Serine Protease Inhibitor-1 Research Articles

Human FAM111A inhibits vaccinia virus replication by degrading viral protein I3 and is antagonized by poxvirus host range factor SPI-1

Zoonotic poxviruses such as mpox virus (MPXV) continue to threaten public health safety since the eradication of smallpox. Vaccinia virus (VACV), the prototypic poxvirus used as the vaccine strain for smallpox eradication, is the best-characterized member of the poxvirus family. VACV encodes a serine protease inhibitor 1 (SPI-1) conserved in all orthopoxviruses, which has been recognized as a host range factor for modified VACV Ankara (MVA), an approved smallpox vaccine and a promising vaccine vector. FAM111A (family with sequence similarity 111 member A), a nuclear protein that regulates host DNA replication, was shown to restrict the replication of a VACV SPI-1 deletion mutant (VACV-ΔSPI-1) in human cells. Nevertheless, the detailed antiviral mechanisms of FAM111A were unresolved. Here, we show that FAM111A is a potent restriction factor for VACV-ΔSPI-1 and MVA. Deletion of FAM111A rescued the replication of MVA and VACV-ΔSPI-1 and overexpression of FAM111A significantly reduced viral DNA replication and virus titers but did not affect viral early gene expression. The antiviral effect of FAM111A necessitated its trypsin-like protease domain and DNA-binding domain but not the PCNA-interacting motif. We further identified that FAM111A translocated into the cytoplasm upon VACV infection by degrading the nuclear pore complex via its protease activity, interacted with VACV DNA-binding protein I3, and promoted I3 degradation through autophagy. Moreover, SPI-1 from VACV, MPXV, or lumpy skin disease virus was able to antagonize FAM111A by prohibiting its nuclear export. Our findings reveal the detailed mechanism by which FAM111A inhibits VACV and provide explanations for the immune evasive function of VACV SPI-1.

The susceptibility of SERPINE1 rs1799889 SNP in diabetic vascular complications: a meta-analysis of fifty-one case-control studies

BackgroundThe serine protease inhibitor-1 (SERPINE1) rs1799889 single nucleotide polymorphism (SNP) has been constantly associated with diabetes mellitus (DM) and its vascular complications. The aim of this meta-analysis was to evaluate this association with combined evidences.MethodsThe systematic search was performed for studies published up to March 2021 which assess the associations between SERPINE1 rs1799889 SNP and the risks of DM, diabetic retinopathy (DR), diabetic cardiovascular disease (CVD) and diabetic nephropathy (DN). Only case-control studies were identified, and the linkage between SERPINE1 rs1799889 polymorphism and diabetic vascular risks were evaluated using genetic models.Results51 comparisons were enrolled. The results revealed a significant association with diabetes risk in overall population (allelic: OR = 1.34, 95 % CI = 1.14–1.57, homozygous: OR = 1.66, 95 % CI = 1.23–2.14, heterozygous: OR = 1.35, 95 % CI = 1.08–1.69, dominant: OR = 1.49, 95 % CI = 1.18–1.88, recessive: OR = 1.30, 95 % CI = 1.06–1.59) as well as in Asian descents (allelic: OR = 1.45, 95 % CI = 1.16–1.82, homozygous: OR = 1.88, 95 % CI = 1.29–2.75, heterozygous: OR = 1.47, 95 % CI = 1.08-2.00, dominant: OR = 1.64, 95 % CI = 1.21–2.24, recessive: OR = 1.46, 95 % CI = 1.09–1.96). A significant association was observed with DR risk (homozygous: OR = 1.25, 95 % CI = 1.01–1.56, recessive: OR = 1.20, 95 % CI = 1.01–1.43) for overall population, as for the European subgroup (homozygous: OR = 1.32, 95 % CI = 1.02–1.72, recessive: OR = 1.38, 95 % CI = 1.11–1.71). A significant association were shown with DN risk for overall population (allelic: OR = 1.48, 95 % CI = 1.15–1.90, homozygous: OR = 1.92, 95 % CI = 1.26–2.95, dominant: OR = 1.41, 95 % CI = 1.01–1.97, recessive: OR = 1.78, 95 % CI = 1.27–2.51) and for Asian subgroup (allelic: OR = 1.70, 95 % CI = 1.17–2.47, homozygous: OR = 2.46, 95 % CI = 1.30–4.66, recessive: OR = 2.24, 95 % CI = 1.40–3.59) after ethnicity stratification. No obvious association was implied with overall diabetic CVD risk in any genetic models, or after ethnicity stratification.ConclusionsSERPINE1 rs1799889 4G polymorphism may outstand for serving as a genetic synergistic factor in overall DM and DN populations, positively for individuals with Asian descent. The association of SERPINE1 rs1799889 SNP and DR or diabetic CVD risks was not revealed.

