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Background: Infections with respiratory syncytial virus (RSV) cause significant morbidity and hospitalization in older adults. We studied the humoral, mucosal and B cell responses of an investigational adjuvanted RSV sF vaccine, MEDI7510, in older adults. Methods: In a substudy of a randomized (1:1), double-blind, placebo-controlled study of MEDI7510 in adults ≥60 years of age, we collected blood and nasal secretions at days 0, 8, 29, 91 and 180 post-vaccination to measure F-specific IgG and IgA antibodies by ELISA, and plasmablasts and memory B cells by IgA/IgG dual-color fluorospot. Results: The 27 vaccine- and 18 placebo-recipients had a mean age of 73 years and included 24 women. Among vaccinees, 93% had significant increases in F-specific plasma IgG 85% had increased plasma IgA; 74% had increased nasal IgG and 26% nasal IgA; 93% had IgG and 89% IgA plasmablasts on Day 8 post-immunization; and 82% had IgG and 7.4% IgA memory B cell responses to the vaccine. Vaccinees

Budigalimab, a novel anti-PD-1 monoclonal antibody, demonstrated efficacy and biomarker pharmacodynamics in patients with head and neck squamous cell carcinoma (HNSCC) or non-small cell lung cancer (NSCLC) consistent with those reported by other PD-1 inhibitors. Herein are presented additional outcomes of biomarker analyses from the phase 1 study of budigalimab monotherapy in patients with HNSCC and NSCLC (NCT03000257). PD-1 inhibitor naive patients with advanced HNSCC (n=41) or NSCLC (n=40) received budigalimab intravenously at 250 mg every 2 weeks (Q2W) or 500 mg Q4W until progression. Archival tumor specimens were evaluated by immunohistochemistry for CD8 and tumor PD-1 ligand 1 (PD-L1) expression, RNA, and whole-exome sequencing. Serum and whole blood samples were acquired at baseline and at select on-treatment time points. As of October 2019, best overall response of 15% in HNSCC and 18% in NSCLC was observed in all treated patients; both cohorts reported responses in PD-L1+ and PD-L1- tumors. Treatment with budigalimab was associated with increases in multiple soluble biomarkers including interferon gamma-induced chemokines. Expanded overall T-cell counts, total CD8 T-cell counts, and percentages of CD8+CD45RA-CD62L- effector memory T cells were observed at cycle 1, day 15 in responders. Univariate analysis demonstrated an association between prolonged progression-free survival and higher tumor mutational burden/neoantigen load, smaller tumor size, lower platelet-lymphocyte ratios, lower CCL23, lower colony-stimulating factor 1, and lower interleukin-6 levels at baseline. The biomarker analysis presented herein identified additional early pharmacodynamic biomarkers associated with anti-PD-1 activity and improved clinical responses to budigalimab in patients with advanced HNSCC and NSCLC.

