D-dimer and CoV-2 spike-immune complexes contribute to the production of PGE2 and proinflammatory cytokines in monocytes.

An overreactive inflammatory response and coagulopathy are observed in patients with severe form of COVID-19. Since increased levels of D-dimer (DD) are associated with coagulopathy in COVID-19, we explored whether DD contributes to the aberrant cytokine responses. Here we show that treatment of healthy human monocytes with DD induced a dose dependent increase in production of pyrogenic mediator, Prostaglandin E2 (PGE2) and inflammatory cytokines, IL-6 and IL-8. The DD-induced PGE2 and inflammatory cytokines were enhanced significantly by co-treatment with immune complexes (IC) of SARS CoV-2 recombinant S protein or of pseudovirus containing SARS CoV-2 S protein (PVCoV-2) coated with spike-specific chimeric monoclonal antibody (MAb) containing mouse variable and human Fc regions. The production of PGE2 and cytokines in monocytes activated with DD and ICs was sensitive to the inhibitors of ß2 integrin and FcγRIIa, and to the inhibitors of calcium signaling, Mitogen-Activated Protein Kinase (MAPK) pathway, and tyrosine-protein kinase. Importantly, strong increase in PGE2 and in IL-6/IL-8/IL-1ß cytokines was observed in monocytes activated with DD in the presence of IC of PVCoV-2 coated with plasma from hospitalized COVID-19 patients but not from healthy donors. The IC of PVCoV-2 with convalescent plasma induced much lower levels of PGE2 and cytokines compared with plasma from hospitalized COVID-19 patients. PGE2 and IL-6/IL-8 cytokines produced in monocytes activated with plasma-containing IC, correlated well with the levels of spike binding antibodies and not with neutralizing antibody titers. Our study suggests that a combination of high levels of DD and high titers of spike-binding antibodies that can form IC with SARS CoV-2 viral particles might accelerate the inflammatory status of lung infiltrating monocytes leading to increased lung pathology in patients with severe form of COVID-19.

Pyrogenic and inflammatory mediators are produced by polarized M1 and M2 macrophages activated with D-dimer and SARS-CoV-2 spike immune complexes.

Lung macrophages are the first line of defense against invading respiratory pathogens including SARS-CoV-2, yet activation of macrophage in the lungs can lead to hyperinflammatory immune response seen in severe COVID-19. Here we used human M1 and M2 polarized macrophages as a surrogate model of inflammatory and regulatory macrophages and explored whether immune complexes (IC) containing spike-specific IgG can trigger aberrant cytokine responses in macrophages in the lungs and associated lymph nodes. We show that IC of SARS-CoV-2 recombinant S protein coated with spike-specific monoclonal antibody induced production of Prostaglandin E2 (PGE2) in non-polarized (M0) and in M1 and M2-type polarized human macrophages only in the presence of D-dimer (DD), a fibrinogen degradation product, associated with coagulopathy in COVID-19. Importantly, an increase in PGE2 was also observed in macrophages activated with DD and IC of SARS-CoV-2 pseudovirions coated with plasma from hospitalized COVID-19 patients but not from healthy subjects. Overall, the levels of PGE2 in macrophages activated with DD and IC were as follows: M1≫M2>M0 and correlated with the levels of spike binding antibodies and not with neutralizing antibody titers. All three macrophage subsets produced similar levels of IL-6 following activation with DD+IC, however TNFα, IL-1ß, and IL-10 cytokines were produced by M2 macrophages only. Our study suggests that high titers of spike or virion containing IC in the presence of coagulation byproducts (DD) can promote inflammatory response in macrophages in the lungs and associated lymph nodes and contribute to severe COVID-19.

Postchallenge administration of brincidofovir protects healthy and immune-deficient mice reconstituted with limited numbers of T cells from lethal challenge with IHD-J-Luc vaccinia virus.