SPI-1 is a missing host-range factor required for replication of the attenuated modified vaccinia Ankara (MVA) vaccine vector in human cells

Modified vaccinia virus Ankara (MVA) is the leading poxvirus vector for development of vaccines against diverse infectious diseases. This distinction is based on high expression of proteins and good immunogenicity despite an inability to assemble infectious progeny in human cells, which together promote efficacy and safety. Nevertheless, the basis for the host-range restriction is unknown despite past systematic attempts to identify the relevant missing viral gene(s). The search for host-range factors is exacerbated by the large number of deletions, truncations and mutations that occurred during the long passage history of MVA in chicken embryo fibroblasts. By whole genome sequencing of a panel of recombinant host-range extended (HRE) MVAs generated by marker rescue with 40 kbp segments of vaccinia virus DNA, we identified serine protease inhibitor 1 (SPI-1) as one of several candidate host-range factors present in those viruses that gained the ability to replicate in human cells. Electron microscopy revealed that the interruption of morphogenesis in human cells infected with MVA occurred at a similar stage as that of a vaccinia virus strain WR SPI-1 deletion mutant. Moreover, the introduction of the SPI-1 gene into the MVA genome led to more than a 2-log enhancement of virus spread in human diploid MRC-5 cells, whereas deletion of the gene diminished the spread of HRE viruses by similar extents. Furthermore, MRC-5 cells stably expressing SPI-1 also enhanced replication of MVA. A role for additional host range genes was suggested by the restoration of MVA replication to a lower level relative to HRE viruses, particularly in other human cell lines. Although multiple sequence alignments revealed genetic changes in addition to SPI-1 common to the HRE MVAs, no evidence for their host-range function was found by analysis thus far. Our finding that SPI-1 is host range factor for MVA should simplify use of high throughput RNAi or CRISPR/Cas single gene methods to identify additional viral and human restriction elements.

Vaccine potential of antigen cocktails composed of recombinant Toxoplasma gondii TgPI-1, ROP2 and GRA4 proteins against chronic toxoplasmosis in C3H mice

The development of an effective and safe vaccine to prevent Toxoplasma gondii infection is an important aim due to the great clinical and economic impact of this parasitosis. We have previously demonstrated that immunization with the serine protease inhibitor-1 (TgPI-1) confers partial protection to C3H/HeN and C57BL/6 mice. In order to improve the level of protection, in this work, we combined this novel antigen with ROP2 and/or GRA4 recombinant proteins (rTgPI-1+rROP2, rTgPI-1+rGRA4, rTgPI-1+rROP2+rGRA4) to explore the best combination against chronic toxoplasmosis in C3H/HeN mice. All tested vaccine formulations, administered following a homologous prime-boost protocol that combines intradermal and intranasal routes, conferred partial protection as measured by the reduction of brain cyst burden following oral challenge with tissue cysts of Me49 T. gondii strain. The highest level of protection was achieved by the mixture of rTgPI-1 and rROP2 proteins with an average parasite burden reduction of 50% compared to the unvaccinated control group. The vaccine-induced protective effect was related to the elicitation of systemic cellular and humoral immune responses that included antigen-specific spleen cell proliferation, the release of Th1/Th2 cytokines, and the generation of antigen-specific antibodies in serum. Additionally, mucosal immune responses were also induced, characterized by secretion of antigen-specific IgA antibodies in intestinal lavages and specific mesenteric lymph node cell proliferation. Our results demonstrate that rTgPI-1+rROP2 antigens seem a promising mixture to be combined with other immunogenic proteins in a multiantigenic vaccine formulation against toxoplasmosis.