With the goal of developing a virus-like particle-based vaccine based on dense bodies (DB) produced by human cytomegalovirus (HCMV) infections, we evaluated scalable culture, isolation, and inactivation methods and applied technically advanced assays to determine the relative purity, composition, and immunogenicity of DB particles. Our results increase our understanding of the benefits and disadvantages of methods to recover immunogenic DB and inactivate contaminating viral particles. Our results indicate that (i) HCMV strain Towne replicates in MRC-5 fibroblasts grown on microcarriers, (ii) DB particles recovered from 2-bromo-5,6-dichloro-1-beta- d -ribofuranosyl benzimidazole riboside (BDCRB)-treated cultures and purified by tangential flow filtration (TFF-DB) or glycerol tartrate gradient sedimentation (GT-DB) constitute 92% or 98%, respectively, of all particles in the final product, (iii) epithelial cell-tropic DB particles are recovered from a single round of coinfection by AD169 and Towne strain viruses, consistent with complementation between the UL130 and UL131A expressed by these strains and restoration of gH/gL/UL128-UL131A (gH pentamer), (iv) equivalent neutralizing antibody titers are induced in mice following immunization with epithelial cell-tropic DB or gH pentamer-deficient DB preparations, (v) UV-inactivated residual virus in GT-DB or TFF-DB preparations retained immunogenicity and induced neutralizing antibody, preventing viral entry into epithelial cells, and (vi) GT-DB and TFF-DB induced cellular immune responses to multiple HCMV peptides. Collectively, this work provides a foundation for future development of DB as an HCMV-based particle vaccine. IMPORTANCE Development of a vaccine to prevent congenital HCMV infection remains a high priority. Vaccination with human cytomegalovirus-derived noninfectious particles, or dense bodies, may constitute a safe vaccination strategy that mimics natural infection. The standard approach for purification of virus particles has been to use a multiple-step, complex gradient that presents a potential barrier to production scale-up and commercialization. In the study described here, we employed an approach that combines treatment with an antiviral terminase inhibitor and purification by a simplified process to produce a vaccine candidate providing broad antiviral humoral and cellular immunity as a foundation for future development.

Background Mass cytometry, or CyTOF (Cytometry by Time-of-Flight), permits the simultaneous detection of over 40 phenotypic and functional immune markers in individual cells without the issues of spectral overlap seen in traditional flow cytometry. Methods In this study, we applied CyTOF to comprehensively characterize the circulating immune cell populations in elderly individuals both before and after administration of an investigational adjuvanted protein vaccine against respiratory syncytial virus (RSV) in a Phase 1a trial. Antigen-specific T cell responses to RSV by IFNγ ELISPOT had been observed in most but not all recipients in the highest dose cohort in this trial. Here, CyTOF was used to characterize the cellular response profile of ELISPOT responders and non-responders in this vaccine dose cohort. Results Both CD4+ and CD8+ T cell antigen-specific IFNγ responses were observed. Principal components analysis revealed baseline differences between responders and non-responders, including differences in activated (HLA-DR+) CD4+ and CD8+ T cells, which were higher in non-responders versus responders. Using viSNE to analyze RSV-responsive CD4+ and CD8+ T cells, we also found increased expression of HLA-DR, CCR7, CD127 and CD69 in non-responders versus responders. Conclusions High parameter CyTOF can help profile immune components associated with differential vaccine responsiveness. Electronic supplementary material The online version of this article (10.1186/s12967-018-1528-1) contains supplementary material, which is available to authorized users.

Neutralizing antibodies specific for respiratory syncytial virus (RSV) represent a major protective mechanism against RSV infection, as demonstrated by the efficacy of the immune-prophylactic monoclonal antibody palivizumab in preventing RSV-associated lower respiratory tract infections in premature infants. Accordingly, the RSV neutralization assay has become a key functional method to assess the neutralizing activity of serum antibodies in preclinical animal models, epidemiology studies, and clinical trials. In this study, we qualified a 24-h, fluorescent focus-based microneutralization (RSVA FFA-MN) method that requires no medium exchange or pre- or postinfection processing to detect green fluorescent protein-expressing RSV strain A2 (RSVA-GFP)-infected cells, using a high-content imaging system for automated image acquisition and focus enumeration. The RSVA FFA-MN method was shown to be sensitive, with a limit of detection (LOD) and limit of quantitation (LOQ) of 1:10, or 3.32 log 2 ; linear over a range of 4.27 to 9.65 log 2 50% inhibitory concentration (IC 50 ); and precise, with intra- and interassay coefficients of variation of n = 1,720). This high-throughput and reliable RSV microneutralization assay has proven useful for testing sera from preclinical vaccine candidate evaluation studies, epidemiology studies, and both pediatric and adult vaccine clinical trials.

