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1.  Immunogenicity of Novel Consensus-based DNA Vaccines Against Avian Influenza 
Vaccine  2007;25(16):2984-2989.
The frequency of H5N1 avian influenza outbreaks in China and Eastern Europe has raised concern in the world health community regarding the potential for an influenza pandemic. Efforts to monitor the disease will only provide minimal warning in a global society, and steps must be taken to prevent the morbidity and mortality associated with past pandemics. The current stockpiling of antibody-inducing “bird flu” vaccines assumes the strain that emerges will be the same as strains currently circulating. We propose a novel consensus-based approach to vaccine development, employing a DNA vaccine strategy that can provide more highly cross-reactive cellular immunity against lethal influenza infection. We show such constructs can induce strong cellular immunity against H5 influenza antigens.
PMCID: PMC4477802  PMID: 17306909
Avian influenza; DNA vaccine; consensus
2.  Induction of Antitumor Immunity In vivo Following Delivery of a Novel HPV-16 DNA Vaccine Encoding an E6/E7 Fusion antigen 
Vaccine  2008;27(3):431-440.
Human papillomavirus type 16 (HPV-16) infection is associated with a majority of cervical cancers and a significant proportion of head and neck cancers. Here, we describe a novel engineered DNA vaccine that encodes a HPV-16 consensus E6/E7 fusion gene (pConE6E7) with the goal of increasing its antitumor cellular immunity. Compared to an early stage HPV-16 E7 DNA vaccine (pE7), this construct was up to five times more potent in driving E7-specific cellular immune responses. Prophylactic administration of this vaccine resulted in 100% protection against HPV E6 and E7-expressing tumors. Therapeutic studies indicated that vaccination with pConE6E7 prevented or delayed the growth of tumors. Moreover, immunization with pConE6E7 could also partially overcome immune tolerance in E6/E7 transgenic mice. Such DNA immunogens are interesting candidates for further study to investigate mechanisms of tumor immune rejection in vivo.
PMCID: PMC4477831  PMID: 19022315
HPV-16; E6 and E7 protein; DNA vaccine
3.  Immunogenicity of a novel engineered HIV-1 clade C synthetic consensus-based envelope DNA vaccine 
Vaccine  2011;29(41):7173-7181.
DNA vaccines require significant engineering in order to generate strong CTL responses in both non-human primates and humans. In this study, we designed a clade C env gene (EY3E1-C) to decrease the genetic distances of virus isolates within clade C and focus the induced T cell responses to conserved clade C epitopes. After generating a consensus sequence by analyzing full-length clade C env early transmitter sequences, several modifications were performed to increase the expression of the EY3E1-C, including codon/RNA optimization, addition of Kozak sequence and addition of an IgE leader sequence. We also shortened the V1 and V2 loops to approximate early transmitter isolate sequences and the cytoplasmic tail was truncated to prevent envelope recycling. When studied as a DNA vaccine in Balb/c mice, compared to a primary codon-optimized clade C envelope DNA vaccine (p96ZM651gp140-CD5), this novel construct is up to three times more potent in driving CTL responses. Importantly this construct not only induces stronger cross-reactive cellular responses within clade C, it also induces stronger immune responses against clade B and group M envelope peptide pools than p96ZM651gp140-CD5. Epitope mapping demonstrated that EY3E1-C was able to induce clade C envelope-specific immune responses against 15 peptide pools, clade B envelope-specific immune responses against 19 peptide pools and group M envelope-specific immune responses against 16 peptide pools out of 29, respectively, indicating that a significant increase in the breadth of induced immune responses. The analysis of antibody responses suggested that vaccination of pEY3E1-C could induce a clade C envelope-specific antibody response. The cellular immune responses of pEY3E1-C could be further enhanced when the DNA was delivered by using electroporation (EP). Thus, the synthetic engineered consensus EY3E1-C gene is capable of eliciting stronger and broader CTL responses than primary clade C envelopes. This finding suggests that such synthetic immunogens could be important for examination of their potential as part of an efficient HIV DNA vaccine.
PMCID: PMC4012758  PMID: 21651948
HIV-1; Clade C; Envelope; Consensus; Cellular immune responses
4.  SPRR2A enhances p53 deacetylation through HDAC1 and down regulates p21 promoter activity 
BMC Molecular Biology  2012;13:20.
Small proline rich protein (SPRR) 2A is one of 14 SPRR genes that encodes for a skin cross-linking protein, which confers structural integrity to the cornified keratinocyte cell envelope. New evidence, however, shows that SPRR2A is also a critical stress and wound repair modulator: it enables a variety of barrier epithelia to transiently acquire mesenchymal characteristics (EMT) and simultaneously quench reactive oxygen species during wound repair responses. p53 is also widely recognized as the node in cellular stress responses that inhibits EMT and triggers cell-cycle arrest, apoptosis, and cellular senescence. Since some p53-directed processes would seem to impede wound repair of barrier epithelia, we hypothesized that SPRR2A up regulation might counteract these effects and enable/promote wound repair under stressful environmental conditions.