Triad of human cellular proteins, IRF2, FAM111A, and RFC3, restrict replication of orthopoxvirus SPI-1 host-range mutants

Viruses and their hosts can reach balanced states of evolution ensuring mutual survival, which makes it difficult to appreciate the underlying dynamics. To uncover hidden interactions, virus mutants that have lost defense genes may be used. Deletion of the gene that encodes serine protease inhibitor 1 (SPI-1) of rabbitpox virus and vaccinia virus, two closely related orthopoxviruses, prevents their efficient replication in human cells, whereas certain other mammalian cells remain fully permissive. Our high-throughput genome-wide siRNA screen identified host factors that prevent reproduction and spread of the mutant viruses in human cells. More than 20,000 genes were interrogated with individual siRNAs and those that prominently increased replication of the SPI-1 deletion mutant were subjected to a secondary screen. The top hits based on the combined data-replication factor C3 (RFC3), FAM111A, and interferon regulatory factor 2 (IRF2)-were confirmed by custom assays. The siRNAs to RFC1, RFC2, RFC4, and RFC5 mRNAs also enhanced spread of the mutant virus, strengthening the biological significance of the RFC complex as a host restriction factor for poxviruses. Whereas association with proliferating cell nuclear antigen and participation in processive genome replication are common features of FAM111A and RFC, IRF2 is a transcriptional regulator. Microarray analysis, quantitative RT-PCR, and immunoblotting revealed that IRF2 regulated the basal level expression of FAM111A, suggesting that the enhancing effect of depleting IRF2 on replication of the SPI-1 mutant was indirect. Thus, the viral SPI-1 protein and the host IRF2, FAM111A, and RFC complex likely form an interaction network that influences the ability of poxviruses to replicate in human cells.

Homologous prime-boost strategy with TgPI-1 improves the immune response and protects highly susceptible mice against chronic Toxoplasma gondii infection

Subunit-based vaccines are safer than live or attenuated pathogen vaccines, although they are generally weak immunogens. Thus, proper combination of immunization strategies and adjuvants are needed to increase their efficacy. We have previously protected C3H/HeN mice from Toxoplasma gondii infection by immunization with the serine protease inhibitor-1 (TgPI-1) in combination with alum. In this work, we explore an original vaccination protocol that combines administration of recombinant TgPI-1 by intradermal and intranasal routes in order to enhance protection in the highly susceptible C57BL/6 strain. Mice primed intradermally with rTgPI-1 plus alum and boosted intranasally with rTgPI-1 plus CpG-ODN elicited a strong specific Th1/Th2 humoral response, along with a mucosal immune response characterized by specific-IgA in intestinal lavages. A positive cellular response of mesentheric lymph node cells and Th1/Th2 cytokine secretion in the ileon were also detected. When immunized mice were challenged with the cystogenic Me49 T. gondii strain, they displayed up to 62% reduction in brain parasite burden. Moreover, adoptive transfer of mesenteric lymph node cells from vaccinated to naïve mice induced significant protection against infection. These results demonstrate that this strategy that combines the administration of TgPI-1 by two different routes, intradermal priming and intranasal boost, improves protective immunity against T. gondii chronic infection in highly susceptible mice.

Serine Protease Inhibitor-6 Differentially Affects the Survival of Effector and Memory Alloreactive CD8-T Cells

The clonal expansion of effector T cells and subsequent generation of memory T cells are critical in determining the outcome of transplantation. While cytotoxic T lymphocytes induce direct cytolysis of target cells through secretion of Granzyme-B (GrB), they also express cytoplasmic serine protease inhibitor-6 (Spi6) to protect themselves from GrB that has leaked from granules. Here, we studied the role of GrB/Spi6 axis in determining clonal expansion of alloreactive CD8-T cells and subsequent generation of memory CD8-T cells in transplantation. CD8-T cells from Spi6(-/-) mice underwent more GrB mediated apoptosis upon alloantigen stimulation in vitro and in vivo following adoptive transfer into an allogeneic host. Interestingly, while OT1.Spi6(-/-) CD8 T cells showed significantly lower clonal expansion following skin transplants from OVA mice, there was no difference in the size of the effector memory CD8-T cells long after transplantation. Furthermore, lack of Spi6 resulted in a decrease of short-lived-effector-CD8-cells but did not impact the pool of memory-precursor-effector-CD8-cells. Similar results were found in heart transplant models. Our findings suggest that the final alloreactive CD8-memory-pool-size is independent from the initial clonal-proliferation as memory precursors express low levels of GrB and therefore are independent of Spi6 for survival. These data advance our understanding of memory T cells generation in transplantation and provide basis for Spi6 based strategies to target effector T cells.