UNLABELLED: Protection from lethality by postchallenge administration of brincidofovir (BCV, CMX001) was studied in normal and immune-deficient (nude, nu/nu) BALB/c mice infected with vaccinia virus (VACV). Whole-body bioluminescence imaging was used to record total fluxes in the nasal cavity, lungs, spleen, and liver and to enumerate pox lesions on tails of mice infected via the intranasal route with 10(5) PFU of recombinant IHD-J-Luc VACV expressing luciferase. Areas under the flux curve (AUCs) were calculated for individual mice to assess viral loads. A three-dose regimen of 20 mg/kg BCV administered every 48 h starting either on day 1 or day 2 postchallenge protected 100% of mice. Initiating BCV treatment earlier was more efficient in reducing viral loads and in providing protection from pox lesion development. All BCV-treated mice that survived challenge were also protected from rechallenge with IHD-J-Luc or WRvFire VACV without additional treatment. In immune-deficient mice, BCV protected animals from lethality and reduced viral loads while animals were on the drug. Viral recrudescence occurred within 4 to 9 days, and mice succumbed ∼10 to 20 days after treatment termination. Nude mice reconstituted with 10(5) T cells prior to challenge with 10(4) PFU of IHD-J-Luc and treated with BCV postchallenge survived the infection, cleared the virus from all organs, and survived rechallenge with 10(5) PFU of IHD-J-Luc VACV without additional BCV treatment. Together, these data suggest that BCV protects immunocompetent and partially T cell-reconstituted immune-deficient mice from lethality, reduces viral dissemination in organs, prevents pox lesion development, and permits generation of VACV-specific memory. IMPORTANCE: Mass vaccination is the primary element of the public health response to a smallpox outbreak. In addition to vaccination, however, antiviral drugs are required for individuals with uncertain exposure status to smallpox or for whom vaccination is contraindicated. Whole-body bioluminescence imaging was used to study the effect of brincidofovir (BCV) in normal and immune-deficient (nu/nu) mice infected with vaccinia virus, a model of smallpox. Postchallenge administration of 20 mg/kg BCV rescued normal and immune-deficient mice partially reconstituted with T cells from lethality and significantly reduced viral loads in organs. All BCV-treated mice that survived infection were protected from rechallenge without additional treatment. In immune-deficient mice, BCV extended survival. The data show that BCV controls viral replication at the site of challenge and reduces viral dissemination to internal organs, thus providing a shield for the developing adaptive immunity that clears the host of virus and builds virus-specific immunological memory.

An evaluation of novel real-time technology as a tool for measurement of radiobiological and radiation-induced bystander effects.

The xCELLigence real-time cell impedance system uses a non-invasive and label-free method to create a cell index that is a composite measure of cell proliferation. The aim of this study was to evaluate xCELLigence against clonogenic assay (gold standard) for measuring radiobiological effects and radiation-induced bystander effects (RIBE). A radiobiological study was conducted by irradiating EMT6.5, 4T1.2 and NMUMG cell lines with different radiation doses, while a RIBE study was done using transfer of conditioned media (CM) harvested from donor to the same type of recipient cell (EMT6.5, 4T1.2, NMUMG, HACAT and SW48). CM was harvested using two protocols which differed in the dose chosen and the exposure to the recipient cells. Results showed that xCELLigence measured a radiobiological effect which correlated with the clonogenic assay. For the RIBE study, no statistically significant differences were observed between xCELLigence or clonogenic survival in control or recipient cells incubated with CM in protocol one. However, there was a significant increase in cell index slope using CM from EMT-6.5 cells irradiated at 7.5 Gy compared with the control group under the second protocol. No other evidence of RIBE was detected by either xCELLigence or clonogenic assay. In conclusion, xCELLigence methods can measure radiobiological effects and the results correlate with clonogenic assay. We observed a lack of RIBE in all tested cell lines with the clonogenic assay; however, we observed a RIBE effect in EMT6.5 cells under one particular protocol that showed RIBE is cell type dependent, is not universally observed and can be detected in different assays.

Effects of postchallenge administration of ST-246 on dissemination of IHD-J-Luc vaccinia virus in normal mice and in immune-deficient mice reconstituted with T cells.

