Immunogenic Form Research Articles

Human Monoclonal Antibodies: The Benefits of Humanization.

The major reasons for developing human monoclonal antibodies were to be able to efficiently manipulate their effector functions while avoiding immunogenicity seen with rodent antibodies. Those effector functions involve interactions with the complement system and naturally occurring Fc receptors on diverse blood white cells. Antibody immunogenicity results from the degree to which the host immune system can recognize and react to these therapeutic agents. Thus far, there is still no generally applicable technology guaranteed to render therapeutic antibodies antigenically silent. This is not to say that the task is impossible, but rather that we need to train the immune system to help us. This can be achieved if we take advantage of natural mechanisms by which an individual can be rendered tolerant of "foreign" antigens, and as a corollary minimize the potential immunogenicity of any contaminating protein aggregates, or "aggregates" arising from antibodies complexing with their antigen. I here summarize our efforts to engineer antibodies to harness optimal effector functions, while also minimizing their immunogenicity. Potential avenues to achieve the latte are predicted from classical work showing that monomeric "foreign" immunoglobulins are good tolerogens, while aggregates of immunoglobulins ate intrinsically immunogenic. Consequently, I argue that one solution to the immunogenicity problem lies in ensuring a temporal quantitative advantage of tolerogenic non-cell-boundmonomer over the cell-binding immunogenic form.

Biological Activity of the Carrier as a Factor in Immunogen Design for Haptens.

Immunoanalytical methods are frequently employed in the detection of hazardous small molecular weight compounds. However, antibody development for these molecules is a challenge, because they are haptens and cannot induce a humoral immune response in experimental animals. Immunogenic forms of haptens are usually prepared by conjugating them to a protein carrier which serves as an immune stimulator. However, the carrier is usually considered merely as a bulk mass, and its biological activity is ignored. Here, we induced an endocytic receptor, transferrin receptor, by selecting its ligand as a carrier protein to enhance antibody production. We conjugated aflatoxin, a potent carcinogenic food contaminant, to transferrin and evaluated its potential to stimulate antibody production with respect to ovalbumin conjugates. Transferrin conjugates induced aflatoxin-specific immune responses in the second immunization, while ovalbumin conjugates reached similar antibody titers after 5 injections. Monoclonal antibodies were successfully developed with mice immunized with either of the conjugates.

Therapeutically Active RIG-I Agonist Induces Immunogenic Tumor Cell Killing in Breast Cancers.

Cancer immunotherapies that remove checkpoint restraints on adaptive immunity are gaining clinical momentum but have not achieved widespread success in breast cancers, a tumor type considered poorly immunogenic and which harbors a decreased presence of tumor-infiltrating lymphocytes. Approaches that activate innate immunity in breast cancer cells and the tumor microenvironment are of increasing interest, based on their ability to induce immunogenic tumor cell death, type I IFNs, and lymphocyte-recruiting chemokines. In agreement with reports in other cancers, we observe loss, downregulation, or mutation of the innate viral nucleotide sensor retinoic acid-inducible gene I (RIG-I/DDX58) in only 1% of clinical breast cancers, suggesting potentially widespread applicability for therapeutic RIG-I agonists that activate innate immunity. This was tested using an engineered RIG-I agonist in a breast cancer cell panel representing each of three major clinical breast cancer subtypes. Treatment with RIG-I agonist resulted in upregulation and mitochondrial localization of RIG-I and activation of proinflammatory transcription factors STAT1 and NF-κB. RIG-I agonist triggered the extrinsic apoptosis pathway and pyroptosis, a highly immunogenic form of cell death in breast cancer cells. RIG-I agonist also induced expression of lymphocyte-recruiting chemokines and type I IFN, confirming that cell death and cytokine modulation occur in a tumor cell-intrinsic manner. Importantly, RIG-I activation in breast tumors increased tumor lymphocytes and decreased tumor growth and metastasis. Overall, these findings demonstrate successful therapeutic delivery of a synthetic RIG-I agonist to induce tumor cell killing and to modulate the tumor microenvironment in vivo Significance: These findings describe the first in vivo delivery of RIG-I mimetics to tumors, demonstrating a potent immunogenic and therapeutic effect in the context of otherwise poorly immunogenic breast cancers. Cancer Res; 78(21); 6183-95. ©2018 AACR.