This is the second phase 1 study of a respiratory syncytial virus (RSV) vaccine containing RSV fusion protein (sF) adjuvanted with glucopyranosyl lipid A (GLA) in a squalene-based 2% stable emulsion (GLA-SE). In this randomized, double-blind study, 261 subjects aged ≥60 years received inactivated influenza vaccine (IIV), a vaccine containing 120 μg sF with escalating doses of GLA (1, 2.5, or 5 μg) in SE, or a vaccine containing 80 μg sF with 2.5 μg GLA in SE. Subjects receiving 120 μg sF with 2.5 or 5 μg GLA were also randomized to receive IIV or placebo. Immunity to RSV was assessed by detection of microneutralizing, anti-F immunoglobulin G, and palivizumab-competitive antibodies and F-specific gamma interferon enzyme-linked immunosorbent spot assay T-cell responses. Higher adjuvant doses increased injection site discomfort, but at the highest dose, the reactogenicity was similar to that of IIV. Significant humoral and cellular immune responses were observed. The 120 μg sF plus 5.0 μg GLA formulation resulted in the highest responses in all subjects and in older subjects. These results confirm previous observations of vaccine tolerability, safety, and immunogenicity and were used to select the 120 μg sF plus 5.0 μg GLA formulation for phase 2 evaluation. (This study has been registered at ClinicalTrials.gov under registration no. NCT02289820.)

Respiratory syncytial virus (RSV) causes significant disease in elderly adults, and we have previously reported that individuals 65 years of age and older have reduced RSV F protein-specific gamma interferon (IFN-γ)-producing T cells compared to healthy younger adults. To measure RSV F-specific memory T cell responses in the elderly following infection or vaccination, we optimized and qualified an IFN-γ enzyme-linked immunospot (ELISPOT) assay. Since peripheral blood mononuclear cells (PBMC) from the elderly could be more fragile, we established optimal cryopreservation techniques and minimal viability acceptance criteria. The number of cells per well, types and concentrations of stimulation antigens, and incubation times were evaluated to maximize assay sensitivity and precision. The optimized assay uses 300,000 cells/well, 2 μg/ml of an RSV F peptide pool (RSV Fpp), and incubation for 22 ± 2 h in serum-free CTL-Test medium. The assay was qualified by 3 analysts using 3 RSV F-responding donor PBMC samples (high, medium, and low responders) tested on 5 different assay days. The assay sensitivity or limit of detection (LOD) was determined to be 21 spot-forming cells (SFC) per 106PBMC, and the lower limit of quantitation (LLOQ) was estimated to be 63 SFC/106PBMC. The intra- and interassay percent coefficients of variation (CV) were

Epstein Barr virus (EBV) is associated with B-cell lymphomas in posttransplant lymphoproliferative disease (PTLD). Latent membrane protein 1 (LMP1), the major oncogenic protein of EBV, promotes tumorigenesis through activation of NF-kappaB, Erk, p38, JNK and Akt. The Jak/STAT signal transduction pathway is also constitutively active in PTLD-associated EBV(+) B-cell lymphomas. Here we determine the mechanism of Jak/STAT activation in EBV(+) B-cell lymphomas and the role of LMP1 in this process. Immunoprecipitation studies revealed no direct interaction of LMP1 and JAK3, but known associations between JAK3 and common gamma chain, and between LMP1 and TRAF3, were readily detected in EBV(+) B cell lines from patients with PTLD. An inducible LMP1 molecule expressed in EBV(-) BL41 Burkitt's cells demonstrated STAT activation only after prolonged LMP1 signaling. While LMP1 induced IFN-gamma production in BL41 cells, IFN-gamma receptor blockade and IFN-gamma neutralization prior to LMP1 activation markedly decreased STAT1 activation and expression of LMP1-driven IFN-gamma inducible genes. Understanding the mechanisms by which EBV induces cellular signal transduction pathways may facilitate development of new treatments for PTLD.

