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Current human papillomavirus (HPV) vaccines provide substantial protection against the most common HPV types responsible for oral and anogenital cancers, but many circulating cancer-causing types remain for which vaccine coverage is lacking. In addition, all current HPV vaccines rely on aluminum salt-based adjuvant formulations that function through unclear mechanisms with few substitutes available. In an effort to expand the toolbox of available adjuvants suitable for HPV vaccines, we compared the immunogenicity of the RG1-VLP (virus-like particle) vaccine in BALB/c mice when formulated with either the aluminum hydroxide adjuvant Alhydrogel or the novel polyphosphazene macromolecular adjuvant poly[di (carboxylatoethylphenoxy) phosphazene] (PCEP). PCEP-formulated RG1-VLPs routinely outperformed VLP/Alhydrogel in several measurements of VLP-specific humoral immunity, including consistent improvements in the magnitude of antibody (Ab) responses to both HPV16-L1 and the L2 RG1 epitope as well as neutralizing titers to HPV16 and cross-neutralization of pseudovirion (PsV) types HPV18 and HPV39. Dose-sparing studies indicated that RG1-VLPs could be reduced in dose by 75% and the presence of PCEP ensured activity comparable to a full VLP dose adjuvanted by Alhydrogel. In addition, levels of HPV16-L1 and -L2-specific Abs were achieved after two vaccinations with PCEP as adjuvant that were equivalent to or greater than levels achieved with three vaccinations with Alhydrogel alone, indicating that the presence of PCEP resulted in accelerated immune responses that could allow for a decreased dose schedule. Given the extensive clinical track record of polyphosphazenes, these data suggest that substitution of alum-based adjuvants with PCEP for the RG1-VLP vaccine could lead to rapid seropositivity requiring fewer boosts, the dose-sparing of commercial VLP-based vaccines, and the establishment of longer-lasting humoral responses to HPV.

Current human papilloma virus (HPV) vaccines provide substantial protection against the most common HPV types responsible for oral and anogenital cancers, but many circulating cancer-causing types remain that lack vaccine coverage. The novel RG1-VLP (virus-like particle) vaccine candidate utilizes the HPV16-L1 subunit as a backbone to display an inserted HPV16-L2 17–36 a.a. “RG1” epitope; the L2 RG1 epitope is conserved across many HPV types and the generation of cross-neutralizing antibodies (Abs) against which has been demonstrated. In an effort to heighten the immunogenicity of the RG1-VLP vaccine, we compared in BALB/c mice adjuvant formulations consisting of novel bacterial enzymatic combinatorial chemistry (BECC)-derived toll-like receptor 4 (TLR4) agonists and the aluminum hydroxide adjuvant Alhydrogel. In the presence of BECC molecules, consistent improvements in the magnitude of Ab responses to both HPV16-L1 and the L2 RG1 epitope were observed compared to Alhydrogel alone. Furthermore, neutralizing titers to HPV16 as well as cross-neutralization of pseudovirion (PsV) types HPV18 and HPV39 were augmented in the presence of BECC agonists as well. Levels of L1 and L2-specific Abs were achieved after two vaccinations with BECC/Alhydrogel adjuvant that were equivalent to or greater than levels achieved with 3 vaccinations with Alhydrogel alone, indicating that the presence of BECC molecules resulted in accelerated immune responses that could allow for a decreased dose schedule for VLP-based HPV vaccines. In addition, dose-sparing studies indicated that adjuvantation with BECC/Alhydrogel allowed for a 75% reduction in antigen dose while still retaining equivalent magnitudes of responses to the full VLP dose with Alhydrogel. These data suggest that adjuvant optimization of HPV VLP-based vaccines can lead to rapid immunity requiring fewer boosts, dose-sparing of VLPs expensive to produce, and the establishment of a longer-lasting humoral immunity.

Poly[di(carboxylatomethylphenoxy)phosphazene] (PCMP), a new member of polyphosphazene immunoadjuvant family, is synthesized. In vitro assessment of a new macromolecule revealed hydrolytic degradation profile and immunostimulatory activity comparable to its clinical stage homologue PCPP; however, PCMP was characterized by a beneficial reduced sensitivity to the ionic environment. In vivo evaluation of PCMP potency was conducted with human papillomavirus (HPV) virus-like particles (VLPs) based RG1-VLPs vaccine. In contrast with previously reported self-assembly of polyphosphazene adjuvants with proteins, which typically results in the formation of complexes with multimeric display of antigens, PCMP surface modified VLPs in a composition dependent pattern, which at a high polymer-to VLPs ratio led to stabilization of antigenic particles. Immunization experiments in mice demonstrated that PCMP adjuvanted RG1-VLPs vaccine induced potent humoral immune responses, in particular, on the level of highly desirable protective cross-neutralizing antibodies, and outperformed PCPP and Alhydrogel adjuvanted formulations.