Whole-body bioimaging was used to study dissemination of vaccinia virus (VACV) in normal and in immune deficient (nu(-)/nu(-)) mice protected from lethality by postchallenge administration of ST-246. Total fluxes were recorded in the liver, spleen, lungs, and nasal cavities of live mice after intranasal infection with a recombinant IHD-J-Luc VACV expressing luciferase. Areas under the flux curve were calculated for individual mice to assess viral loads. Treatment for 2 to 5 days of normal BALB/c mice with ST-246 at 100 mg/kg starting 24 h postchallenge conferred 100% protection and reduced viral loads in four organs compared to control mice. Mice also survived after 5 days of treatment with ST-246 at 30 mg/kg, and yet the viral loads and poxes were higher in these mice compared to 100-mg/kg treatment group. Nude mice were not protected by ST-246 alone or by 10 million adoptively transferred T cells. In contrast, nude mice that received T cells and 7-day treatment with ST-246 survived infection and exhibited reduced viral loads compared to nonreconstituted and ST-246-treated mice after ST-246 was stopped. Similar protection of nude mice was achieved using adoptively transferred 1.0 and 0.1 million, but not 0.01 million, purified T cells or CD4(+) or CD8(+) T cells in conjunction with ST-246 treatment. These data suggest that ST-246 protects immunocompetent mice from lethality and reduces viral dissemination in internal organs and poxvirus lesions. Furthermore, immune-deficient animals with partial T cell reconstitution can control virus replication after a course of ST-246 and survive lethal vaccinia virus challenge.

Clonality of smooth muscle and fibroblast cell populations isolated from human fibroid and myometrial tissues.

Uterine fibroids are conventionally defined as clonally derived benign tumours from the proliferation of a single smooth muscle cell (SMC). We have previously identified fibroblast-like cells in fibroids, the presence of which raises the question as to whether all cells within the fibroid have the same clonal origin. The first aim of this study was to develop a fluorescence-activated cell sorting (FACS)-based method to isolate different cell types from human myometrium and fibroid tissues. Secondly, we aimed to use X chromosome inactivation analysis to determine the clonality of cell subpopulations isolated from myometrial and fibroid tissues. Human myometrium and fibroid tissues were collected from women undergoing hysterectomy. Immunohistochemistry (IHC) and flow cytometry confirmed that in addition to SMCs, fibroblasts constitute a significant proportion of cells in human myometrium and fibroid tissues. FACS based on CD90 and ALDH1 reliably separated cells into three myometrial and four fibroid subpopulations: SMCs, vascular smooth muscle cells and two fibroblast subsets. Clonality was first determined by X chromosome inactivation using the classic DNA methylation-sensitive HUMARA assay. Data from this assay were highly variable, with only a quarter of samples meeting the definition of clonal fibroid and non-clonal myometrium. However, using an RNA-based X chromosome inactivation HUMARA assay, we were able to demonstrate clonality of all cellular constituents of most fibroids. Our results confirm that most fibroids are derived from a single cell, and for the first time demonstrates that these clonal cells differentiate into fibroblast and SMC subpopulation as the fibroid grows.

Common fibroid-associated genes are differentially expressed in phenotypically dissimilar cell populations isolated from within human fibroids and myometrium.

Uterine fibroids are a prevalent gynaecological condition in reproductive-aged women and are the commonest reason for hysterectomy. The cellular composition of clonal fibroids are heterogeneous, with phenotypically dissimilar cells that include smooth muscle cells (SMC), vascular SMC (VSMC) and fibroblasts. The aim of our study was to investigate genes that are commonly differentially expressed between fibroid and myometrial whole tissues in phenotypically different sub-populations of cells isolated from fibroid and myometrium. Genes to be investigated by fluorescence-activated cell sorting, quantitative real-time PCR and immunocytochemistry include transforming growth factor ß (TGFB) and retinoic acid (RA) signalling families and steroid hormone receptors. We hypothesised that each cell population isolated from fibroid and myometrium would differ in the expression of fibroid-associated genes. We demonstrated that phenotypically different cellular constituents of uterine fibroids differentially express cellular RA-binding protein 2 (CRABP2), progesterone receptor B (PRB) and TGFB receptor 2 mRNA in fibroid-derived cells of VSMC and SMC phenotype. CRABP2 mRNA was also differentially expressed in fibroblasts and VSMC sub-populations from within clonal fibroid tumours. We conclude that differential regulation of RA, TGFB and PR pathway transcription occurs in fibroid-associated SMC and -fibroblasts and that investigation of paracrine interactions between different cell types within the fibroid microenvironment provides an important new paradigm for understanding the pathophysiology of this common disease.