Plant-produced Recombinant Influenza A Vaccines Based on the M2e Peptide

Influenza is a widely distributed infection that almost annually causes seasonal epidemics. The current egg-based platforms for influenza vaccine production are facing a number of challenges and are failing to satisfy the global demand in the case of pandemics due to the long production time. Recombinant vaccines are an alternative that can be quickly produced in high quantities in standard expression systems. Plants may become a promising biofactory for the large-scale production of recombinant proteins due to low cost, scalability, and safety. Plant-based expression systems have been used to produce recombinant vaccines against influenza based on two targets; the major surface antigen hemagglutinin and the transmembrane protein M2. Different forms of recombinant hemagglutinin were successfully expressed in plants, and some plantproduced vaccines based on hemagglutinin were successfully tested in clinical trials. However, these vaccines remain strain specific, while the highly conserved extracellular domain of the M2 protein (M2e) could be used for the development of a universal influenza vaccine. In this review, the state of the art in developing plant-produced influenza vaccines based on M2e is presented and placed in perspective. A number of strategies to produce M2e in an immunogenic form in plants have been reported, including its presentation on the surface of plant viruses or virus-like particles formed by capsid proteins, linkage to bacterial flagellin, and targeting to protein bodies. Some M2e-based vaccine candidates were produced at high levels (up to 1 mg/g of fresh plant tissue) and were shown to be capable of stimulating broad-range protective immunity.

PO-413 Immune modulating properties of cyclophosphamide synergise with immunotherapy in preclinical models of neuroblastoma

IntroductionNeuroblastoma (NB) is one of the most common childhood cancers accounting for 15% of paediatric cancer deaths. Current therapies, which include chemotherapy, are highly toxic with significant treatment related mortality. There is little scope for further intensification therefore alternatives strategies such as immunotherapy, are a priority for improving patient outcomes. Classically, chemotherapy is regarded as immunosuppressive but recent work has highlighted that some may illicit an immunogenic form of cell death. This work sets out to identify the immunomodulating effects of cyclophosphamide (CPM) on tumour infiltrating immune cells and whether these properties can synergise with immunomodulatory antibodies in preclinical models of NB.Material and methodsThe effects of CPM and doxorubicin on tumour cells were investigated in vitro by analyses of immunogenic cell death (ICD) markers. Two different in vivo subcutaneous murine NB models (NXS2 +NB9464D), were treated i.p with different doses of CPM. Tissues were harvested and detailed immunophenotyping performed. Combination of CPM and anti-PD-1 therapy was investigated in both models using tumour growth and survival as end points. Combination therapy was also assessed in TH-MYCN mice which develop spontaneous neuroblastoma.Results and discussionsChemotherapy application in vitro led to an increase in expression of the ICD markers, ecto-calreticulin and Hsp-70. CPM was found to have numerous immune modulating activities in vivo, including the reduction of intratumoral Treg cells, even at low doses (20 mg/kg), in both NB models. Combination therapy was shown to increase CD8+ and CD4+ percentages within tumours, along with an increase CD4+ effector and memory cell proportions. Anti-PD-1 therapy synergised with CPM to improve median survival and slow tumour growth. Metronomic dosing of CPM and anti-PD-1 antibody led to tumour regression in TH-MYCN tumour bearing mice.ConclusionThis work supports combining low dose CPM to enhance immunomodulatory antibody therapy, to generate therapeutic anti-neuroblastoma immunity. Ongoing work is focused on elucidating the mechanism of action of the CPM and anti-PD-1 combination therapy, along with identifying the optimal chemotherapy and antibody dosing strategies.

