–41. [PubMed]
  • KIM S, POURSINE-LAURENT J, TRUSCOTT SM, LYBARGER L, SONG YJ, YANG L, FRENCH AR, SUNWOO JB, LEMIEUX S, HANSEN TH, YOKOYAMA WM. Licensing of natural killer cells by host major histocompatibility complex class I molecules. Nature. 2005;436:709–713. [PubMed]
  • KUTZLER MA, WEINER DB. DNA vaccines: ready for prime time? Nat Rev Genet. 2008;9:776–788. [PMC free article] [PubMed]
  • LEE AH, SUH YS, SUNG JH, YANG SH, SUNG YC. Comparison of various expression plasmids for the induction of immune response by DNA immunization. Molecules and cells. 1997;7:495–501. [PubMed]
  • LILIENFELD BG, CREW MD, FORTE P, BAUMANN BC, SEEBACH JD. Transgenic expression of HLA-E single chain trimer protects porcine endothelial cells against human natural killer cell-mediated cytotoxicity. Xenotransplantation. 2007;14:126–134. [PubMed]
  • LUESCHER IF, VIVIER E, LAYER A, MAHIOU J, GODEAU F, MALISSEN B, ROMERO P. CD8 modulation of T-cell antigen receptor-ligand interactions on living cytotoxic T lymphocytes. Nature. 1995;373:353–356. [PubMed]
  • LYBARGER L, YUYY, MILEY MJ, FREMONT DH, MYERS N, PRIMEAU T, TRUSCOTT SM, CONNOLLY JM, HANSEN TH. Enhanced immune presentation of a single-chain major histocompatibility complex class I molecule engineered to optimize linkage of a C-terminally extended peptide. J Biol Chem. 2003;278:27105–27111. [PubMed]
  • MACGREGOR RR, BOYER JD, UGEN KE, LACY KE, GLUCKMAN SJ, BAGARAZZI ML, CHATTERGOON MA, BAINE Y, HIGGINS TJ, CICCARELLI RB, CONEY LR, GINSBERG RS, WEINER DB. First human trial of a DNA-based vaccine for treatment of human immunodeficiency virus type 1 infection: safety and host response. J Infect Dis. 1998;178:92–100. [PubMed]
  • MANNA PP, JARAMILLO A, MAJUMDER K, CAMPBELL LG, FLEMING TP, DIETZ JR, DIPERSIO JF, MOHANAKUMAR T. Generation of CD8+ cytotoxic T lymphocytes against breast cancer cells by stimulation with mammaglobin-A-pulsed dendritic cells. Breast Cancer Res Treat. 2003;79:133–136. [PubMed]
  • MITAKSOV V, TRUSCOTT SM, LYBARGER L, CONNOLLY JM, HANSEN TH, FREMONT DH. Structural engineering of pMHC reagents for T cell vaccines and diagnostics. Chem Biol. 2007;14:909–922. [PMC free article] [PubMed]
  • MITCHISON NA, O’MALLEY C. Three-cell-type clusters of T cells with antigen-presenting cells best explain the epitope linkage and noncognate requirements of the in vivo cytolytic response. Eur J Immunol. 1987;17:1579–1583. [PubMed]
  • NARAYANAN K, JARAMILLO A, BENSHOFF ND, CAMPBELL LG, FLEMING TP, DIETZ JR, MOHANAKUMAR T. Response of established human breast tumors to vaccination with mammaglobin-A cDNA. J Natl Cancer Inst. 2004;96:1388–1396. [PubMed]
  • NCHINDA G, KUROIWA J, OKS M, TRUMPFHELLER C, PARK CG, HUANG Y, HANNAMAN D, SCHLESINGER SJ, MIZENINA O, NUSSENZWEIG MC, UBERLA K, STEINMAN RM. The efficacy of DNA vaccination is enhanced in mice by targeting the encoded protein to dendritic cells. J Clin Invest. 2008;118:1427–1436. [PubMed]
  • OHS, PERERA LP, TERABE M, NIL, WALDMANN TA, BERZOFSKY JA. IL-15 as a mediator of CD4+ help for CD8+ T cell longevity and avoidance of TRAIL-mediated apoptosis. Proc Natl Acad Sci U S A. 2008;105:5201–5206. [PubMed]