Serine protease inhibitor-6 inhibits granzyme B-mediated injury of renal tubular cells and promotes renal allograft survival.

Protease inhibitor 9 (PI-9) is an intracellular serpin that specifically inhibits granzyme B, a cytotoxic serine protease found in the cytosolic granules of cytotoxic T lymphocytes and natural killer cells. Enhanced cortical expression of PI-9 has been observed in kidney allografts with subclinical rejection, suggesting that the tubular epithelial cell (TEC) expression of this protein may have a protective role and attenuate overt allograft rejection. We demonstrate that TEC express SPI-6 protein, the murine homolog of PI-9, basally with a modest increase after cytokine exposure. Tubular epithelial cell expression of SPI-6 blocks granzyme B-mediated death because TEC from SPI-6 null kidneys have increased susceptibility to cytotoxic CD8+ cells in vitro. The role of SPI-6 was tested in a mouse kidney transplant model using SPI-6 null or wild type donor kidneys (H-2) into nephrectomized recipients (H-2). SPI-6 null kidney recipients demonstrated reduced renal function at day 8 after transplantation compared to controls (creatinine, 113±23 vs. 28±3 μmol/L; n=5; P<0.01), consistent with observed tubular injury and extensive mononuclear cell infiltration. Loss of donor kidney SPI-6 shortened graft survival time (20±19 vs. 66±33 days; n=8-10; P<0.001). Our data show for the first time that resistance of kidney TEC to cytotoxic T-cell granzyme B-induced death in vitro and in vivo is mediated by the expression of SPI-6. We suggest that SPI-6 is an important endogenous mechanism to prevent rejection injury from perforin or granzyme B effectors and enhanced PI-9 or SPI-6 expressions by TEC may provide protection from diverse forms of inflammatory kidney injury and promote long-term allograft survival.

DN Treg Cells Specifically Suppress Cognate Alloreactive Memory T Cell By Granzyme B, and Prolong Allograft Survival.

[Background] Since the emphasis of clinical organ transplant survival moved from short term to long term, memory T (Tm) cell, by mediating several key-mechanisms such as transplant vasculopathy and allograft nephropathy, has been recognized as an essential culprit of long term organ rejection. It is inherently resistant to conventional therapies such as apoptosis-induction and co-stimulation-blockade, yet indispensable to the recipient's protective immunity against environmental pathogens. In this study, we show that double-negative (DN) Treg cell might be a delicate solution to this dilemma. [Methods and Results] DN Tregs specifically suppress cognate B6-anti-Balb/c CD8+ Tm both in vitro (suppression rate: 53.9±1.2%), and in vivo, with the CD8+ Tm cell-mediated graft rejection delayed from (21.5±1.7)-day to (63.0±4.7)-day in a BALB/c to B6Rag1-/- skin transplantation, but with no effect on anti-C3H Tm. Both CD8+Tem and Tcm compartments are significantly suppressed by DN Treg. We further identified the suppression as Granzyme B (GzmB)- and perforin (PFN)-dependent. Intriguingly, CD4+ Tm cells resist DN Treg both in vitro and in vivo. For the first time, we found CD4+ Tm highly express Serine Protease Inhibitor-6 (Spi6) ((5.03±1.15)-fold of CD4+ Teff, protein level), an inhibitor of GzmB. The deficiency of Spi6 sensitized CD4+ Tm both in vitro (suppression rate: 10.5±5.8% to 37.5±6.2%) and in vivo (skin-allograft survival from (28.3±5.7)-day to (75.5±7.9)-day) to killing by DN Tregs. Whereas the restored sensitivity can be again abrogated in vitro (suppression rate: 4.5±3.2%) and in vivo (skin-allograft survival (22.5±2.4)-day) by PFN-deficiency in DN Tregs. [Conclusion] In summary, DN Tregs specifically suppress cognate allo-reactive CD8+ Tm cells by GzmB-PFN pathway, and effectively delay CD8+ Tm cell-mediated graft rejection without compromising Tm cell-dependent protective immunity. The fact that CD4+ Tm resists DN Treg by highly expressing Spi6 and can be sensitized by Spi6-deficiency underlines the essential role of Spi6 in CD4+ Tm cell fate, which also shed light to the possibility of combining DN Treg and Spi6-targeting therapy to even further improve long-term allograft survival.