Respiratory syncytial virus (RSV) causes significant disease in elderly adults, but an effective vaccine is not yet available. We have previously reported that vaccines consisting of engineered respiratory syncytial virus soluble fusion protein (RSV sF) adjuvanted with glucopyranosyl lipid A (GLA) in an oil-in-water emulsion (stable emulsion [SE]) induce RSV F-specific T and B cell responses in mice and rats that protect from viral challenge. Here, we evaluated the immunogenicity of GLA-SE adjuvanted RSV sF vs unadjuvanted RSV sF vaccines in cynomolgus macaques (Macaca fascicularis). RSV F-specific IgG, RSV neutralizing antibodies, and RSV F-specific T cell IFNγ ELISPOT responses induced by GLA-SE adjuvanted RSV sF peaked at week 6 at significantly higher levels than achieved by unadjuvanted RSV sF and remained detectable through week 24, demonstrating response longevity. Two weeks after a week 24 booster immunization, humoral and cellular responses reached levels similar to those seen at the earlier peak response. Importantly, the GLA-SE adjuvanted RSV sF vaccine induced cross-neutralizing antibodies to other RSV A and B strains as well as F-specific IgA and IgG memory B cells. GLA-SE adjuvanted RSV sF was also demonstrated to drive a Th1-biased response characterized by more IFNγ than IL-4. This study indicates that a GLA-SE adjuvanted RSV sF vaccine induces robust humoral and Th1-biased cellular immunity in non-human primates and may benefit human populations at risk for RSV disease.

The relationship between EBV infection and sensitivity to death receptor (DR)-induced apoptosis is poorly understood. Using EBV− and EBV+ BJAB cells, we provide the first evidence that EBV can protect latently infected B cell lymphomas from apoptosis triggered through Fas or TRAIL receptors. Caspase 8 activation was impaired and cellular FLIP recruitment was enriched in death-inducing signaling complexes formed in EBV-infected BJAB cells relative to parent BJAB cells. Furthermore, latent membrane protein 1 expression alone could reduce caspase activation and confer partial resistance to DR apoptosis in BJAB cells. This protective effect was dependent on C-terminal activating region 2-driven NF-κB activation, which in turn up-regulated cellular FLIP expression in latent membrane protein 1+ BJAB cells. Thus, the ability of latent EBV to block DR apoptosis may help to ensure the survival of host cells during B cell differentiation, and contribute to the development of B cell lymphomas, especially in immunocompromised individuals.

Tumor-associated proteins can act as effective immunotherapeutic targets. Immunization with tumor TCR protein conjugated to the immunogenic protein keyhole limpet hemocyanin (KLH) protects mice from tumor challenge with the murine T cell lymphoma C6VL. The immune mechanisms responsible for this tumor protection are of interest for designing more effective vaccine strategies. Previous studies using depletion experiments had suggested a CD8-mediated component of protection induced by TCR-KLH vaccines. In this study we used CD8α knockout, μMT, and FcγR knockout mice to investigate the relative roles of CD8+ T cells and Ab in protective immunity induced by TCR-KLH immunization. We found that CD8+ T cells are not required for tumor protection, although they may contribute to protection. Vaccine-induced Abs are sufficient to mediate protection against this murine T cell lymphoma through an FcR-dependent mechanism. This was confirmed with Ab transfers, which protect challenged mice. Additionally, recombinase-activating gene 1−/− splenocytes can mediate Ab-dependent cellular cytotoxicity against this tumor in the presence of bound anti-TCR Abs. IFN-γ knockout mice demonstrated a requirement for IFN-γ, probably via generation of IgG2c Abs, in vaccine-induced tumor protection. IFN-γ knockout mice were not protected by immunization and had a severe impairment in IgG2c Ab production in response to immunization. Although mock-depleted anti-TCR Abs could transfer tumor protection, IgG2c-deficient anti-TCR Abs were unable to transfer tumor protection to wild-type mice. These results suggest that TCR-KLH vaccine-induced tumor protection in the C6VL system is primarily attributable to the induction of IgG2c Abs and humoral immunity.

