Daniela Verthelyi

Differential signaling by CpG DNA in DCs and B cells: not just TLR9

CpG-containing oligodeoxynucleotides (CpG ODNs) act on Toll-like receptor 9 (TLR9) that is expressed on B cells and plasmacytoid dendritic cells (pDCs) to stimulate the innate immune system, however, different types of CpG ODNs induce distinct responses. Recent papers suggest some CpG ODNs could require a second receptor or cofactor to signal. The different signaling complexes assembled might impact on the affinity with which CpG ODNs signal to TLR9 or activate additional pathways that lead to distinct immune responses.

Immunoregulatory activity of CpG oligonucleotides in humans and nonhuman primates.

Oligodeoxynucleotides (ODN) containing CpG motifs mimic the ability of microbial DNA to activate the innate immune system. The resultant response limits the early spread of infectious organisms while promoting the development of adaptive immunity. CpG ODN show promise as vaccine adjuvants and in the treatment of asthma, allergy, infection, and cancer. Due to evolutionary divergence in CpG recognition between species, CpG ODN that are most active in rodents are poorly immunostimulatory in primates. Thus, evidence that CpG ODN have therapeutic activity in mice must be confirmed in primates. Two distinct types of CpG ODN were identified that stimulate primate PBMC. D-type ODN trigger plasmacytoid DC to secrete IFNalpha, monocytes to mature into functionally active DC, and NK cells to secrete IFNgamma. K-type ODN stimulate B cells and monocytes to proliferate and secrete IgM, IL-10, and/or IL-6. In vivo studies in nonhuman primates indicate that proinflammatory or humoral immune responses can be selectively facilitated by judicious use of these distinct types of ODN.

Inhibition of Taq polymerase as a method for screening heparin for oversulfated contaminants

Heparin and low molecular heparins are extensively used in the treatment of a wide range of diseases in addition to their classic anticoagulant activity and can be found coating medical devices such as catheters, stents and filters. Early in 2008, a sharp increase in heparin-associated severe adverse events, including over 80 deaths, was linked to the presence of a contaminant identified as hypersulfated chondroitin sulfate (OS-CS). OS-CS is one of several oversulfated glycosaminoglycans (GAGs) of different origins that can potentially cause similar clinical problems underscoring the need to develop robust screening methods for contaminants in existing and future lots of heparin. This study demonstrates that oversulfated GAGs block the activity of Taq polymerase used for real time PCR. Based on this finding we developed a simple, rapid, sensitive and high throughput screening method to detect and quantify oversulfated chondroitin sulfate (OS-CS) and other potential oversulfated contaminants in commercial lots of heparin. This method requires less than 100 miliUnits (mU) of heparin as starting material, therefore avoiding the need to lyophilize and concentrate samples, and has a limit of detection of <1 ng for all oversulfated GAGs tested.

Prior Exposure to Zika Virus Significantly Enhances Peak Dengue-2 Viremia in Rhesus Macaques

Structural and functional homologies between the Zika and Dengue viruses’ envelope proteins raise the possibility that cross-reactive antibodies induced following Zika virus infection might enhance subsequent Dengue infection. Using the rhesus macaque model we show that prior infection with Zika virus leads to a significant enhancement of Dengue-2 viremia that is accompanied by neutropenia, lympocytosis, hyperglycemia, and higher reticulocyte counts, along with the activation of pro-inflammatory monocyte subsets and release of inflammatory mediators. Zika virus infection induced detectable Dengue cross-reactive serum IgG responses that significantly amplified after Dengue-2 virus infection. Serum from Zika virus immune animals collected prior to Dengue-2 infection showed significant capacity for in vitro antibody dependent enhancement of Dengue-1, 2, 3 and 4 serotypes suggesting that pre-existing immunity to Zika virus could potentially enhance infection by heterologous Dengue serotypes. Our results provide first in vivo evidence that prior exposure to Zika virus infection can enhance Dengue infection, which has implications for understanding pathogenesis and the development of vaccines.

Adjuvant Properties of CpG Oligonucleotides in Primates

Unlike mammalian DNA, bacterial, plasmid, and synthetic DNA containing unmethylated CpG dinucleotides in specific sequence contexts are recognized by the Toll-like receptor 9 expressed by B cells and plasmacytoid dendritic cells, and trigger the activation of the innate and adaptive immune system. Upon signaling, CpG DNA induces B cells, natural killer cells, macrophages, and dendritic cells to proliferate, differentiate, take up, and present antigen and secrete a variety of immunoglobulins, chemokines, and predominantly Th1-type cytokines. Preclinical studies in mice and primates show that DNA sequences containing CpG motifs can selectively promote cellular and/or humoral immune responses in vivo. Early results from ongoing clinical studies indicate that CpG oligonucleotides (ODN) are well tolerated and improve the immune response to microbial vaccines. This work examines the progress in utilizing CpG ODN as adjuvants in conventional and DNA vaccines.