Evidence for Necroptosis as a Mechanism of Islet Amyloid–Induced Beta-Cell Death

Islet amyloid deposition occurs in the majority of individuals with type 2 diabetes (T2D) and contributes to beta-cell death by mechanisms which are not fully understood. Necroptosis is an immunogenic form of cell death dependent on tumor necrosis factor receptor 1 (TNFR1) signaling, receptor interacting protein kinase 3 (RIPK3) oligomerization, and mixed lineage kinase domain-like (MLKL) phosphorylation. We previously showed mRNA and protein levels of TNFa, a TNFR1 ligand, are increased in islets by amyloid deposition in vitro and in vivo, raising the possibility that amyloid formation induces necroptosis. Thus, we sought to determine whether the components of necroptosis are present in beta cells, and if islet amyloid-induced TNFa expression elicits beta-cell necroptosis. We observed protein expression of TNFR1, RIPK3, and MLKL in the INS-1 beta-cell line, indicating necroptosis components are present in beta cells. In human islet amyloid polypeptide (hIAPP) transgenic mouse islets cultured in 16.7 mM glucose, an in vitro model of islet amyloidosis, we found necroptosis signaling to be increased compared to amyloid-free wild type (WT) islets. This manifested as increased oligomerization of RIPK3 protein (trimers: 2.7 ± 0.2 [hIAPP] vs. 1.0 [WT], n=3, p<0.01) and phosphorylation of the RIPK3 target MLKL (1.8 ± 0.3 vs. 1.0, n=3, p=0.06), without a change in TNFR1 protein expression (1.1 ± 0.1 vs. 1.0, n=2, p=0.74). As further support that amyloid induces immunogenic cell death, immune cell mRNA abundance was increased in vivo in amyloid containing hIAPP transgenic islets vs. WT islets (macrophages: Emr1, 1.6 ± 0.2 [hIAPP] vs. 1.1 ± 0.1 [WT], n=4, p=0.04, T cells: Cd3, 2.7 ± 0.5 vs. 1.0 ± 0.2, n=4, p=0.03, and B cells: Cd20, 2.3 ± 0.2 vs. 1.2 ± 0.5, n=2, p=0.17). These data suggest that necroptosis may be a previously unrecognized mechanism of beta-cell death in response to islet amyloid formation. Further studies are needed to determine whether inhibition of necroptosis signaling may reduce beta-cell death in T2D. Disclosure A.T. Templin: None. M.F. Hogan: None. N. Esser: None. S. Zraika: None. R.L. Hull: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company. S.E. Kahn: Advisory Panel; Self; Boehringer Ingelheim GmbH, Elcelyx Therapeutics, Inc., Eli Lilly and Company, Intarcia Therapeutics, Inc., Janssen Research & Development, Merck & Co., Inc., Novo Nordisk A/S.

VaxArray for hemagglutinin and neuraminidase potency testing of influenza vaccines

Practical methods to measure the potency of influenza vaccines are needed as alternatives for the standard single radial immunodiffusion (SRID) assay. VaxArray assays for influenza hemagglutinin (HA) and neuraminidase (NA) have been developed to address this need. In this report, we evaluate the use of these assays to assess the potency of HA and NA of an A/H3N2 subunit vaccine by determining the correlation between the amounts measured by VaxArray and the immunogenicity in mice. The antibody response after one and two doses of five formulations of the vaccine ranging from 5 µg/mL to 80 µg/mL of HA, was measured by hemagglutination inhibition (HAI) and neuraminidase inhibition (NAI) assays. For hemagglutinin, vaccine potency determined by VaxArray was equivalent to potency measured SRID and these amounts were predictive of immunogenicity, with excellent correlation between potency measured by VaxArray and the HAI geometric mean titers (GMT). Likewise, the amount of NA measured by VaxArray was predictive of the NAI GMT. The VaxArray NA assay reported non-detectable levels of intact NA for a sample that had been heat degraded at 56 °C for 20 h, demonstrating that the assay measures the native, active form of NA. Similarly, the HA potency measured by VaxArray in this heat-treated sample was very low when a monoclonal antibody was used to detect the amount of antigen bound. Importantly, the force degraded sample induced low HAI titers and the NAI titers were not measurable, supporting the conclusion that the VaxArray HA and NA assays measure the immunogenic forms of these A/H3N2 antigens. This study indicates that VaxArray assays can be used to assess the potency of HA and NA components in influenza vaccines as a proxy for immunogenicity.