Aberrant expression and regulation of NR2F2 and CTNNB1 in uterine fibroids.

Uterine fibroids are the most common benign tumour afflicting women of reproductive age. Despite the large healthcare burden caused by fibroids, there is only limited understanding of the molecular mechanisms that drive fibroid pathophysiology. Although a large number of genes are differentially expressed in fibroids compared with myometrium, it is likely that most of these differences are a consequence of the fibroid presence and are not causal. The aim of this study was to investigate the expression and regulation of NR2F2 and CTNNB1 based on their potential causal role in uterine fibroid pathophysiology. We used real-time quantitative RT-PCR, western blotting and immunohistochemistry to describe the expression of NR2F2 and CTNNB1 in matched human uterine fibroid and myometrial tissues. Primary myometrial and fibroid smooth muscle cell cultures were treated with progesterone and/or retinoic acid (RA) and sonic hedgehog (SHH) conditioned media to investigate regulatory pathways for these proteins. We showed that NR2F2 and CTNNB1 are aberrantly expressed in fibroid tissue compared with matched myometrium, with strong blood vessel-specific localisation. Although the SHH pathway was shown to be active in myometrial and fibroid primary cultures, it did not regulate NR2F2 or CTNNB1 mRNA expression. However, progesterone and RA combined regulated NR2F2 mRNA, but not CTNNB1, in myometrial but not fibroid primary cultures. In conclusion, we demonstrate aberrant expression and regulation of NR2F2 and CTNNB1 in uterine fibroids compared with normal myometrium, consistent with the hypothesis that these factors may play a causal role uterine fibroid development.

Passive immunotherapies protect WRvFire and IHD-J-Luc vaccinia virus-infected mice from lethality by reducing viral loads in the upper respiratory tract and internal organs.

Whole-body bioimaging was employed to study the effects of passive immunotherapies on lethality and viral dissemination in BALB/c mice challenged with recombinant vaccinia viruses expressing luciferase. WRvFire and IHD-J-Luc vaccinia viruses induced lethality with similar times to death following intranasal infection, but WRvFire replicated at higher levels than IHD-J-Luc in the upper and lower respiratory tracts. Three types of therapies were tested: licensed human anti-vaccinia virus immunoglobulin intravenous (VIGIV); recombinant anti-vaccinia virus immunoglobulin (rVIG; Symphogen, Denmark), an investigational product containing a mixture of 26 human monoclonal antibodies (HuMAbs) against mature virion (MV) and enveloped virion (EV); and HuMAb compositions targeting subsets of MV or EV proteins. Bioluminescence recorded daily showed that pretreatment with VIGIV (30 mg) or with rVIG (100 µg) on day -2 protected mice from death but did not prevent viral replication at the site of inoculation and dissemination to internal organs. Compositions containing HuMAbs against MV or EV proteins were protective in both infection models at 100 µg per animal, but at 30 µg, only anti-EV antibodies conferred protection. Importantly, the t statistic of the mean total fluxes revealed that viral loads in surviving mice were significantly reduced in at least 3 sites for 3 consecutive days (days 3 to 5) postchallenge, while significant reduction for 1 or 2 days in any individual site did not confer protection. Our data suggest that reduction of viral replication at multiple sites, including respiratory tract, spleen, and liver, as monitored by whole-body bioluminescence can be used to predict the effectiveness of passive immunotherapies in mouse models.

Polyclonal antibody cocktails generated using DNA vaccine technology protect in murine models of orthopoxvirus disease.