Using a well characterized cholangiocarcinoma cell line we show that levels of SPRR2A expression, similar to that seen during stressful biliary wound repair responses, disrupts acetylation and subsequent p53 transcriptional activity. p53 deacetylation is accomplished via two distinct, but possibly related, mechanisms: 1) a reduction of p300 acetylation, thereby interfering with p300-p53 binding and subsequent p300 acetylation of K382 in p53; and 2) an increase in histone deacetylase 1 (HDAC1) mRNA and protein expression. The p300 CH3 domain is essential for both the autoacetylation of p300 and transference of the acetyl group to p53 and HDAC1 is a component of several non-p300 complexes that enhance p53 deacetylation, ubiquitination, and proteosomal degradation. HDAC1 can also bind the p300-CH3 domain, regulating p300 acetylation and interfering with p300 mediated p53 acetylation. The importance of this pathway is illustrated by showing complete restoration of p53 acetylation and partial restoration of p300 acetylation by treating SPRR2A expressing cells with HDAC1 siRNA.
Up-regulation of SPRR2A, similar to that seen during barrier epithelia wound repair responses reduces p53 acetylation by interfering with p300-p53 interactions and by increasing HDAC1 expression. SPRR2A, therefore, functions as a suppressor of p53-dependent transcriptional activity, which otherwise might impede cellular processes needed for epithelial wound repair responses such as EMT.
PMCID: PMC3495018  PMID: 22731250
5.  Cooperation of p300 and PCAF in the Control of MicroRNA 200c/141 Transcription and Epithelial Characteristics 
PLoS ONE  2012;7(2):e32449.
Epithelial to mesenchymal transition (EMT) not only occurs during embryonic development and in response to injury, but is an important element in cancer progression. EMT and its reverse process, mesenchymal to epithelial transition (MET) is controlled by a network of transcriptional regulators and can be influenced by posttranscriptional and posttranslational modifications. EMT/MET involves many effectors that can activate and repress these transitions, often yielding a spectrum of cell phenotypes. Recent studies have shown that the miR-200 family and the transcriptional suppressor ZEB1 are important contributors to EMT. Our previous data showed that forced expression of SPRR2a was a powerful inducer of EMT and supports the findings by others that SPRR gene members are highly upregulated during epithelial remodeling in a variety of organs. Here, using SPRR2a cells, we characterize the role of acetyltransferases on the microRNA-200c/141 promoter and their effect on the epithelial/mesenchymal status of the cells. We show that the deacetylase inhibitor TSA as well as P300 and PCAF can cause a shift towards epithelial characteristics in HUCCT-1-SPRR2a cells. We demonstrate that both P300 and PCAF act as cofactors for ZEB1, forming a P300/PCAF/ZEB1 complex on the miR200c/141 promoter. This binding results in lysine acetylation of ZEB1 and a release of ZEB1 suppression on miR-200c/141 transcription. Furthermore, disruption of P300 and PCAF interactions dramatically down regulates miR-200c/141 promoter activity, indicating a PCAF/P300 cooperative function in regulating the transcriptional suppressor/activator role of ZEB1. These data demonstrate a novel mechanism of miRNA regulation in mediating cell phenotype.
PMCID: PMC3284570  PMID: 22384255
6.  mTOR Inhibition and Alloantigen-Specific Regulatory T Cells Synergize to Promote Long-Term Graft Survival in Immunocompetent Recipients1 
Minimization of immunosuppression and donor-specific tolerance to MHC-mismatched organ grafts are important clinical goals. The therapeutic potential of regulatory T cells (Treg) has been demonstrated, but conditions for optimizing their in vivo function post-transplant in nonlymphocyte-depleted hosts remain undefined. Herein, we address mechanisms through which inhibition of the mammalian target of rapamycin (Rapa) (mTOR) synergizes with alloAg-specific Treg (AAsTreg), to permit long-term, donor-specific heart graft survival in immunocompetent hosts. Crucially, immature allogeneic dendritic cells (DC) allowed AAsTreg selection in vitro, with minimal expansion of unwanted (TH17) cells. The rendered Treg potently inhibited T cell proliferation in an Ag-specific manner. However, these AAsTreg remained unable to control T cells stimulated by allogeneic mature DC, – a phenomenon dependent on the release of pro-inflammatory cytokines. In vivo, Rapa administration reduced danger-associated IL-6 production, T cell proliferation and graft infiltration. Based on these observation, AAsTreg were administered post-transplant (d7) in combination with a short course of Rapa and rendered >80% long-term (>150 d) graft survival, a result superior to that achieved with polyclonal Treg. Moreover, graft protection was alloAg-specific. Significantly, long-term graft survival was associated with alloreactive T cell anergy. These findings delineate combination of transient mTOR inhibition with appropriate AAsTreg selection as an effective approach to promote long-term organ graft survival.
PMCID: PMC2923839  PMID: 20007530

Results 1-6 (6)