Background Patients with hereditary diffuse gastric cancer (HDGC), a cancer predisposition syndrome associated with germline mutations of the CDH1 (E-cadherin) gene, have few effective treatment options. Despite marked differences in natural history, histopathology, and genetic profile to patients afflicted by sporadic gastric cancer, patients with HDGC receive, in large, identical systemic regimens. The lack of a robust preclinical in vitro system suitable for effective drug screening has been one of the obstacles to date which has hampered therapeutic advances in this rare disease. Methods In order to identify therapeutic leads selective for the HDGC subtype of gastric cancer, we compared gene expression profiles and drug phenotype derived from an oncology library of 1912 compounds between gastric cancer cells established from a patient with metastatic HDGC harboring a c.1380delA CDH1 germline variant and sporadic gastric cancer cells. Results Unsupervised hierarchical cluster analysis shows select gene expression alterations in c.1380delA CDH1 SB.mhdgc-1 cells compared to a panel of sporadic gastric cancer cell lines with enrichment of ERK1–ERK2 (extracellular signal regulated kinase) and IP3 (inositol trisphosphate)/DAG (diacylglycerol) signaling as the top networks in c.1380delA SB.mhdgc-1 cells. Intracellular phosphatidylinositol intermediaries were increased upon direct measure in c.1380delA CDH1 SB.mhdgc-1 cells. Differential high-throughput drug screening of c.1380delA CDH1 SB.mhdgc-1 versus sporadic gastric cancer cells identified several compound classes with enriched activity in c.1380 CDH1 SB.mhdgc-1 cells including mTOR (Mammalian Target Of Rapamycin), MEK (Mitogen-Activated Protein Kinase), c-Src kinase, FAK (Focal Adhesion Kinase), PKC (Protein Kinase C), or TOPO2 (Topoisomerase II) inhibitors. Upon additional drug response testing, dual PI3K (Phosphatidylinositol 3-Kinase)/mTOR and topoisomerase 2A inhibitors displayed up to >100-fold increased activity in hereditary c.1380delA CDH1 gastric cancer cells inducing apoptosis most effectively in cells with deficient CDH1 function. Conclusion Integrated pharmacological and transcriptomic profiling of hereditary diffuse gastric cancer cells with a loss-of-function c.1380delA CDH1 mutation implies various pharmacological vulnerabilities selective to CDH1-deficient familial gastric cancer cells and suggests novel treatment leads for future preclinical and clinical treatment studies of familial gastric cancer. Electronic supplementary material The online version of this article (doi:10.1186/s12967-017-1197-5) contains supplementary material, which is available to authorized users.

Complement activation contributes to the expression of local and remote organ injury in animal models of ischemia-reperfusion (IR). We demonstrate here that a soluble form of decay-accelerating factor (DAF) protects normal C57Bl/6 and autoimmunity-prone B6.MRL/lpr mice subjected to hindlimb IR from remote intestinal and lung injury without affecting the degree of local skeletal muscle injury. In addition, DAF treatment attenuates remote organ injury in mice subjected to mesenteric IR. Soluble DAF allowed the deposition of complement 3 in local and remote injury sites while it limited the presence of terminal membrane attack complex and did not increase animal susceptibility to sepsis. These data provide evidence that soluble DAF might offer clinical benefit to patients suffering remote intestinal or lung damage in response to muscle or other organ injury.

Regulatory T cells (T Reg ), CD4 + CD25 + FOXP3 + , are implicated in suppressing tumor immune responses. We analyzed peripheral blood lymphocytes (PBL) from breast cancer patients receiving a modified HLA class II HER2/ neu peptide (AE37) vaccine for T Reg cells and correlated their levels with vaccine-specific immune responses. The mean CD4 + CD25 + FOXP3 + T Reg cells decreased in patients with vaccination with no significant difference in serum TGF-β levels. IFN-γ ELISPOT and DTH increased after vaccination with a good correlation between T Reg cell reduction and size of DTH to AE37. The T Reg cell reduction and associated immune response suggest that AE37 may be clinically useful.

