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1.  In vitro and in vivo protective efficacies of antibodies that neutralize the RNA N-glycosidase activity of Shiga toxin 2 
BMC Immunology  2010;11:16.
Shiga toxin 2 (Stx2), one of two Stx liberated by Stx-producing Escherichia coli, is composed of an A subunit monomer and a B subunit pentamer, and is directly linked with hemolytic uremic syndrome in children. The pentameric B subunit binds to its cell surface receptor Gb3 for toxin internalization, and the A subunit follows intracellular retrograde transport to the cytosol where its RNA N-glycosidase activity (RNA-NGA) shuts down the protein synthesis, and leads to cell death. The present study investigated the ability of 19 Stx2 A subunit-specific human monoclonal antibodies (HuMAbs) to neutralize the RNA-NGA, and the association this neutralizing activity with protection of HeLa cells and mice against Stx2-induced death.
The HuMAbs that were stronger inhibitors of RNA-NGA were also better at neutralizing Stx2 mediated HeLa cell death, and those that were weaker inhibitors of RNA-NGA activity were also weaker in protecting HeLa cells. These results suggest that the ability of an A subunit-specific antibody to block the RNA-NGA of the toxin is directly related to its ability to neutralize Stx2-mediated HeLa cell death. However, with the exception of the best RNA-NGA blocking antibodies 5C12 and 2F10, the efficacies of antibody neutralization of RNA-NGA of Stx2 did not correlate with their in vivo protective efficacies. The HuMAb 6C3, which neutralized RNA N-glycosidase activity of Stx2 less effectively than the HuMAbs 6D8 and 6B7, protected 100% of the mice against Stx2 challenge at 50 μg/mouse dose. In contrast, the HuMAbs 6D8 and 6B7, which neutralized RNA N-glycosidase activity of Stx2 more effectively than 6C3, protected 20% and 0% mice at that dose, respectively.
The neutralization efficiency of the RNA-NGA of Stx2 by A subunit-specific antibodies correlate strongly with their abilities to protect HeLa cells against Stx2-mediated toxicity but only the strongest RNA-NGA-neutralizing antibodies correlate very well with both protecting HeLa cells and mice against Stx2 challenge.
PMCID: PMC2859748  PMID: 20334660
2.  CX3CR1 Is Expressed in Differentiated Human Ciliated Airway Cells and Co-Localizes with Respiratory Syncytial Virus on Cilia in a G Protein-Dependent Manner 
PLoS ONE  2015;10(6):e0130517.
Respiratory syncytial virus (RSV) is the principal cause of bronchiolitis in infants and a significant healthcare problem. The RSV Glycoprotein (G) mediates attachment of the virus to the cell membrane, which facilitates interaction of the RSV Fusion (F) protein with nucleolin, thereby triggering fusion of the viral and cellular membranes. However, a host protein ligand for G has not yet been identified. Here we show that CX3CR1 is expressed in the motile cilia of differentiated human airway epithelial (HAE) cells, and that CX3CR1 co-localizes with RSV particles. Upon infection, the distribution of CX3CR1 in these cells is significantly altered. Complete or partial deletion of RSV G results in viruses binding at least 72-fold less efficiently to cells, and reduces virus replication. Moreover, an antibody targeting an epitope near the G protein’s CX3CR1-binding motif significantly inhibits binding of the virus to airway cells. Given previously published evidence of the interaction of G with CX3CR1 in human lymphocytes, these findings suggest a role for G in the interaction of RSV with ciliated lung cells. This interpretation is consistent with past studies showing a protective benefit in immunizing against G in animal models of RSV infection, and would support targeting the CX3CR1-G protein interaction for prophylaxis or therapy. CX3CR1 expression in lung epithelial cells may also have implications for other respiratory diseases such as asthma.
PMCID: PMC4479564  PMID: 26107373
3.  Transfer of maternal cytokines to suckling piglets: in vivo and in vitro models with implications for immunomodulation of neonatal immunity 
Maternal cytokines may play instructive roles in development of the neonatal immune system. However, cytokines in colostrum and milk and their transfer from mothers to neonates have not been well documented, except for TGF-β. Swine provide a unique model to study lactogenic cytokines because the sow's impermeable placenta prohibits transplacental passage. We investigated IL-6 and TNF-α (pro-inflammatory), IFN-γ and IL-12, (Th1), IL-10 and IL-4 (Th2) and TGF-β1 (Th3) concentrations in sow serum and colostrum/milk and serum of their suckling and weaned piglets and in age-matched colostrum-deprived gnotobiotic piglets. All cytokines were detected in colostrum/milk and correlated with concentrations in sow serum except for mammary-derived TNF-α and TGF-β1. Detection of IL-12 and TGF-β1 in pre-suckling and colostrum-deprived gnotobiotic piglet serum suggests constitutive production: other cytokines were undetectable confirming absence of transplacental transfer. Peak median cytokine concentrations in suckling piglet serum occurred at post-partum days 1-2 (IL-4>IL-6>IFN-γ>IL-10). The effects in vitro of physiologically relevant concentrations of the two predominant lactogenic cytokines (TGF-β1 and IL-4) on porcine naive B cell responses to lipopolysaccharide (LPS) and rotavirus (RV) were investigated. High (10ng/ml) TGF-β1 suppressed immunoglobulin secreting cell responses to LPS and rotavirus; low concentrations (0.1ng/ml) promoted isotype switching to IgA antibody. Interleukin-4 induced inverse dose-dependent (0.1>10ng/ml) isotype switching to IgA and enhanced IgM secreting cell responses to LPS and rotavirus. In summary, we documented the transfer and persistence of maternal cytokines from colostrum/milk to neonates and their potential role in Th-2 biased IgA responses and reduced immunologic responsiveness of neonates.