BACKGROUND: Previously we demonstrated that DNA vaccination of nonhuman primates (NHP) with a small subset of vaccinia virus (VACV) immunogens (L1, A27, A33, B5) protects against lethal monkeypox virus challenge. The L1 and A27 components of this vaccine target the mature virion (MV) whereas A33 and B5 target the enveloped virion (EV). RESULTS: Here, we demonstrated that the antibodies produced in vaccinated NHPs were sufficient to confer protection in a murine model of lethal Orthopoxvirus infection. We further explored the concept of using DNA vaccine technology to produce immunogen-specific polyclonal antibodies that could then be combined into cocktails as potential immunoprophylactic/therapeutics. Specifically, we used DNA vaccines delivered by muscle electroporation to produce polyclonal antibodies against the L1, A27, A33, and B5 in New Zealand white rabbits. The polyclonal antibodies neutralized both MV and EV in cell culture. The ability of antibody cocktails consisting of anti-MV, anti-EV, or a combination of anti-MV/EV to protect BALB/c mice was evaluated as was the efficacy of the anti-MV/EV mixture in a mouse model of progressive vaccinia. In addition to evaluating weight loss and lethality, bioimaging technology was used to characterize the spread of the VACV infections in mice. We found that the anti-EV cocktail, but not the anti-MV cocktail, limited virus spread and lethality. CONCLUSIONS: A combination of anti-MV/EV antibodies was significantly more protective than anti-EV antibodies alone. These data suggest that DNA vaccine technology could be used to produce a polyclonal antibody cocktail as a possible product to replace vaccinia immune globulin.

DNA modification of live cell surface.

We report a novel approach for the attachment of DNA fragments to the surface of live cells. By using fluorescence microscopy and flow cytometry we demonstrated that our synthetic conjugates of fatty acid with oligonucleotides can be incorporated in plasma membrane and then hybridized with complementary sequences at the cell surface. Method permits to control amount of immobilized DNA on the cell surface. All procedures can be completed within minutes and do not alter cell viability. Using this approach we tethered floating myeloid HL-60 cells to adherent A431 epitheliocytes in a sequence specific fashion. Thus, this method allows rapid and simple DNA multicoding of the cell surface and, therefore, opens new opportunities in manipulating with cell-cell interactions.

Application of bioluminescence imaging to the prediction of lethality in vaccinia virus-infected mice.

To find an alternative endpoint for the efficacy of antismallpox treatments, bioluminescence was measured in live BALB/c mice following lethal challenge with a recombinant WR vaccinia virus expressing luciferase. Intravenous vaccinia immunoglobulin treatments were used to confer protection on a proportion of animals. Using known lethality outcomes in 200 animals and total fluxes recorded daily in live animals, we performed univariate receiver operating characteristic (ROC) curve analysis to assess whether lethality can be predicted based on bioluminescence. Total fluxes in the spleens on day 3 and in the livers on day 5 generated accurate predictive models; the area under the ROC curve (AUC) was 0.91. Multiple logistic regression analysis utilizing a linear combination of six measurements: total flux in the liver on days 2, 3, and 5; in the spleen on days 1 and 3; and in the nasal cavity on day 4 generated the most accurate predictions (AUC = 0.96). This model predicted lethality in 90% of animals with only 10% of nonsurviving animals incorrectly predicted to survive. Compared with bioluminescence, ROC analysis with 25% and 30% weight loss as thresholds accurately predicted survival on day 5, but lethality predictions were low until day 9. Collectively, our data support the use of bioimaging for lethality prediction following vaccinia virus challenge and for gaining insight into protective mechanisms conferred by vaccines and therapeutics.

Production of fever mediator PGE2 in human monocytes activated with MDP adjuvant is controlled by signaling from MAPK and p300 HAT: Key role of T cell derived factor.