Identification of potential general markers of disease resistance in American oysters, Crassostrea virginica through gene expression studies

Several diseases have a significant impact on American oyster populations in the Atlantic coasts of North America. Knowledge about the responses of oysters to pathogenic challenge could help in identifying potential markers of disease resistance and biomarkers of the health status of an oyster population. A previous analysis of the transcriptome of resistant and susceptible American oysters in response to challenge with the bacterial pathogen Roseovarius crassostreae, as well as sequencing of suppression subtractive hybridization libraries from oysters challenged with the protozoan parasite Perkinsus marinus, provided a list of genes potentially involved in disease resistance or susceptibility. We investigated the patterns of inducible gene expression of several of these genes in response to experimental challenge with the oyster pathogens R. crassostreae, Vibrio tubiashii, and P. marinus. Oysters showing differential susceptibility to R. crassostreae demonstrated differential patterns of expression of genes coding for immune (serine protease inhibitor-1, SPI1) and stress-related (heat shock protein 70, HSP70; arginine kinase) proteins 30 days after challenge with this bacterial pathogen. Differential patterns of expression of immune (spi1, galectin and a matrix metalloproteinase) and stress-related (hsp70, histone H4, and arginine kinase) genes was observed in hemocytes from adult oysters challenged with P. marinus, but not with V. tubiashii. While levels of spi1 expression in hemocytes collected 8 and 21 days after P. marinus challenge were negatively correlated with parasite load in oysters tissues at the end of the challenge (62 days), levels of expression of hsp70 in hemocytes collected 1-day after challenge were positively correlated with oyster parasite load at 62 days. Our results confirm previous research on the role of serine protease inhibitor-1 in immunity and disease resistance in oysters. They also suggest that HSP70 and histone H4 could be used as a markers of health status or disease susceptibility in oysters.

Spi6 Protects Alloreactive CD4+ But Not CD8+ Memory T Cell From Granzyme B Attack by Double-Negative T Regulatory Cell

Memory T (Tm) cells pose a major barrier to long-term transplant survival. Whether regulatory T cells (Tregs)can control them remains poorly defined. Previously,we established that double-negative (DN) Tregs suppress effector T (Teff) cells. Here, we demonstrate that DNTregs effectively suppress CD4+/CD8+Teff and CD8+Tm but not CD4+Tm cells, whereas the suppression on CD8+Tm is abrogated by perforin (PFN) deficiency in DNTregs. Consistently, in a BALB/c to B6-Rag1-/-skin transplantation, transfer of DN Tregs suppressed the rejection mediated by CD4þ/CD8+Teff and CD8+Tmcells (76.0±4.9, 87.5±5.0 and 63.0±4.7 days, respectively)but not CD4þTmcells (25.3±1.4 days). Both CD8þ effector memory T and central memory T compartments significantly reduced after DN Treg transfer. CD4+Tm highly expresses granzyme B (GzmB) inhibitor serine protease inhibitor-6 (Spi6). Spi6 deficiency renders CD4þTm susceptible to DN Treg suppression. In addition,transfer of WT DN Tregs, but not PFN-/-DN Tregs,inhibited the skin allograft rejection mediated by Spi6-/-CD4þTm(75.5±7.9 days). In conclusion, CD4+ and CD8+Tm cells differentially respond toDNTregs’ suppression.The GzmB resistance conferred by Spi6 in CD4þTm cells might hint at the physiological significance of Tmpersistence

Study on cardiovascular diseases marker gene expression profile of familial aggregation hypertension