ICOS activation in combination with electrochemotherapy generates effective anti-cancer immunological responses in murine models of primary, secondary and metastatic disease

Electrochemotherapy is an evolving therapy which has recently been shown to induce an immunogenic form of cell death. It is hypothesized that the immunogenic cell death induced by electrochemotherapy may compliment the responses seen with anti-cancer immunotherapies. We therefore examined the effect of electrochemotherapy in combination with ICOS activation, which promotes the activity of previously activated T cells. In comparison to either monotherapy which resulted in no curative outcomes in any model, in a CT26 primary tumour 50% of mice were cured, with 100% of cured mice surviving tumour rechallenge. In a dual flank CT26 model mimicking secondary disease 20% of mice were cured, and 30% of mice were cured using an aggressively metastatic Lewis Lung Carcinoma model. We have shown the novel combination of electrochemotherapy with ICOS activation can inhibit local and distal tumour growth, including total tumour clearance with long lasting immunological memory.

Attenuated Human Parainfluenza Virus Type 1 Expressing the Respiratory Syncytial Virus (RSV) Fusion (F) Glycoprotein from an Added Gene: Effects of Prefusion Stabilization and Packaging of RSV F.

Human respiratory syncytial virus (RSV) is the most prevalent worldwide cause of severe respiratory tract infection in infants and young children. Human parainfluenza virus type 1 (HPIV1) also causes severe pediatric respiratory illness, especially croup. Both viruses lack vaccines. Here, we describe the preclinical development of a bivalent RSV/HPIV1 vaccine based on a recombinant HPIV1 vector, attenuated by a stabilized mutation, that expresses RSV F protein modified for increased stability in the prefusion (pre-F) conformation by previously described disulfide bond (DS) and hydrophobic cavity-filling (Cav1) mutations. RSV F was expressed from the first or second gene position as the full-length protein or as a chimeric protein with its transmembrane and cytoplasmic tail (TMCT) domains substituted with those of HPIV1 F in an effort to direct packaging in the vector particles. All constructs were recovered by reverse genetics. The TMCT versions of RSV F were packaged in the rHPIV1 particles much more efficiently than their full-length counterparts. In hamsters, the presence of the RSV F gene, and in particular the TMCT versions, was attenuating and resulted in reduced immunogenicity. However, the vector expressing full-length RSV F from the pre-N position was immunogenic for RSV and HPIV1. It conferred complement-independent high-quality RSV-neutralizing antibodies at titers similar to those of wild-type RSV and provided protection against RSV challenge. The vectors exhibited stable RSV F expression in vitro and in vivo In conclusion, an attenuated rHPIV1 vector expressing a pre-F-stabilized form of RSV F demonstrated promising immunogenicity and should be further developed as an intranasal pediatric vaccine.IMPORTANCE RSV and HPIV1 are major viral causes of acute pediatric respiratory illness for which no vaccines or suitable antiviral drugs are available. The RSV F glycoprotein is the major RSV neutralization antigen. We used a rHPIV1 vector, bearing a stabilized attenuating mutation, to express the RSV F glycoprotein bearing amino acid substitutions that increase its stability in the pre-F form, the most immunogenic form that elicits highly functional virus-neutralizing antibodies. RSV F was expressed from the pre-N or N-P gene position of the rHPIV1 vector as a full-length protein or as a chimeric form with its TMCT domain derived from HPIV1 F. TMCT modification greatly increased packaging of RSV F into the vector particles but also increased vector attenuation in vivo, resulting in reduced immunogenicity. In contrast, full-length RSV F expressed from the pre-N position was immunogenic, eliciting complement-independent RSV-neutralizing antibodies and providing protection against RSV challenge.

An influenza A virus vaccine based on an M2e-modified alphavirus.

The 23-residue external domain of the influenza A virus M2 protein (M2e) has significant potential as a vaccine antigen. Here, we describe the construction and characterization of an M2e-modified Sindbis virus designated E2S1-M2e. E2S1-M2e virions contain M2e as an N-terminal extension of the E2 glycoprotein and therefore express 240 copies of the M2e peptide on their surface. The E2S1-M2e virus expressed M2e in an accessible and immunogenic form and induced M2e-specific antibodies when administered to mice. Mice that received an intranasal vaccination with E2S1-M2e were protected against a lethal challenge with a virulent, mouse-adapted strain of influenza A virus.