Fever and inflammatory responses were observed in some subjects in early clinical trials of vaccines adjuvanted with muramyl dipeptide (MDP), a NOD2 agonist. Biosynthesis of Prostaglandin E2 (PGE2) that transmits febrile signals to the brain is controlled by an inducible enzyme, Cyclooxygenase 2 (COX-2). MDP alone was not sufficient to induce expression of COX-2 and PGE2 production in vitro. Conditioned medium prepared from Peripheral Blood Mononuclear Cells (PBMCs)-derived CD3-bead purified human T cells (TCM) dramatically increased COX2 gene transcription, COX-2 protein expression, and PGE2 production in MDP-treated monocytes. We explored epigenetic changes at the COX2 promoter using Chromatin Immunoprecipitation assay (ChIP). Increase in COX2 transcription correlated with increased recruitment of RNA polymerase II (Pol II) and p300 histone acetyl transferase (HAT) to the COX2 promoter in monocytes activated with MDP and TCM. The role of p300 HAT was confirmed by using C646, an inhibitor of p300, that reduced binding of acetylated H3 and H4 histones at the COX2 promoter, COX2 transcription, and PGE2 production in monocytes. Binding of p300, Nuclear Factor Kappa B (NF-κB), and Pol II to the COX2 promoter was also sensitive to inhibitors of Mitogen-Activated Protein Kinase (MAPK) pathway and to antibodies against Macrophage-1 (Mac-1) integrin in MDP/TCM-treated monocytes. Importantly, recombinant Glycoprotein Ib alfa (GPIbα), the recently identified factor in TCM, increased binding of NF-κB, p300, and of Pol II to the COX2 promoter and COX2 transcription in MDP-treated monocytes. Our findings suggest that a second signal through Mac-1 and MAPK is triggered by a T cell derived soluble GPIbα protein leading to the assembly of the transcription machinery at the COX2 promoter and production of PGE2 in human monocytes in response to MDP/NOD2 activation.

T cell-derived soluble glycoprotein GPIbα mediates PGE2 production in human monocytes activated with the vaccine adjuvant MDP.

Vaccine adjuvants containing analogs of microbial products activate pattern recognition receptors (PRRs) on antigen-presenting cells, including monocytes and macrophages, which can cause prostaglandin E2 (PGE2) release and consequently undesired inflammatory responses and fever in vaccine recipients. Here, we studied the mechanism of PGE2 production by human monocytes activated with muramyl dipeptide (MDP) adjuvant, which activates cytosolic nucleotide-binding oligomerization domain 2 (NOD2). In rabbits, administration of MDP elicited an early increase in PGE2 followed by fever. In human monocytes, MDP alone did not induce PGE2 production. However, high amounts of PGE2 and the proinflammatory cytokines IL-1ß and IL-6 were secreted by monocytes activated with MDP in the presence of conditioned medium obtained from CD3 bead-isolated T cells (Tc CM) but not from those isolated without CD3 beads. Mass spectrometry and immunoblotting revealed that the costimulatory factor in Tc CM was glycoprotein Ib α (GPIbα). Antibody-mediated blockade of GPIbα or of its receptor, Mac-1 integrin, inhibited the secretion of PGE2, IL-1ß, and IL-6 in MDP + Tc CM-activated monocytes, whereas recombinant GPIbα protein increased PGE2 production by MDP-treated monocytes. In vivo, COX2 mRNA abundance was reduced in the liver and spleen of Mac-1 KO mice after administration of MDP compared with that of treated wild-type mice. Our findings suggest that the production of PGE2 and proinflammatory cytokines by MDP-activated monocytes is mediated by cooperation between two signaling pathways: one delivered by MDP through NOD2 and a second through activation of Mac-1 by T cell-derived GPIbα.

Retinoids regulate genes involved in retinoic acid synthesis and transport in human myometrial and fibroid smooth muscle cells.