To explore the cardiovascular diseases marker gene expression profile of the familial aggregation hypertension patients,and to screen differentially expressed genes. The patients who had directly related family members for more than three generations suffering from hypertension were selected as experiment group, and healthy individuals as control group. Oligo GEArray gene chip technique was used to detect the expression of cardiovascular diseases marker gene in peripheral blood. The ratio of positive/negative standard value >2.0, or ≤0.5 and >0 was identified as differential gene. Compared with control group, there were 10 up-regulated differential genes in experiment group, composing genes involved in lipid metabolism, immune response-related molecules, cell adhesion molecules, extracellular molecules and coagulation, including apolipoprotein E (ApoE), epithelial V-like antigen-1 (EVA-1), interferon-γ (IFN-γ), interleukin-1β (IL-1β), IL-8, integrin-β1 (ITGB-1), matrix metalloproteinase-9 (MMP-9), nuclear factor-ΚB (NF-ΚB), platelet endothelial cell adhesion molecule-1 (PECAM-1), selectin-P (SEL-P). There were 3 down-regulated genes, including coagulation factors-III (F-III), lectin-like oxidized low density lipoprotein receptor-1 (LOX-1), and serine protease inhibitor-1 (SERPINE-1). This study suggested that familial aggregation hypertension related to a variety of gene markers of cardiovascular disease, especially elements concerning coagulation and extracellular protease inhibitor-related genes.

Proteinase inhibitor–9 expression is induced by maturation in dendritic cells via p38 MAP kinase

Dendritic cells (DC) are crucial for the induction of CD8(+) T cell responses. However, as DC present antigen, they may also be at risk for being killed by recent activated CD8+ T cells. Previously we have shown that lipopolysaccharide (LPS)- or CD40L-stimulated monocyte-derived DC express high levels of the granzyme B-inhibitory serpin proteinase inhibitor-9 (PI-9). Furthermore we found that its murine homolog serine protease inhibitor-6 (SPI-6) protected murine DC against cytotoxic T lymphocyte-induced apoptosis. These data suggest that PI-9/SPI-6 may regulate survival of DC when they are under attack by effector cells. We therefore analyzed how PI-9 is regulated upon stimulation with several Toll-like receptor ligands. We found that the expression of PI-9 correlated with maturation, as measured with CD86 levels, but not with the production of interleukin-12-p70. Using LPS as stimulus, we also showed that the induction of PI-9 is dependent on the p38 mitogen-activated protein kinase signaling pathway. The data suggest that PI-9 is tightly linked to maturation and may allow DC to exert their function in a potentially hostile environment.

The role of serpinb9/serine protease inhibitor 6 in preventing granzyme B–dependent hepatotoxicity

Virally infected hepatocytes are resistant to cytotoxic lymphocyte killing by perforin-dependent and granzyme-dependent effector mechanisms. The present studies were designed to examine the role of serine protease inhibitor 6 (SPI-6) in limiting granzyme B-dependent cytotoxic effector mechanisms in the liver. SPI-6-specific small interfering RNA (siRNA) administration to C57Bl/6J (B6) mice elicited transient alanine aminotransferase (ALT) elevations that were not observed in either granzyme B-deficient B6 (B6.gzmb(-/-)) or natural killer (NK) cell-depleted B6 mice. When SPI-6 expression was abolished by siRNA administration at the time of infection with a recombinant, replication-deficient adenovirus [E1-deleted adenovirus encoding beta-galactosidase (AdCMV-LacZ)], earlier and dramatically increased, and earlier ALT elevations were observed in wild-type B6 but not in B6.gzmb(-/-) or NK cell-depleted mice. When a 3-fold higher dose of AdCMV-LacZ was administered to B6 mice, the coadministration of SPI-6 siRNA resulted in the early onset of lethal, acute liver failure. Of note, the accelerated clearance of AdCMV-LacZ was observed in recipients of SPI-6 siRNA. These results indicate that the regulated expression of SPI-6 in hepatocytes during viral infection or following noninfectious causes of liver injury protects hepatocytes against excessively vigorous granzyme B-dependent killing but may also delay immune clearance of virally infected hepatocytes.