A model for the regulation of follicular dendritic cells predicts invariant reciprocal-time decay of post-vaccine antibody response.

Follicular dendritic cells (FDC) play a crucial role in the regulation of humoral immunity. They are believed to be responsible for long-term persistence of antibody, due to their role in antibody response induction and their ability to retain antigen in immunogenic form for long periods. In this article, a regulatory control model is proposed which links persistence of humoral immunity with cellular processes associated with FDCs. The argument is comprised of three elements. The first is a literature review of population-level studies of post-vaccination antibody persistence. It is found that reciprocal-time (∝ 1/t) decay of antibody levels is widely reported, over a range of ages, observation times, and vaccine types. The second element is a mathematical control model for cell population decay for which reciprocal-time decay is a stable attractor. Additionally, control effectors are easily identified, leading to models of homeostatic control of the reciprocal-time decay rate. The final element is a literature review of FDC functionality. This reveals a striking concordance between cell properties required by the model and those widely observed of FDCs, some of which are unique to this cell type.The proposed model is able to unify a wide range of disparate observations of FDC function under one regulatory principle, and to characterize precisely forms of FDC regulation and dysregulation. Many infectious and immunological diseases are increasingly being linked to FDC regulation, therefore a precise understanding of the underlying mechanisms would be of significant benefit for the development of new therapies.

A time-resolved fluoroimmunoassay to assay the rabies virus glycoprotein: application for estimation of human rabies vaccine potency

Replacement of the in vivo rabies vaccine potency test (NIH test) by in vitro methods had been discussed by several researcher including WHO expert working groups. In this paper, a time-resolved fluoroimmunoassay (TRFIA) for the assay of rabies virus glycoprotein in rabies vaccine was first established to estimate the rabies vaccine potency by using specific monoclonal antibody that only recognized the native, trimeric and immunogenic form of rabies virus glycoprotein. Potency of the rabies virus glycoprotein was assayed with satisfactory performance under optimal conditions, and the method demonstrated satisfactory results when applied in practical samples. The correlation coefficient of potency values obtained from the present TRFIA and ELISA was 0.912, and 0.903 for those from the present TRFIA and NIH test. These preliminary results confirmed that this TRFIA can replace ELISA with higher performance, and could be a promising replacement of the NIH test. Based upon these results, the present TRFIA seemed to be a convenient tool for evaluating rabies vaccine potency and its products at different stages accordingly.

Abstract 5588: Brentuximab vedotin-driven immunogenic cell death enhances antitumor immune responses, and is potentiated by PD1 inhibition in vivo

Abstract Brentuximab vedotin (BV) is an antibody-drug conjugate directed against CD30 consisting of a monoclonal antibody conjugated to monomethyl auristatin E (MMAE), a microtubule-disrupting agent. BV antitumor activity is thought to be primarily the result of intracellular payload release leading to mitotic arrest and apoptotic cell death. We have demonstrated that BV drives apoptosis in a manner consistent with immunogenic cell death (ICD) including activation of the unfolded protein/ER stress response with a concomitant increase in surface expression of calreticulin and HSP70. In this study, we provide evidence that in vivo administration of BV leads to directed proinflammatory immune responses against the tumor and this activity is further potentiated by anti-PD-1 therapy. To examine the immunomodulatory effects of BV-induced ICD in vivo, we employed 3 murine model systems. In the first model, BV treatment induced ICD in subcutaneously-engrafted CD30+ L540cy cells, generated strong chemotactic responses detectable within the tumor and in circulation, and increased infiltration of dendritic cells into the tumor microenvironment. To assess whether BV-driven ICD could confer specific antitumor immunity, we used a model in which ICD was induced in BV-treated human CD30-expressing A20 lymphoma cells in vitro. These cells were used to immunize wild-type BALB/c mice followed by live A20 challenge. Immunization with BV-treated cells, undergoing ICD, delayed tumor growth and improved survival compared to mice immunized with flash-frozen cells. Furthermore, administering anti-PD-1 to mice immunized with BV-treated cells displayed marked combinatorial effects, leading to improved tumor clearance compared to either treatment alone. Additionally, T cells isolated from mice immunized with BV-treated cells and transferred into tumor-bearing immunodeficient mice resulted in tumor regression and survival, demonstrating robust T cell memory. Lastly, we employed a humanized tumor model pairing CD30+ PDL1+ PDL2+ lymphoblastoid cell line (LCL) xenografts with adoptively transferred autologous PBMC. In this setting, mice treated with suboptimal doses of BV showed greatly enhanced cytotoxic T cell and NK cell accumulation in LCL tumors resulting in accelerated immune-mediated tumor clearance. Tumor regression was further accelerated by treating mice with a combination of BV and a human PD1 inhibitor, demonstrating complementary modes of action for these agents. Together, these data indicate that targeted treatment with the MMAE antibody drug conjugate brentuximab vedotin drives an immunogenic form of tumor cell death that enhances innate and adaptive antitumor immunity. Combination of BV with PD1 inhibitors resulted in greater antitumor activity than either agent alone. Multiple clinical trials are ongoing to evaluate the efficacy of this treatment pairing. Citation Format: Anthony T. Cao, Che-Leung Law, Shyra J. Gardai, Ryan A. Heiser. Brentuximab vedotin-driven immunogenic cell death enhances antitumor immune responses, and is potentiated by PD1 inhibition in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5588. doi:10.1158/1538-7445.AM2017-5588