  • PORGADOR A, YEWDELL JW, DENG Y, BENNINK JR, GERMAIN RN. Localization, quantitation, and in situ detection of specific peptide-MHC class I complexes using a monoclonal antibody. Immunity. 1997;6:715–726. [PubMed]
  • PRIMEAU T, MYERS NB, YUYY, LYBARGER L, WANG X, TRUSCOTT SM, HANSEN TH, CONNOLLY JM. Applications of major histocompatibility complex class I molecules expressed as single chains. Immunol Res. 2005;32:109–121. [PubMed]
  • SCHADECK EB, SIDHU M, EGAN MA, CHONG SY, PIACENTE P, MASOOD A, GARCIA-HAND D, CAPPELLO S, ROOPCHAND V, MEGATI S, QUIROZ J, BOYER JD, FELBER BK, PAVLAKIS GN, WEINER DB, ELDRIDGE JH, ISRAEL ZR. A dose sparing effect by plasmid encoded IL-12 adjuvant on a SIVgag-plasmid DNA vaccine in rhesus macaques. Vaccine. 2006;24:4677–4687. [PubMed]
  • SHEDLOCK DJ, SHEN H. Requirement for CD4 T cell help in generating functional CD8 T cell memory. Science. 2003;300:337–339. [PubMed]
  • SUMIDA SM, MCKAY PF, TRUITT DM, KISHKO MG, ARTHUR JC, SEAMAN MS, JACKSON SS, GORGONE DA, LIFTON MA, LETVIN NL, BAROUCH DH. Recruitment and expansion of dendritic cells in vivo potentiate the immunogenicity of plasmid DNA vaccines. J Clin Invest. 2004;114:1334–1342. [PMC free article] [PubMed]
  • SUN JC, BEVAN MJ. Defective CD8 T cell memory following acute infection without CD4 T cell help. Science. 2003;300:339–342. [PMC free article] [PubMed]
  • TANAKA Y, AMOS KD, FLEMING TP, EBERLEIN TJ, GOEDEGEBUURE PS. Mammaglobin-A is a tumor-associated antigen in human breast carcinoma. Surgery. 2003;133:74–80. [PubMed]
  • TANG DC, DEVIT M, JOHNSTON SA. Genetic immunization is a simple method for eliciting an immune response. Nature. 1992;356:152–154. [PubMed]
  • TRUMPFHELLER C, FINKE JS, LOPEZ CB, MORAN TM, MOLTEDO B, SOARES H, HUANG Y, SCHLESINGER SJ, PARK CG, NUSSENZWEIG MC, GRANELLI-PIPERNO A, STEINMAN RM. Intensified and protective CD4+ T cell immunity in mice with anti-dendritic cell HIV gag fusion antibody vaccine. J Exp Med. 2006;203:607–617. [PMC free article] [PubMed]
  • TRUSCOTT SM, LYBARGER L, MARTINKO JM, MITAKSOV VE, KRANZ DM, CONNOLLY JM, FREMONT DH, HANSEN TH. Disulfide bond engineering to trap peptides in the MHC class I binding groove. J Immunol. 2007;178:6280–6289. [PubMed]
  • TRUSCOTT SM, WANG X, LYBARGER L, BIDDISON WE, MCBERRY C, MARTINKO JM, CONNOLLY JM, LINETTE GP, FREMONT DH, HANSEN TH, CARRENO BM. Human major histocompatibility complex (MHC) class I molecules with disulfide traps secure disease-related antigenic peptides and exclude competitor peptides. J Biol Chem. 2008;283:7480–7490. [PMC free article] [PubMed]
  • ULMER JB, DONNELLY JJ, PARKER SE, RHODES GH, FELGNER PL, DWARKI VJ, GROMKOWSKI SH, DECK RR, DEWITT CM, FRIEDMAN A, et al. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science. 1993;259:1745–1749. [PubMed]
  • VIEHL CT, FREY DM, PHOMMALY C, CHEN T, FLEMING TP, GILLANDERS WE, EBERLEIN TJ, GOEDEGEBUURE PS. Generation of mammaglobin-A-specific CD4 T cells and identification of candidate CD4 epitopes for breast cancer vaccine strategies. Breast Cancer Res Treat 2007 [PubMed]
  • VIEHL CT, TANAKA Y, CHEN T, FREY DM, TRAN A, FLEMING TP, EBERLEIN TJ, GOEDEGEBUURE PS. Tat mammaglobin fusion protein transduced dendritic cells stimulate mammaglobin-specific CD4 and CD8 T cells. Breast Cancer Res Treat. 2005;91:271–278. [PubMed]