Increased Sensitivity of Radiated Murine Cervical Cancer Tumors to E7 Subunit Vaccine–driven CTL-mediated Killing Induces Synergistic Anti-tumor Activity

The development of therapeutic vaccines has important implications for the treatment of cancer patients. Here we investigate whether human papillomavirus (HPV) E7 subunit vaccines can enhance tumor radioresponse using an established cervical cancer animal model. Radiation plus E7 subunit vaccines improved complete response, cure, and recurrence rates of tumors dramatically compared with single therapy alone. In particular, both components of the E7 subunit vaccines (E7 protein and CpG-oligodeoxynucleotide) were required for the induction of antigen (Ag)-specific cytotoxic T-lymphocyte (CTL) responses and for therapeutic synergy with radiotherapy. Moreover, with combined therapy the radiation dose could be reduced by 16 Gy to achieve an equivalent anti-tumor efficacy to radiation treatment alone. This therapeutic synergy was found to be mediated by CD8(+) CTLs and was concomitant with histological changes (presence of apoptotic bodies and multinucleated giant cells; heavy infiltration of lymphocytes), as determined by in vivo T-cell depletion and histological analysis. Finally, phenotypic changes of radiated tumors and their increased sensitivity to CTL-mediated killing appeared to be responsible for therapeutic synergy. These results show that E7 subunit vaccines act as a potent enhancer of tumor radioresponse and that this is mediated by increased sensitivity of radiated tumors to CTL-mediated killing. This study further suggests that E7-targeted therapeutic vaccines have the potential to improve radiotherapy in patients with cervical cancer.

672. The Effect of Serine Protease Inhibitor 6 (SPI-6) Gene Transfer on a Mouse Model of Sjögren's Syndrome

Top of pageAbstract Sj|[ouml]|gren's syndrome (SS) is an autoimmune disease characterized by a focal and diffuse lymphoid cell infiltration into the salivary and lacrimal glands. This chronic immune cell activation leads to reduced secretory function with symptoms of xerostomia (dry mouth) and keratoconjunctivitis sicca (dry eyes). It has been hypothesized that these sequelae are due to apoptosis of the exocrine epithelial cells. Apoptosis can be induced by cytotoxic T-lymphocytes (CTLs), which contain the enzyme Granzyme B (GrB) in cytoplasmic granules. GrB initiates a caspase-independent apoptosis pathway and is inhibited by serine proteinase inhibitor 6 (SPI-6) in the mouse. In this study, we evaluated the effect of transgenic SPI-6 produced in salivary glands after gene transfer to non-obese diabetic (NOD) mice, which develop exocrine gland infiltrates and are a commonly used disease model for SS. We constructed two serotype 2 rAAV vectors, rAAVSPI-6 and rAAVLacZ (encoding |[beta]|-galactosidase; vector control) using the CMV promoter. rAAVs (1010 particles/gland) were administered directly into both submandibular glands of 8-week old female NOD mice (n=8) via retroductal delivery. Salivary flow rates were determined before vector delivery and at time of sacrifice (16-weeks old). Serum glucose levels and weight were measured weekly and diabetic mice (serum glucose|[ge]|400 mg/dL) were treated with insulin. After sacrifice, submandibular glands were harvested and analyzed for inflammatory infiltrates (focus scores), as well as SPI-6 expression and cytokine profile [mouse interleukins (IL)-2, 4, 6, 12, 18, interferon (IFN)-|[gamma]|, tumor necrosis factor-|[alpha]| and RANTES] in aqueous gland extracts. At 8 weeks, salivary flow rates were not different between mice assigned to the rAAVSPI-6 and rAAVLacZ groups. In contrast, at 16 weeks flow rates were significantly higher in the rAAVSPI-6-treated mice (p=0.018). Submandibular gland IL-10 levels were significantly increased (p=0.009), while IFN-|[gamma]| levels were significantly decreased (p=0.029), in the rAAVSPI-6 mice compared to the rAAVLacZ group. There were no differences in focus scores, serum glucose levels or animal weights between the two treatment groups at 16 weeks. Overall, these results suggest that SPI-6 gene transfer may be beneficial in limiting the autoimmune exocrinopathy of NOD mice.