Engineering of Genetically Arrested Parasites (GAPs) For a Precision Malaria Vaccine.

Continuous stage conversion and swift changes in the antigenic repertoire in response to acquired immunity are hallmarks of complex eukaryotic pathogens, including Plasmodium species, the causative agents of malaria. Efficient elimination of Plasmodium liver stages prior to blood infection is one of the most promising malaria vaccine strategies. Here, we describe different genetically arrested parasites (GAPs) that have been engineered in Plasmodium berghei, P. yoelii and P. falciparum and compare their vaccine potential. A better understanding of the immunological mechanisms of prime and boost by arrested sporozoites and experimental strategies to enhance vaccine efficacy by further engineering existing GAPs into a more immunogenic form hold promise for continuous improvements of GAP-based vaccines. A critical hurdle for vaccines that elicit long-lasting protection against malaria, such as GAPs, is safety and efficacy in vulnerable populations. Vaccine research should focus on solutions toward turning malaria into a vaccine-preventable disease, which would offer an exciting new path of malaria control.

Nano-Pulse Stimulation is a physical modality that can trigger immunogenic tumor cell death

BackgroundWe have been developing a non-thermal, drug-free tumor therapy called Nano-Pulse Stimulation (NPS) that delivers ultrashort electric pulses to tumor cells which eliminates the tumor and inhibits secondary tumor growth. We hypothesized that the mechanism for inhibiting secondary tumor growth involves stimulating an adaptive immune response via an immunogenic form of apoptosis, commonly known as immunogenic cell death (ICD). ICD is characterized by the emission of danger-associated molecular patterns (DAMPs) that serve to recruit immune cells to the site of the tumor. Here we present evidence that NPS stimulates both caspase 3/7 activation indicative of apoptosis, as well as the emission of three critical DAMPs: ecto-calreticulin (CRT), ATP and HMGB1.MethodsAfter treating three separate cancer cell lines (MCA205, McA-RH7777, Jurkat E6-1) with NPS, cells were incubated at 37 °C. Cell-culture supernatants were collected after three-hours to measure for activated caspases 3/7 and after 24 h to measure CRT, ATP and HMGB1 levels. We measured the changes in caspase-3 activation with Caspase-Glo® by Promega, ecto-CRT with anti-CRT antibody and flow cytometry, ATP by luciferase light generation and HMGB1 by ELISA.ResultsThe initiation of apoptosis in cultured cells is greatest at 15 kV/cm and requires 50 A/cm2. Reducing this current inhibits cell death. Activated caspase-3 increases 8-fold in Jurkat E6-1 cells and 40% in rat hepatocellular carcinoma and mouse fibrosarcoma cells by 3 h post treatment. This increase is non-linear and peaks at 15–20 J/mL for all field strengths. 10 and 30 kV/cm fields exhibited the lowest response and the 12 and 15 kV/cm fields stimulated the largest amount of caspase activation. We measured the three DAMPs 24 h after treatment. The expression of cell surface CRT increased in an energy-dependent manner in the NPS treated samples. Expression levels reached or exceeded the expression levels in the majority of the anthracycline-treated samples at energies between 25 and 50 J/mL. Similar to the caspase response at 3 h, secreted ATP peaked at 15 J/mL and then rapidly declined at 25 J/mL. HMGB1 release increased as treatment energy increased and reached levels comparable to the anthracycline-treated groups between 10 and 25 J/mL.ConclusionNano-Pulse Stimulation treatment at specific energies was able to trigger the emission of three key DAMPs at levels comparable to Doxorubicin and Mitoxantrone, two known inducers of immunogenic cell death (ICD). Therefore NPS is a physical modality that can trigger immunogenic cell death in tumor cells.