Elements of the innate and adaptive immune response have been implicated in the development of tissue damage after ischemic reperfusion (I/R). Here we demonstrate that T cells infiltrate the intestine of C57BL/6 mice subjected to intestinal I/R during the first hour of reperfusion. The intensity of the T cell infiltration was higher in B6.MRL/lpr mice subjected to intestinal I/R and reflected more severe tissue damage than that observed in control mice. Depletion of T cells limited I/R damage in B6.MRL/lpr mice, whereas repletion of B6.MRL/lpr lymph node-derived T cells into the I/R-resistant Rag-1−/− mouse reconstituted tissue injury. The tissue-infiltrating T cells were found to produce IL-17. Finally, IL-23 deficient mice, which are known not to produce IL-17, displayed significantly less intestinal damage when subjected to I/R. Our data assign T cells a major role in intestinal I/R damage by virtue of producing the pro-inflammatory cytokine IL-17.

Natural Abs and autoantibodies bind antigens displayed by ischemia-conditioned tissues, followed by complement activation and enhanced tissue injury during reperfusion. Anti-ribonucleoprotein (RNP) Ab is associated with lung disease in patients with autoimmune disease but it is not known whether these abs contribute to lung injury. Mesenteric I/R in mice leads to local and remote lung injury. Accordingly, we used this model to investigate whether anti-RNP Abs would reconstitute I/R damage with prominent lung damage in injury-resistant Rag1(-/-) animals. Rag1(-/-) mice injected with anti-RNP Ab containing serum and subjected to mesenteric I/R suffered greater intestinal injury than control-treated and sham-operated animals. The magnitude of the reconstituted damage was anti-RNP Ab titer-dependent. Anti-RNP Ab-treated animals demonstrated a dose-dependent increase in lung histologic injury scores compared to control and sham animals. Anti-RNP mediated injury was shown to be complement dependent. These experiments reveal a novel mechanism whereby anti-RNP Abs contributes to the development of pulmonary pathology in patients with autoimmune diseases following exposure of remote organs to I/R injury.

Ischemia-reperfusion (IR) is a frequent clinical entity encountered in diverse fields such as vascular surgery, trauma, myocardial infarction, hemorrhage, stroke, and multi-organ failure. The acute inflammatory response following an ischemic insult instigates pathology in local and remote organs leading to significant morbidity and mortality. IR induces injury in a biphasic pattern. The acute phase is characterized by oxidative tissue injury resulting in activation of intracellular damage pathway and antibody-mediated complement activation. The sub acute phase is characterized by leukocyte infiltration. It has been shown previously in several models of IR that complement inhibitors reduce tissue injury. In this study the role of G-protein-coupled receptor signaling was examined. Studies in the mesenteric IR model a global Gi inhibitor, pertussis toxin, attenuated local intestinal tissue injury and production of LTB4 and PGE2. In these animals the systemic inflammatory response was also inhibited, assessed by decrease lung tissue injury and blocked cellular infiltrate. Next specific receptor inhibitors such as blocking antibodies to examine the effectiveness of inhibiting chemokine receptors such as CXCR2 or blocking adhesion molecules such as ICAM-1 or P-selectin were utilized. The data indicate specific chemokine receptors are more efficient in inhibiting tissue damage. Comparison of these inhibitors will be useful in developing therapeutics which can control deleterious effects of systemic inflammation after an initial trauma. USAMRAA W81xWH0610485

Introduction: We are conducting a Phase II clinical trial of 2 HER2 peptide vaccines, GP2 (MHC Class I restricted) and AE37 (MHC Class II restricted), for the prevention of breast cancer (BCa) recurrence in disease-free, high risk patients (pts). We present analysis of T cell populations in trial patients at the time of enrollment and differences based on time since chemotherapy. We describe a “reconstitution” of the immune system after immunosuppressive chemotherapy. Methods: After completion of standard therapy, disease-free, BCa pts were enrolled. Demographic data was collected. Blood was collected prior to administration of their first vaccine. Peripheral blood mononuclear cells from 50 pts were isolated and evaluated for CD8+, CD4+CD8+, CD4-CD8- and CD4+ T cell populations. T cell proliferation responses were measured in patients of both arms of the trial; generically with FluM-specific CD8 cells (HLA-A2:Ig dimer assay) in the GP2 arm (n=58), and then with proliferation response to AE36 and AE37 in the AE37 arm (n=85). Linear regression analyses evaluated the relationship between time from chemotherapy and each T cell population. Immune responses of pts enrolled less than one year from chemotherapy (1yr group) using a t-test. Results: Chemotherapy regimens were determined by the treating oncologist and consisted primarily of anthracycline-based regimens with a taxane. Regression analysis revealed a significant correlation between time from chemotherapy and both CD4+ and CD8+ Tcell counts (R= .433, p=0.015 and R=.439, p=.014, respectively). Total T cell, CD4-CD8- and CD4+CD8+ populations, however, did not significantly correlate with increased time from chemotherapy (R=.28, p=0.128, R=.068, p=.715 and R=.058, p=.755, repectively). Comparison between the 1yr group in the GP2 arm (n=43 and n=15) for FluM-specific CD8 cells revealed a non-significant increase in immune response in the >1yr group (2.74 vs 3.57, p=0.15). A similar comparison in the AE37 arm (n=56 1yr) revealed increased proliferation in the >1yr group (AE36: 1110 vs 2167, p=0.034, AE37: 983 vs 2179, p=0.001). There were no significant differences between the >1yr and Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-130. doi:1538-7445.AM2012-LB-130