BACKGROUND: Despite the fact that uterine fibroids are the most common benign tumors in women, their etiology is poorly understood. We have previously shown that multiple members of the retinoic acid (RA) pathway have altered expression in fibroids compared with normal myometrium. The aims of the present study were: to investigate regulation of genes involved in the RA pathway in vitro; and to identify genes that can be used as markers to distinguish myometrial and fibroid smooth muscle cells in culture. METHODS AND RESULTS: We demonstrate here for the first time that differential expression of aldehyde dehydrogenase 1 (ALDH1) between fibroids and myometrium is maintained in cell culture (without endothelial cells), and that this gene is differentially regulated by retinoids in myometrial compared with fibroid cells. RA and retinol also regulate expression of ADH1, cellular retinol binding protein 1 and cellular RA binding protein 2 in fibroid and myometrial cells. We show that many of the RA pathway genes tested maintain expression levels and differences in vitro. We also identify nine genes that are differentially expressed between myometrium and fibroids and maintain these differences and expression levels in cultured cells isolated from the same tissues. These genes can be used as markers to distinguish myometrial and fibroid cells in culture. CONCLUSIONS: Based on these findings, we propose that the RA pathway has an important and possible causative role in fibroid growth, as evidenced by the large number of genes with significantly altered expression in uterine fibroids that can be regulated by RA.

What Is the Predictive Value of Animal Models for Vaccine Efficacy in Humans? The Importance of Bridging Studies and Species-Independent Correlates of Protection.

Animal models have played a pivotal role in all stages of vaccine development. Their predictive value for vaccine effectiveness depends on the pathogen, the robustness of the animal challenge model, and the correlates of protection (if known). This article will cover key questions regarding bridging animal studies to efficacy trials in humans. Examples include human papillomavirus (HPV) vaccine in which animal protection after vaccination with heterologous prototype virus-like particles (VLPs) predicted successful efficacy trials in humans, and a recent approval of anthrax vaccine in accordance with the "Animal Rule." The establishment of animal models predictive of vaccine effectiveness in humans has been fraught with difficulties with low success rate to date. Challenges facing the use of animal models for vaccine development against Ebola and HIV will be discussed.

Development of an animal model of progressive vaccinia in nu/nu mice and the use of bioluminescence imaging for assessment of the efficacy of monoclonal antibodies against vaccinial B5 and L1 proteins.

Bioluminescence imaging (BLI) was used to follow dissemination of recombinant vaccinia virus (VACV) expressing luciferase (IHD-J-Luc) in BALB/c nu/nu mice treated post-challenge with monoclonal antibodies (MAbs) against L1 and B5 VACV proteins in a model of Progressive Vaccinia (PV). Areas Under the flux Curve (AUC) were calculated for viral loads in multiple organs in individual mice. Following scarification with 105 pfu, IHD-J-Luc VACV undergoes fast replication at the injection site and disseminates rapidly to the inguinal lymph nodes followed by spleen, liver, and axillary lymph nodes within 2-3 days and before primary lesions are visible at the site of scarification. Extension of survival in nude mice treated with a combination of anti-B5 and anti-L1 MAbs 24 h post challenge correlated with a significant reduction in viral load at the site of scarification and delayed systemic dissemination. Nude mice reconstituted with 104 T cells prior to challenge with IHD-J-Luc, and treated with MAbs post-challenge, survived infection, cleared the virus from all organs and scarification site, and developed anti-VACV IgG and VACV-specific polyfunctional CD8+ T cells that co-expressed the degranulation marker CD107a, and IFNγ and TNFα cytokines. All T cell reconstituted mice survived intranasal re-challenge with IHD-J-Luc (104 pfu) two months after the primary infection. Thus, using BLI to monitor VACV replication in a PV model, we showed that anti-VACV MAbs administered post challenge extended survival of nude mice and protected T cell reconstituted nude mice from lethality by reducing replication at the site of scarification and systemic dissemination of VACV.

Increased CXCR4-dependent HIV-1 fusion in activated T cells: role of CD4/CXCR4 association.