268. Enhancement of DNA Vaccine Potency by Coadministration of a Tumor Antigen Gene and DNA Encoding Serine Protease Inhibitor-6

Top of pageAbstract Serine protease inhibitor 6 (SPI-6), also called Serpinb9, inhibits granzyme B and thus may provide a method for delaying apoptotic cell death in dendritic cells. We have previously enhanced DNA vaccine potency by targeting antigen to MHC antigen presentation pathways, using proteins such as Mycobacterium tuberculosis heat shock protein 70, calreticulin, domain II of Pseudomonas aeruginosa exotoxin A, or the sorting signal of the lysosome-associated membrane protein type 1. In this study, we explored intradermal coadministration of DNA encoding SPI-6 with DNA constructs encoding human papillomavirus type 16 E7 linked to these intracellular targeting molecules for its ability to generate E7-specific CD8+ T-cell immune responses and E7-specific antitumor effects. This combination of strategies resulted in significantly increased E7-specific CD8+ T-cell and CD4+ Th1-cell responses, enhanced tumor treatment ability, and stronger tumor protection when compared with vaccination without SPI-6. Among these targeting strategies tested, mice vaccinated with Sig/E7/lysosome-associated membrane protein type 1 mixed with SPI-6 showed the greatest fold increase in E7-specific CD8+ T cells ( approximately 5-fold). Vaccination with a nonfunctional mutant of SPI-6 did not result in immune enhancement, indicating that enhancement was dependent on the antiapoptotic function of SPI-6. Our results suggest that DNA vaccines combining strategies that enhance MHC class I and II antigen processing with SPI-6 have potential clinical implications for control of viral infection and neoplasia.

Enhancement of DNA vaccine potency by coadministration of a tumor antigen gene and DNA encoding serine protease inhibitor-6.

Serine protease inhibitor 6 (SPI-6), also called Serpinb9, inhibits granzyme B and thus may provide a method for delaying apoptotic cell death in dendritic cells. We have previously enhanced DNA vaccine potency by targeting antigen to MHC antigen presentation pathways, using proteins such as Mycobacterium tuberculosis heat shock protein 70, calreticulin, domain II of Pseudomonas aeruginosa exotoxin A, or the sorting signal of the lysosome-associated membrane protein type 1. In this study, we explored intradermal coadministration of DNA encoding SPI-6 with DNA constructs encoding human papillomavirus type 16 E7 linked to these intracellular targeting molecules for its ability to generate E7-specific CD8+ T-cell immune responses and E7-specific antitumor effects. This combination of strategies resulted in significantly increased E7-specific CD8+ T-cell and CD4+ Th1-cell responses, enhanced tumor treatment ability, and stronger tumor protection when compared with vaccination without SPI-6. Among these targeting strategies tested, mice vaccinated with Sig/E7/lysosome-associated membrane protein type 1 mixed with SPI-6 showed the greatest fold increase in E7-specific CD8+ T cells ( approximately 5-fold). Vaccination with a nonfunctional mutant of SPI-6 did not result in immune enhancement, indicating that enhancement was dependent on the antiapoptotic function of SPI-6. Our results suggest that DNA vaccines combining strategies that enhance MHC class I and II antigen processing with SPI-6 have potential clinical implications for control of viral infection and neoplasia.

Spi-1: an hepatic serine protease inhibitor regulated by GH and other hormones

A sensitive RNAse protection method was used to show that serine protease inhibitor-1 (Spi-1) is expressed in rat liver and heart, but not in kidney or brain. Bovine somatotropin (bGH) and placental lactogen (bPL) induced rat hepatocyte cultures to express both Spi-1 and IGF-1 mRNA, with bPL approximately 100-fold more potent than bGH. Bovine prolactin (bPrL) did not induce hepatocyte Spi-1 mRNA, demonstrating lack of involvement of lactogenic receptors. Albumin mRNA levels were stable during hepatocyte culturing and were unaffected by growth hormone (GH) treatment, showing that neither culture conditions nor GH treatment affected cellular differentiation. Eliminating serum-free medium hormone supplements one at a time, estradiol, testosterone and T 3 were shown to be unnecessary for GH induction of Spi-1, while dexamethasone removal decreased Spi-1 mRNA levels to 10% of GH-stimulated controls. bGH induction of Spi-1 mRNA in the presence of only dexamethasone and glucagon was 75% higher ( p< 0.01) than levels seen with insulin also present.