  • WANG B, UGEN KE, SRIKANTAN V, AGADJANYAN MG, DANG K, REFAELI Y, SATO AI, BOYER J, WILLIAMS WV, WEINER DB. Gene inoculation generates immune responses against human immunodeficiency virus type 1. Proc Natl Acad Sci U S A. 1993;90:4156–4160. [PubMed]
  • WANG R, DOOLAN DL, LETP, HEDSTROM RC, COONAN KM, CHAROENVIT Y, JONES TR, HOBART P, MARGALITH M, NGJ, WEISS WR, SEDEGAH M, DE TAISNE C, NORMAN JA, HOFFMAN SL. Induction of antigen-specific cytotoxic T lymphocytes in humans by a malaria DNA vaccine. Science. 1998;282:476–480. [PubMed]
  • WATSON MA, DARROW C, ZIMONJIC DB, POPESCU NC, FLEMING TP. Structure and transcriptional regulation of the human mammaglobin gene, a breast cancer associated member of the uteroglobin gene family localized to chromosome 11q13. Oncogene. 1998;16:817–824. [PubMed]
  • WATSON MA, DINTZIS S, DARROW CM, VOSS LE, DIPERSIO J, JENSEN R, FLEMING TP. Mammaglobin expression in primary, metastatic, and occult breast cancer. Cancer Res. 1999;59:3028–3031. [PubMed]
  • WATSON MA, FLEMING TP. Isolation of differentially expressed sequence tags from human breast cancer. Cancer Res. 1994;54:4598–4602. [PubMed]
  • WATSON MA, FLEMING TP. Mammaglobin, a mammary-specific member of the uteroglobin gene family, is overexpressed in human breast cancer. Cancer Res. 1996;56:860–865. [PubMed]
  • WOOLDRIDGE L, LISSINA A, VERNAZZA J, GOSTICK E, LAUGEL B, HUTCHINSON SL, MIRZA F, DUNBAR PR, BOULTER JM, GLICK M, CERUNDOLO V, VAN DEN BERG HA, PRICE DA, SEWELL AK. Enhanced immunogenicity of CTL antigens through mutation of the CD8 binding MHC class I invariant region. Eur J Immunol. 2007;37:1323–1333. [PMC free article] [PubMed]
  • WOOLDRIDGE L, VAN DEN BERG HA, GLICK M, GOSTICK E, LAUGEL B, HUTCHINSON SL, MILICIC A, BRENCHLEY JM, DOUEK DC, PRICE DA, SEWELL AK. Interaction between the CD8 coreceptor and major histocompatibility complex class I stabilizes T cell receptor-antigen complexes at the cell surface. J Biol Chem. 2005;280:27491–27501. [PMC free article] [PubMed]
  • XIANG J, HUANG H, LIU Y. A new dynamic model of CD8+ T effector cell responses via CD4+ T helper-antigen-presenting cells. J Immunol. 2005;174:7497–7505. [PubMed]
  • YEWDELL JW, DEL VAL M. Immunodominance in TCD8+ responses to viruses: cell biology, cellular immunology, and mathematical models. Immunity. 2004;21:149–153. [PubMed]
  • YUYY, NETUSCHIL N, LYBARGER L, CONNOLLY JM, HANSEN TH. Cutting edge: single-chain trimers of MHC class I molecules form stable structures that potently stimulate antigen-specific T cells and B cells. J Immunol. 2002;168:3145–3149. [PubMed]
  • YUZ, THEORET MR, TOULOUKIAN CE, SURMAN DR, GARMAN SC, FEIGENBAUM L, BAXTER TK, BAKER BM, RESTIFO NP. Poor immunogenicity of a self/tumor antigen derives from peptide-MHC-I instability and is independent of tolerance. J Clin Invest. 2004;114:551–559. [PMC free article] [PubMed]
  • ZHANG Y, LIS, SHAN M, PAN X, ZHUANG K, HEL, GOULD K, TIEN P. Hepatitis B virus core antigen epitopes presented by HLA-A2 single-chain trimers induce functional epitope-specific CD8+ T-cell responses in HLA-A2.1/Kb transgenic mice. Immunology. 2007;121:105–112. [PubMed]