Activation of peripheral CD4+ T cells resulted in augmented fusion with X4 human immunodeficiency virus type 1 (HIV-1) envelope-expressing cells without parallel increases in the surface expression of CD4 or CXC chemokine receptor 4 (CXCR4). Our study used biochemical methods and biological assays to correlate the increased fusion potential of activated T cells with changes in CXCR4 isoforms and CD4-CXCR4 association. Western blot analyses of CXCR4, precipitated from resting T cells, identified several CXCR4 species with molecular weights of 47, 50, 62, and 98 kDa. After 24 h stimulation with phytohemagglutinin/interleukin-2, a marked reduction was seen in the 47-kDa, with a concomitant increase in the amounts of 50 and 62-64 kDa CXCR4. T cell activation also induced an increase in the coprecipitation of CXCR4 with CD4. The 62-kDa CXCR4 predominantly coprecipitated with CD4 and was shown to be ubiquitinated. Stripping of CD4 from the cell surface with pronase treatment prior to cell lysis only partially reduced coprecipitation of CD4 with the 62-kDa CXCR4, revealing a pool of intracellular CD4-CXCR4 complexes. Coprecipitation of CXCR4 with CD4 was reduced in activated cells treated with Brefeldin A and Monensin, suggesting that late endosomes play a role in intracellular association of CXCR4 with CD4. Confocal microscopy confirmed the colocalization of CD4 and CXCR4 within CD63+ endocytic compartments. These findings demonstrated a correlation between the enhanced susceptibility of activated T cells to HIV-1 fusion and accumulation of ubiquitinated 62-64 kDa CXCR4 species, which preferentially associated with CD4. The CD4-CXCR4 complexes may shuttle between late endosomes and the cell surface.

HIV coreceptors: role of structure, posttranslational modifications, and internalization in viral-cell fusion and as targets for entry inhibitors.

The human immunodeficiency virus (HIV) envelope glycoprotein forms trimers on the virion surface, with each monomer consisting of two subunits, gp120 and gp41. The gp120 envelope component binds to CD4 on target cells and undergoes conformational changes that allow gp120 to interact with certain G-protein-coupled receptors (GPCRs) on the same target membranes. The GPCRs that function as HIV coreceptors were found to be chemokine receptors. The primary coreceptors are CCR5 and CXCR4, but several other chemokine receptors were identified as "minor coreceptors", indicating their ability support entry of some HIV strains in tissue cultures. Formation of the tri-molecular complexes stabilizes virus binding and triggers a series of conformational changes in gp41 that facilitate membrane fusion and viral cell entry. Concerted efforts are underway to decipher the specific interactions between gp120/CD4, gp120/coreceptors, and their contributions to the subsequent membrane fusion process. It is hoped that some of the transient conformational intermediates in gp120 and gp41 would serve as targets for entry inhibitors. In addition, the CD4 and coreceptors are primary targets for several classes of inhibitors currently under testing. Our review summarizes the current knowledge on the interactions of HIV gp120 with its receptor and coreceptors, and the important properties of the chemokine receptors and their regulation in primary target cells. We also summarize the classes of coreceptor inhibitors under development.

CXCR4 heterogeneity in primary cells: possible role of ubiquitination.

The chemokine receptor CXCR4 is a primary coreceptor for the HIV-1 virus. The predicted molecular weight (MW) of glycosylated CXCR4 is 45-47 kDa. However, immunoblots of whole cell lysates from human lymphocytes, monocytes, macrophages, and the Jurkat T-lymphocyte line revealed multiple MW isoforms of CXCR4. Three of the bands could be precipitated by anti-CXCR4 monoclonal antibodies (101 and 47 kDa) or coprecipitated with CD4 (62 kDa). Expression of these isoforms was enhanced by infection with a recombinant vaccinia virus encoding CXCR4. In immunoblots of two-dimensional gels, antiubiquitin antibodies reacted with the 62-kDa CXCR4 species from monocytes subsequent to coprecipitation with anti-CD4 antibodies. Culturing of monocytes and lymphocytes with lactacystin enhanced the amount of the 101-kDa CXCR4 isoform in immunoblots by three- to sevenfold. In lymphocytes, lactacystin also increased cell-surface expression of CXCR4, which correlated with enhanced fusion with HIV-1 envelope-expressing cells. Similar increases in the intensity of the 101-kDa isoform were seen after treatment with the lysosomal inhibitors monensin and ammonium chloride. Antiubiquitin antibodies reacted with multiple proteins above 62 kDa, which were precipitated with anti-CXCR4 antibodies. Our data indicate that ubiquitination may contribute to CXCR4 heterogeneity and suggest roles for proteasomes and lysosomes in the constitutive turnover of CXCR4 in primary human cells.