Background Illness associated with Respiratory Syncytial Virus (RSV) remains an unmet medical need in both full-term infants and older adults. The fusion glycoprotein (F) of RSV, which plays a key role in RSV infection and is a target of neutralizing antibodies, is an attractive vaccine target for inducing RSV-specific immunity. Methodology and Principal Findings BALB/c mice and cotton rats, two well-characterized rodent models of RSV infection, were used to evaluate the immunogenicity of intramuscularly administered RSV vaccine candidates consisting of purified soluble F (sF) protein formulated with TLR4 agonist glucopyranosyl lipid A (GLA), stable emulsion (SE), GLA-SE, or alum adjuvants. Protection from RSV challenge, serum RSV neutralizing responses, and anti-F IgG responses were induced by all of the tested adjuvanted RSV sF vaccine formulations. However, only RSV sF + GLA-SE induced robust F-specific TH1-biased humoral and cellular responses. In mice, these F-specific cellular responses include both CD4 and CD8 T cells, with F-specific polyfunctional CD8 T cells that traffic to the mouse lung following RSV challenge. This RSV sF + GLA-SE vaccine formulation can also induce robust RSV neutralizing titers and prime IFNγ-producing T cell responses in Sprague Dawley rats. Conclusions/Significance These studies indicate that a protein subunit vaccine consisting of RSV sF + GLA-SE can induce robust neutralizing antibody and T cell responses to RSV, enhancing viral clearance via a TH1 immune-mediated mechanism. This vaccine may benefit older populations at risk for RSV disease.

Vaccination with tumor-specific immunoglobulin or idiotype (Id) is a promising new form of immunotherapy for B-cell malignancies. Id protein vaccination has demonstrated clinical activity in B-cell lymphomas, yet it requires the laborious and time-consuming procedures of tumor–myeloma cell hybridization, large-scale in vitro culture, and protein purification. Recombinant adenoviruses are highly efficient and immunogenic gene transfer vehicles from which individualized vaccines can be rapidly assembled using polymerase chain reaction–amplified tumor Id genes. Id-encoding adenoviruses were evaluated as vaccines in 2 murine B-cell lymphoma models. A single injection of recombinant Id adenovirus provided protection from subsequent tumor challenge that was equivalent or superior to that afforded by Id protein vaccination. Protected mice had substantial serum titers of Id-specific antibodies. When used in conjunction with chemotherapy, vaccination also prolonged the survival of mice bearing pre-existing tumor. Mechanistic studies demonstrated that tumor protection was not dependent upon T cells. Importantly, in mice prevaccinated with an irrelevant adenovirus, tumor protection following vaccination with Id adenovirus was not significantly impaired. These findings have implications for the design of future lymphoma immunotherapy trials.

Post-transplant lymphoproliferative disorder (PTLD)-associated Epstein Barr virus (EBV)+ B-cell lymphomas are serious complications of solid organ and bone marrow transplantation. The EBV protein LMP2a, a B-cell receptor (BCR) mimic, provides survival signals to virally-infected cells through Syk tyrosine kinase. Therefore, we explored whether Syk inhibition is a viable therapeutic strategy for EBV-associated PTLD. We have shown that R406, the active metabolite of the Syk inhibitor fostamatinib, induces apoptosis and cell cycle arrest while decreasing downstream phosphatidylinositol-3′-kinase (PI3K)/Akt signaling in EBV+ B-cell lymphoma PTLD lines in vitro. However, Syk inhibition did not inhibit or delay the in vivo growth of solid tumors established from EBV-infected B-cell lines. Instead, we observed tumor growth in adjacent inguinal lymph nodes exclusively in fostamatinib-treated animals. In contrast, direct inhibition of PI3K/Akt significantly reduced tumor burden in a xenogeneic mouse model of PTLD without evidence of tumor growth in adjacent inguinal lymph nodes. Taken together, our data indicate that Syk activates PI3K/Akt signaling which is required for survival of EBV+ B-cell lymphomas. PI3K/Akt signaling may be a promising therapeutic target for PTLD, and other EBV-associated malignancies.