A bioluminescent caspase-1 activity assay rapidly monitors inflammasome activation in cells

Inflammasomes are protein complexes induced by diverse inflammatory stimuli that activate caspase-1, resulting in the processing and release of cytokines, IL-1β and IL-18, and pyroptosis, an immunogenic form of cell death. To provide a homogeneous method for detecting caspase-1 activity, we developed a bioluminescent, plate-based assay that combines a substrate, Z-WEHD-aminoluciferin, with a thermostable luciferase in an optimized lytic reagent added directly to cultured cells. Assay specificity for caspase-1 is conferred by inclusion of a proteasome inhibitor in the lytic reagent and by use of a caspase-1 inhibitor to confirm activity. This approach enables a specific and rapid determination of caspase-1 activation. Caspase-1 activity is stable in the reagent thereby providing assay convenience and flexibility. Using this assay system, caspase-1 activation has been determined in THP-1 cells following treatment with α-hemolysin, LPS, nigericin, gramicidin, MSU, R848, Pam3CSK4, and flagellin. Caspase-1 activation has also been demonstrated in treated J774A.1 mouse macrophages, bone marrow-derived macrophages (BMDMs) from mice, as well as in human primary monocytes. Caspase-1 activity was not detected in treated BMDMs derived from Casp1−/− mice, further confirming the specificity of the assay. Caspase-1 activity can be measured directly in cultured cells using the lytic reagent, or caspase-1 activity released into medium can be monitored by assay of transferred supernatant. The caspase-1 assay can be multiplexed with other assays to monitor additional parameters from the same cells, such as IL-1β release or cell death. The caspase-1 assay in combination with a sensitive real-time monitor of cell death allows one to accurately establish pyroptosis. This assay system provides a rapid, convenient, and flexible method to specifically and quantitatively monitor caspase-1 activation in cells in a plate-based format. This will allow a more efficient and effective assessment of inflammasome activation as well as enable high-throughput screening for inflammasome modulators.

Chemo-Immunotherapy: Role of Indoleamine 2,3-Dioxygenase in Defining Immunogenic Versus Tolerogenic Cell Death in the Tumor Microenvironment.

In certain settings, chemotherapy can trigger an immunogenic form of tumor cell death. More often, however, tumor cell death after chemotherapy is not immunogenic, and may be actively tolerizing. However, even in these settings the dying tumor cells may be much more immunogenic than previously recognized, if key suppressive immune checkpoints such as indoleamine 2,3-dioxygenase (IDO) can be blocked. This is an important question, because a robust immune response to dying tumor cells could potentially augment the efficacy of conventional chemotherapy, or enhance the strength and duration of response to other immunologic therapies. Recent findings using preclinical models of self-tolerance and autoimmunity suggest that IDO and related downstream pathways may play a fundamental role in the decision between tolerance versus immune activation in response to dying cells. Thus, in the period of tumor cell death following chemotherapy or immunotherapy, the presence of IDO may help dictate the choice between dominant immunosuppression versus inflammation, antigen cross-presentation, and epitope spreading. The IDO pathway thus differs from other checkpoint-blockade strategies, in that it affects early immune responses, at the level of inflammation, activation of antigen-presenting cells, and initial cross-presentation of tumor antigens. This "up-stream" position may make IDO a potent target for therapeutic inhibition.