BACKGROUND: Regulatory T cells (TReg) are elevated in breast cancer patients with the number of TReg cells being inversely associated with clinical outcome. We are conducting a prospective, randomized phase II trial of the HER-2/neu peptide vaccines GP2 and AE37. GP2 is an HLA-A2+ -restricted peptide (HER-2/neu aa: 654–662) capable of eliciting a CD8+ cytotoxic T cell response directed against HER-2/neu expressing tumors. AE37 is the Ii-Key hybrid of the HER-2/neu peptide AE36 (HER-2/neu aa:776–790) and is capable of stimulating CD4+ helper T-cells with anti-tumor activity. Given the ability of AE37 to activate CD4+ helper T cells, concern exists that unwanted stimulation of TReg cells may occur with vaccination; although, this was not seen in our phase I trial. We have sought to confirm this finding by evaluating peripheral TReg cell levels in patients from our larger phase II breast cancer vaccine trial. METHODS: Node positive or high-risk node negative breast cancer patients with any level of HER-2/neu expression rendered disease-free by standard treatments were enrolled. HLA-A2+ patients were placed in the GP2 arm and randomized to either GP2 + GM-CSF (immunoadjuvant) or GM-CSF alone. HLA-A2− patients were placed in the AE37 arm and randomized to either AE37 + GM-CSF or GM-CSF alone. Vaccinations were given as six monthly intradermal inoculations. CD4+CD25high CD127low (CD127low) TReg cell levels were measured before (R0), mid-series (R3), upon completion (R6), and at six (RC6) and twelve (RC12) months after completion of the vaccine series using antibody staining and flow cytometry analysis. p values were calculated using the Student's t-test. RESULTS: TReg cells were measured in 65 patients in the AE37 arm (34 vaccine, 31 adjuvant) and 33 patients in the GP2 arm (17 vaccine, 16 adjuvant). Patients receiving the AE37 vaccine exhibited a decrease in CD127low TReg cells from baseline at all measured timepoints (R0=4.2, R3=3.7, p CONCLUSIONS: Administration of a CD4 T cell-eliciting HER-2/neu peptide (AE37) vaccine to breast cancer patients induced a sustained decrease in circulating CD127low TReg cells. No such decrease was seen in randomized adjuvant-treated patients. Use of the CD8-eliciting, GP2 vaccine did not change circulating TReg cell levels. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-413. doi:10.1158/1538-7445.AM2011-LB-413

BACKGROUND: We are conducting a prospective, randomized, single-blinded phase II trial evaluating the HER-2/neu peptide vaccines AE37 and GP2 in the adjuvant setting to prevent breast cancer recurrence. AE37 is the Ii-Key hybrid of the HER-2/neu peptide AE36 (HER-2/neu aa:776–790) and is capable of stimulating CD4+ helper T-cells with anti-tumor activity. GP2 is an HLA-A2+ -restricted immunogenic peptide from the HER-2/neu protein (HER-2/neu aa: 654–662) which is capable of stimulating CD8+ cytotoxic T-lymphocytes with the ability to recognize and destroy HER-2/neu-expressing tumor cells. Here we present data comparing the immunological profiles between these CD4 and CD8 eliciting vaccines METHODS: Node positive or high-risk node negative breast cancer patients with any level of HER-2/neu expression rendered disease-free by standard treatments were enrolled. Patients were HLA-typed at enrollment. HLA-A2+ patients were placed in the GP2 arm and randomized to either GP2 + GM-CSF (immunoadjuvant) or GM-CSF alone. HLA-A2− patients were placed in the AE37 arm and randomized to either AE37 + GM-CSF or GM-CSF alone. Vaccinations were given in six monthly intradermal inoculations. In vitro (3H-thymidine proliferation for AE37, HLA-A2:immunoglobulin dimer for GP2) and in vivo (delayed-type hypersensitivity (DTH)) immune responses were measured before (R0), mid-series (R3), upon completion (R6), and at six (RC6) and twelve (RC12) months after completion of the vaccine series. RESULTS: We have enrolled 247 patients (AE37=171, 87 vaccine and 84 control) (GP2=76, 41 vaccine and 35 control). Proliferative responses to both AE37 and AE36 were increased from baseline in the vaccine group at all time points (AE37: R0=0.98, R3=3.29, R6=3.38, RC6=3.64, RC12=3.68, p CONCLUSIONS: In a novel prospective, randomized, single-blinded phase II vaccine trial of the AE37 and GP2 peptide vaccines with a GM-CSF only control group, breast cancer patients vaccinated with either a CD4 or CD8 eliciting peptide vaccine exhibit high levels of HER-2/neu-specific in vitro and in vivo immune responses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-414. doi:10.1158/1538-7445.AM2011-LB-414