Background Respiratory Syncytial Virus (RSV) causes significant disease in the elderly, in part, because immunosenescence impairs protective immune responses to infection in this population. Despite previous and current efforts, there is no RSV vaccine currently licensed in infants or elderly adults. Adjuvanted RSV subunit vaccines have the potential to boost waning immune responses and reduce the burden of RSV disease in the elderly population. Results We used an aged BALB/c mouse model to evaluate immune responses to RSV Fusion (F) protein in the absence and presence of an alum adjuvant. We demonstrate that aged BALB/c mice immunized with alum-adjuvanted RSV F protein had significantly reduced lung viral titers at day 4 following challenge with wild-type (wt) RSV. Serum neutralizing antibody titers measured on day 27 correlated with protection in both young and aged vaccinated mice, although the magnitude of antibody titers was lower in aged mice. Unlike young mice, in aged mice, alum-adjuvanted RSV F did not induce lung TH2-type cytokines or eosinophil infiltration compared to non-adjuvanted F protein following wt RSV challenge. Conclusion Our studies demonstrate that neutralizing anti-RSV antibody titers correlate with protection in both young and aged BALB/c mice vaccinated with RSV F protein vaccines. The F + alum formulation mediated greater protection compared to the non-adjuvanted F protein in both young and aged mice. However, while alum can boost F-specific antibody responses in aged mice, it does not completely overcome the reduced ability of a senescent immune system to respond to the RSV F antigen. Thus, our data suggest that a stronger adjuvant may be required for the prevention of RSV disease in immunosenescent populations, to achieve the appropriate balance of protective neutralizing antibodies and effective TH1-type cytokine response along with minimal lung immunopathology.

The B lymphotrophic γ-herpesvirus EBV is associated with a variety of lymphoid- and epithelial-derived malignancies, including B cell lymphomas in immunocompromised and immunosuppressed individuals. The primary oncogene of EBV, latent membrane protein 1 (LMP1), activates the PI3K/Akt pathway to induce the autocrine growth factor, IL-10, in EBV-infected B cells, but the mechanisms underlying PI3K activation remain incompletely understood. Using small molecule inhibition and siRNA strategies in human B cell lines expressing a chimeric, signaling-inducible LMP1 protein, nerve growth factor receptor (NGFR)-LMP1, we show that NGFR-LMP1 utilizes Syk to activate PI3K/Akt signaling and induce IL-10 production. NGFR-LMP1 signaling induces phosphorylation of BLNK, a marker of Syk activation. Whereas Src kinases are often required for Syk activation, we show here that PI3K/Akt activation and autocrine IL-10 production by NGFR-LMP1 involves the Src family kinase Fyn. Finally, we demonstrate that NGFR-LMP1 induces phosphorylation of c-Cbl in a Syk- and Fyn-dependent fashion. Our results indicate that the EBV protein LMP1, which lacks the canonical ITAM required for Syk activation, can nevertheless activate Syk, and the Src kinase Fyn, resulting in downstream c-Cbl and PI3K/Akt activation. Fyn, Syk, and PI3K/Akt antagonists thus may present potential new therapeutic strategies that target the oncogene LMP1 for treatment of EBV+ B cell lymphomas.

Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is a proven oncogene that is essential for transformation of human B cells by the virus. LMP1 induces constitutive activation of several signal transduction pathways involving nuclear factor κB, phosphatidylinositol 3-kinase/Akt, and the mitogen-activated protein kinases (MAPK) p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (Erk). Sequencing of LMP1 isolated from a panel of EBV+ B cell lymphomas identified three different variants of LMP1, each distinct from the B95.8 prototype isoform. All tumor variants of LMP1 as well as the B95.8 LMP1 isoform were able to induce rapid p38 phosphorylation as well as Akt and JNK activation. Additionally all variants showed similar ability to activate nuclear factor κB. In contrast, only tumor-derived LMP1 variants induced prolonged Erk activation and c-Fos expression. Sequence analysis revealed only two amino acids, 212 and 366, shared by the tumor variants but distinct from B95.8. Point mutation of either amino acids 212 (glycine to serine) or 366 (serine to threonine) from the B95.8 isoform to the tumor variant version of LMP1 was sufficient for gain of function characterized by sustained activation of Erk and subsequent c-Fos induction and binding to the AP1 site. Our results indicate that the enhanced ability of tumor-derived LMP1 to induce and stabilize the c-Fos oncogene can be localized to two amino acids in the C terminus of LMP1.

EBV is a B lymphotrophic γ-herpesvirus that is associated with multiple human malignancies, including posttransplant lymphoproliferative disorder. The EBV-encoded protein, latent membrane protein 1 (LMP1), is required for oncogenic transformation of human B cells by EBV. An important consequence of LMP1 expression in EBV-infected B cells is the induction of cellular IL-10, which acts as an autocrine growth factor for B cell lymphomas. However, the mechanisms by which LMP1 induces IL-10 are incompletely understood. We previously showed that rapamycin, a clinically relevant immunosuppressant and mammalian target of rapamycin inhibitor, could suppress IL-10 production by EBV-infected B cell lines. To test the hypothesis that PI3K, which acts upstream of mammalian target of rapamycin, might also be involved in LMP1-dependent IL-10 production, we generated B cell lines expressing signaling-inducible chimeric LMP1. Our results show that induced LMP1 signaling elicits both p38- and PI3K-dependent IL-10 production in EBV− B cells. Moreover, distinct regions of the LMP1 signaling tail are associated with p38- vs PI3K-dependent IL-10 induction. We also demonstrate that the LMP1-dependent p38 and PI3K activation regulates IL-10 induction through discrete mechanisms. Whereas p38 activation is critical for the phosphorylation of the transcription factor CREB, PI3K activation is required for the inactivation of glycogen synthase kinase 3β (GSK3β), an inhibitory kinase that can regulate CREB function. We find that GSK3β regulates LMP1-dependent IL-10 induction, with GSK3β inhibition by pharmacologic or small interfering RNA strategies enhancing LMP1-induced IL-10 induction. These findings demonstrate that LMP1 uses both p38 and PI3K activation for maximal up-regulation of IL-10.

3086 Background: Immunomodulatory antibodies targeting specific T cell costimulatory pathways have shown great potential as cancer therapeutics. OX40 agonists can enhance T cell proliferation and s...

Epstein Barr virus is a B lymphotrophic gammaherpes virus that is associated with multiple human malignancies. The EBV-encoded protein, latent membrane protein 1 (LMP1), is required for transformation of human B cells by EBV in vitro. LMP1 usurps multiple cellular signal transduction pathways to promote cell survival and to induce expression of cellular IL-10, an autocrine growth factor for EBV+ B cell lymphomas. However, the mechanisms by which LMP1 induces IL-10 are not understood. We previously showed that rapamycin, an mTOR inhibitor, could suppress IL-10 production by EBV-infected B cell lines. To test whether LMP1 activates rapamycin-sensitive signaling pathways to induce IL-10, we stably transfected EBV− BL41 cells to express a chimeric NGFR.LMP1 protein that mimics LMP1 signaling upon crosslinking. Treatment with either the p38 inhibitor SB203580 or with inhibitors that affect PI3K/AKT/mTOR signaling diminished (25–75% decrease) IL-10 production in EBV+ B cell lines. While induction of LMP1 signaling through NGFR.LMP1 resulted in phosphorylation of MAPK and PI3K pathway intermediates and production of IL-10, inhibition of either the p38 or PI3K/AKT/mTOR signaling axis markedly suppressed LMP1-mediated IL-10 production. These data indicate that p38 and PI3K/AKT/mTOR signaling contributes to LMP1-induced IL-10 production. Moreover, targeting these pathways may be an effective strategy to prevent IL-10 production and inhibit the growth of EBV+ B cell lymphomas.