Expression and regulation in the brain of the chemokine CCL27 gene locus

The chemokine CCL27 has chemoattractant properties for memory T cells and has been implicated in skin allergic reactions. The present study reports the expression in the brain of two CCL27 splice variants localized in the cerebral cortex and limbic regions. CCL27-like immunoreactivity was identified mainly in neurons. Variant 1 was found elevated in the olfactory bulbs during allergic inflammation induced by intranasal challenge with allergen. This was accompanied by the presence of T cells in the olfactory bulbs. Intranasal administration of neutralizing antibodies against CCL27 reduced the presence of T cells in the olfactory bulbs suggesting a function in T cell activity in the brain.

PF4-HIT antibody (KKO) complexes activate broad innate immune and inflammatory responses

INTRODUCTIONnHeparin-induced thrombocytopenia (HIT) is an immune-mediated complication of heparin anticoagulation therapy resulting in thrombocytopenia frequently accompanied by thrombosis. Current evidence suggests that HIT is associated with antibodies developed in response to multi-molecular complexes formed by platelet factor 4 (PF4) bound to heparin or cell surface glycosaminoglycans. These antibody complexes activate platelets and monocytes typically through FcγRIIA receptors increasing the production of PF4, inflammatory mediators, tissue factor and thrombin. The influence of underlying events in HIT including complex-induced pro-inflammatory cell activation and structural determinants leading to local inflammatory responses are not fully understood.nnnMETHODSnThe stoichiometry and complex component requirements were determined by incubating fresh peripheral blood mononuclear cells (PBMC) with different concentrations of unfractionated heparin (H), low molecular weight heparin (LMWH), PF4- and anti-PF4-H complex antibodies (KKO). Cytokine mRNA or protein were measured by qRT-PCR or Meso Scale Discovery technology, respectively. Gene expression profile analysis for 594 genes was performed using Nanostring technology and analyzed using Ingenuity Pathway Analysis software.nnnRESULTS AND CONCLUSIONSnThe data show that antibodies magnify immune responses induced in PBMCs by PF4 alone or in complex with heparin or LMWH. We propose that following induction of HIT antibodies by heparin-PF4 complexes, binding of the antibodies to PF4 is sufficient to induce a local pro-inflammatory response which may play a role in the progression of HIT. In vitro assays using PBMCs may be useful in characterizing local inflammatory and innate immune responses induced by HIT antibodies in the presence of PF4 and different sources of heparins.nnnFDA DISCLAIMERnThe findings and conclusions in this article are solely the responsibility of the authors and are not being formally disseminated by the Food and Drug Administration. Thus, they should not be construed to represent any Agency determination or policy.

High-throughput real-time PCR for early detection and quantitation of arenavirus Tacaribe

Arenaviruses merit significant attention both as causative agents of endemic hemorrhagic fevers and as model systems to study the immune response to acute and persistent viral infections. Development of highly sensitive quantitative screening methods to detect arenavirus is critical for early diagnosis of patients, to screen the rodent population in endemic areas, and as a research tool to confirm effective tissue clearance during the development of anti-viral strategies. This study describes a novel sensitive and reproducible method to quantify prototypic new world arenavirus Tacaribe RNA in cell cultures and tissues using a real-time TaqMan PCR-based detection system. The method has a sensitivity of 100 RNA copies per 200ng of total RNA, making it 2 logs more sensitive than the currently utilized TCID(50) method, and a linear range from 10(2) to 10(9) copies/reaction. The qRT-PCR method is high-throughput and screening can be achieved in <2h allowing for diagnosis of infected patients before the onset of symptoms. This new method is a powerful tool to screen populations for infection and monitor the clearance achieved by available therapies, and serves as a model diagnostic tool for other arenaviruses.

The IP10 (CXCL10) specific cDNA probe of the mCK-5c multiprobe RNase protection assay kit carries two nucleotide insertions that complicate the interpretation of results

RNase protection assays (RPA) employing multiprobe sets are powerful tools to simultaneously measure transcription of several different genes. We used BD Biosciences/Pharmingens mouse chemokine probeset mCK-5c to measure chemokine gene expression in brain and spleen tissue of mice. Depending on the RPA protocol used, we observed differences in the relative amounts of transcripts for interferon-inducible protein 10 (IP-10) and T-cell activation-3 (TCA-3). Isolation and sequencing of the IP-10 specific gene from the mCK-5c probeset revealed two nucleotide insertions in the probe that are not present in the natural IP-10 cDNA. We show that these insertions cause RNase A-dependent degradation of the protected IP-10 mRNA yielding a fragment indistinguishable in size from that specific for TCA-3, thus leading to over-interpretation of TCA-3 expression as well as underestimation of IP-10 gene expression levels.