Posttranslational forms of beta 2-glycoprotein I in the pathogenesis of the antiphospholipid syndrome.

The antiphospholipid syndrome (APS) is an autoimmune disease characterised by a procoagulant state that predisposes to recurrent thrombosis and miscarriages. Two major discoveries have advanced our understanding of the underlying complex pathogenesis of the APS. The first was the discovery that beta-2 glycoprotein-1 (β2GPI) is the major auto antigen in APS. The second was the discovery in more recent years that β2GPI contains allosteric disulphide bonds susceptible to posttranslational modification that may be involved in the development of autoantibodies in APS. The main allosteric disulphide bond in the fifth domain of β2GPI can exist in two redox states: free thiol or oxidised. It is the conformational transformation of β2GPI from its free thiol form to its more immunogenic oxidised form that exposes neo-epitopes on the first and fifth domains. The purpose of this review is to highlight the recent findings on the posttranslational forms of β2GPI in the pathogenesis of APS. We suggest that novel assays quantitating the different redox forms of β2GPI in plasma or serum may be used to supplement existing clinical and laboratory assays to more accurately stratify risk of thrombosis or miscarriage in APS patients.

A direct, bioluminescent caspase-1 assay for monitoring inflammasome activation

Abstract Caspase-1, an essential component of the inflammasome, is activated in response to inflammatory stimuli, resulting in 1) the processing and release of IL-1b and IL-18, and 2) pyroptosis, an immunogenic form of cell death. To provide a homogeneous method for detecting caspase-1 activity, we developed a bioluminescent assay that combines a substrate, Z-WEHD-aminoluciferin, with a thermostable luciferase in an optimized reagent added directly to cultured cells. The assay can also measure caspase-1 activity released into the medium. Assay specificity for caspase-1 is conferred by inclusion of a proteasome inhibitor, MG-132, in the reagent and by use of a caspase-1 inhibitor, Ac-YVAD-CHO, to confirm activity. Using this assay system, caspase-1 activity has been monitored in THP-1 cells, J774A.1 mouse macrophages, mouse bone marrow-derived macrophages (BMDMs), and human primary monocytes treated with known inflammasome inducers. Caspase-1 activity was not detected in BMDMs derived from Casp 1−/−mice. Measuring caspase-1 activity released into medium enables multiplexing of the sample to monitor additional cell parameters. Multiplexing to monitor caspase-1 activity, cell death, and IL-1b release revealed that unprimed, undifferentiated THP-1 cells stimulated with nigericin or a-hemolysin activate caspase-1 and undergo pyroptosis, but do not release any IL-1b. This suggests that K+ efflux alone can engage the inflammasome and activate caspase-1 in THP-1 cells, providing an interesting system to assess pyroptosis in the absence of IL-1b. This convenient caspase-1 assay will allow a more efficient and effective assessment of inflammasome activation as well as enable high-throughput screening for inflammasome modulators.

Production of an enzymatically active and immunogenic form of ectodomain of Porcine rubulavirus hemagglutinin-neuraminidase in the yeast Pichia pastoris

Blue-eye disease (BED) of swine is a viral disease endemic in Mexico. The etiological agent is a paramyxovirus classified as Porcine rubulavirus (PoRV-LPMV), which exhibits in its envelope the hemagglutinin-neuraminidase (HN) glycoprotein, the most immunogenic and a major target for vaccine development. We report in this study the obtaining of ectodomain of PoRV HN (eHN) through the Pichia pastoris expression system. The expression vector (pPICZαB-HN) was integrated by displacement into the yeast chromosome and resulted in a Mut+ phenotype. Expressed eHN in the P. pastoris X33 strain was recovered from cell-free medium, featuring up to 67nmol/min/mg after 6 days of expression. eHN was recognized by the serum of infected pigs with strains currently circulating in the Mexican Bajio region. eHN induces antibodies in mice after 28 days of immunization with specific recognition in ELISA test. These antibodies were able to inhibit >80% replication by viral neutralization assays in cell culture. These studies show the obtaining of a protein with similar characteristics to the native HN and which may be a candidate to propose a vaccine or to use the antigen in a serologic diagnostic test.