Introduction: The affinity between tumor-associated antigens and MHC Class I complexes directly affects the degree of CD8+ T cell (CTL) response. GP2 (IISAVVGIL, p654-662) is a HER2/neu-derived peptide, that has poor binding affinity to HLA-A2 (A2) and HLA-A3 (A3). We conducted a preclinical study to determine if amino acid substitutions in the GP2 peptide, predicted to increase binding affinity to A2 and A3, enhance CTL recognition and stimulation. Methods: Based on computer modeling algorithms (BIMAS, SYFPEITHI), two GP2 peptide variants were predicted to have higher binding affinity to A2: SPA-12(2L9V-GP2) and SPA-13(2L5L9V-GP2). Freshly isolated PBMCs were obtained from randomly chosen, disease-free, node positive (NP) and high risk node negative (NN) breast cancer (BCa) patients, who had completed standard adjuvant therapy and are enrolled in the E75 vaccine booster trial. Peptide-specific CD8+ T cells were measured by HLA-A2: IgG dimer (A2 pts) and by interferon-γ ELISPOT (A2, A3 pts) assays. Groups were compared by t-test (dimer) and Mann-Whitney U (ELISPOT) tests. Results: Thirty-five A2 (21 NP, 14 NN) and thirteen A3 (13 NP) samples were included in the study. Among A2 pts, the mean level of SPA-12-specific CTLs was greater than the GP2- (2.8±0.3% vs. 1.7±0.1%, p Conclusion: The substituted sequences SPA-12 (2L9V-GP2) and SPA-13 (2L5L9V-GP2), both have higher predicted binding affinity to A2 and A3 than GP2. While SPA-12 showed significantly increased recognition and functional CTL responses compared to GP2, SPA-13 demonstrated lower responses. These data suggest that SPA-12 may be a more effective cancer vaccine than GP2, particularly in NP BCa patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-322.

Background: Regulatory T cells (Treg) are widely accepted to play a key role in the immune tolerance and escape of cancer cells. Originally Tregs were identified as being CD4+ T cells expressing high levels of CD25 (IL2 receptor). The identification of the transcription factor FoxP3 resulted in defining Tregs as CD4CD25hiFoxP3+ T cells by staining permeablized cells. Recently Tregs have been identified as CD4CD25hi T cells with low levels of CD127 (IL7 receptor) expressing a CD4+CD25hiCD127- cell surface phenotype. In an ongoing clinical trial with a MHC class II HER2/neu pep tide vaccine we have evaluated these various definitions of Treg phenotypes for immunological monitoring. Methods: Tregs were quantified in disease-free, high risk BCa patients enrolled in adjuvant peptide-based breast cancer vaccine trials. Peripheral blood mononuclear cells (PBMCs) were both permeabilized and stained with CD4, CD25, and FoxP3 monoclonal antibodies and surface stained with CD4, CD25, and CD127. Samples were then analyzed by flow cytometry. The means are compared with t-test and correlation is determined with Pearson's correlation coefficient. Results: 196 PBMC samples from 72 patients were available for Treg quantification. There was a greater mean percentage of cells identified as Treg in permeabilized than nonpermeabilized cells by staining with CD4+CD25hi (5.9±0.1% vs 4.8 ±0.1%, p Conclusion: There was good correlation between various methods of defining Tregs with and without cell membrane permeabilization. Defining Treg cells as either CD4+CD25hiFoxP3+ or CD4+CD25+CD127- appear to be more specific than defining Tregs as CD4+CD25hi alone. Furthermore, defining Treg populations by surface staining as CD4+CD25hiCD127- yields similar levels with good correlation as CD4+CD25hiFoxP3+ and may be the preferred method for immune monitoring. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-329.