BACKGROUND: Toll-like receptor (TLR)4 agonists are known potent immunostimulatory compounds. These compounds can be formulated as part of novel adjuvants to enhance vaccine medicated immune responses. However, the contribution of the formulation to the innate in vivo activity of TLR4 agonist compounds is not well understood. METHODOLOGY AND PRINCIPAL FINDINGS: We evaluated synthetic TLR4 agonist Glucopyranosyl Lipid A (GLA) for its effects on molecular and cellular innate immune responses in the murine model. Microarray techniques were used to compare the responses to GLA in an aqueous formulation or in an oil-in-water Stable Emulsion formulation (GLA-SE) versus either SE alone or the mineral salt aluminum hydroxide (alum) at the muscle injection site over multiple timepoints. In contrast to the minimal gene upregulation induced by SE and alum, both GLA and GLA-SE triggered MyD88- and TRIF-dependent gene expression. Genes for chemokines, cytokine receptors, signaling molecules, complement, and antigen presentation were also strongly upregulated by GLA and GLA-SE. These included chemokines for T(H)1-type T cells (CXCL9 and CXCL10) and mononuclear leukocytes (CCL2, CCL3) among others. GLA-SE induced stronger and more sustained gene upregulation than GLA in the muscle; GLA-SE induced genes were also detected in local draining lymph nodes and at lower levels in peripheral blood. Both GLA and GLA-SE resulted in increased cellular trafficking to the draining lymph nodes and upregulated MHC molecules and ICAM1 on local dendritic cells. GLA and GLA-SE transiently upregulated circulating MCP-1, TNFα, IFNγ and IP-10 in blood. CONCLUSIONS/SIGNIFICANCE: While GLA and GLA-SE activate a large number of shared innate genes and proteins, GLA-SE induces a quantitatively and qualitatively stronger response than GLA, SE or alum. The genes and proteins upregulated could be used to facilitate selection of appropriate adjuvant doses in vaccine formulations.

Background Respiratory syncytial virus (RSV) causes significant illness in older adults resulting in substantial health and economic impact. A successful vaccine would reduce morbidity in this growing segment of the population. Methods In this double-blind phase 1 study, subjects 60 years of age and older were enrolled by cohort and randomized to receive vaccines containing escalating doses (20, 50, or 80 μg) of soluble RSV fusion protein (sF) alone or adjuvanted with 2.5 μg of glucopyranosyl lipid A, a toll-like receptor-4 agonist, in 2% stable emulsion (GLA-SE). Each cohort included 20 vaccine and 4 placebo recipients. Immune responses were evaluated using assays for RSV microneutralizing, anti-F IgG, and palivizumab competitive antibodies and for F-specific interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) responses. Results The inclusion of adjuvant increased local reactogenicity, with the majority of subjects who received sF and adjuvant reporting low-grade injection site pain or tenderness. At all doses, the safety profile was acceptable for further development. Immune responses were antigen dose-dependent, and the inclusion of adjuvant increased both humoral and cellular immune responses, with responses statistically higher than for placebo recipients in all 4 assays. At the highest dosage level with adjuvant, half of the subjects had a ≥3-fold rise from day 0 in RSV neutralizing antibody titers, and all had a ≥3-fold rise in antibody levels by anti-F IgG and palivizumab competitive antibody assays on day 29. For the day 8 IFNγ ELISPOT assay, 74% of subjects in the highest dosing cohort had a ≥3-fold rise from baseline. Conclusions The safety and immunogenicity results from this study support inclusion of the GLA-SE adjuvant in this RSV vaccine for older adults and also support assessment of the efficacy of the vaccine in a larger clinical trial. Clinicaltrials.gov NCT02115815 .