PMCID: PMC4094377  PMID: 17403542
Cytokines; Milk; Neonate; Swine; Th2 bias
4.  Biodistribution and elimination kinetics of systemic Stx2 by the Stx2A and Stx2B subunit-specific human monoclonal antibodies in mice 
BMC Immunology  2012;13:27.
Hemolytic uremic syndrome (HUS) leading to acute kidney failure, is a condition linked to the production of primarily Shiga toxin 2 (Stx2) by some E. coli serotypes. We have previously shown that Stx2 A subunit-specific human monoclonal antibody (HuMAb) 5C12, and B subunit-specific HuMAb 5H8 inhibit cultured cell death, and protect mice and piglets from fatal Stx2-intoxication. We have also shown that 5H8 blocks binding of Stx2 to its cell-surface receptor globotriaosyl ceramide (Gb3), whereas Stx2 when complexed with 5C12 binds Gb3 with higher affinity than Stx2. The mechanism by which 5C12 neutralizes Stx2 in vitro involves trapping of Stx2 in the recycling endosomes and releasing it into the extracellular environment. Because of the clinical implications associated with the formation of Stx2/antibody complexes and the potential for trapping and clearance through a severely damaged kidney associated with HUS, we investigated the likely site(s) of Stx2/antibody localization and clearance in intoxicated mice treated with antibody or placebo.
Mice were injected with radiolabeled Stx2 (125I-Stx2) 4 hours after administration of 5C12, 5H8, or phosphate buffered saline (PBS) and the sites of localization of labeled Stx2, were investigated 3, 24 and 48 hours later. The liver recorded statistically much higher concentrations of labeled Stx2 for groups receiving 5C12 and 5H8 antibodies after 3, 24 and 48 hours, as compared with the PBS group. In contrast, highest levels of labeled Stx2 were detected in the kidneys of the PBS group at all 3 sampling times. Mice receiving either of the two HuMAbs were fully protected against the lethal effect of Stx2, as compared with the fatal outcome of the control group.
The results suggest that HuMAbs 5C12 and 5H8 promoted hepatic accumulation and presumably clearance of toxin/antibody complexes, significantly diverting Stx2 localization in the kidneys, the target of Stx2 and the cause of HUS. This is in contrast to the fatal outcome of the control group receiving PBS. The results also confirm earlier observations that both HuMAbs are highly and equally protective against Stx2 intoxication in mice.
PMCID: PMC3436740  PMID: 22655967
Shiga toxin; Radiolabel; Antibody; Toxin elimination; Toxin concentration; Pharmacokinetic; Human monoclonal antibody
5.  Stx2- but not Stx1-specific human monoclonal antibody protects piglets challenged with enterohemorrhagic Escherichia coli producing Stx1 and Stx2 
The Journal of infectious diseases  2010;201(7):1081-1083.
Shiga toxin 2 (Stx2) producing Escherichia coli (STEC) strains are more frequently isolated from hemolytic-uremic syndrome cases than strains that produce Stx1 and Stx2, and rarely the strains that produce only Stx1. Studies have implicated Stx2 as the sole contributor to acute kidney failure and other systemic complications in humans, and our study adds further support to this assumption since Stx2- and Stx1-specific antibodies protected 100% and 0% of piglets, respectively, against an oral challenge with a Stx1- and Stx2-producing STEC strain. We conclude that Stx2-specific antibody is sufficient to protect piglets, and possibly humans, against STEC producing both toxins.
PMCID: PMC2846416  PMID: 20196656
Shiga toxin; Stx; antibody; enterohemorrhagic; piglet; hemolytic uremic syndrome; kidney failure; E. coli
6.  A piglet Model of Acute gastroenteritis induced by Shigella dysenteriae type 1 
The Journal of infectious diseases  2010;201(6):903-911.
The lack of a standardized laboratory animal model that mimics key aspects of human shigellosis remains major obstacle to addressing questions on pathogenesis, screening therapeutics and evaluating vaccines.
We characterize a piglet model for Shigella dysenteriae type 1.
Piglets developed acute diarrhea, anorexia, dehydration and often fatal, with severity depending on age and dose. Bacteria were apparent in the lumen and on surface epithelium throughout the gut initially, but severe mucosal damage and bacterial cellular invasion were most profound in the colon. Detached necrotic colonocytes were present in the lumen, with inflammatory cells outpouring from damaged mucosa. High levels of IL-8 and IL-12, were followed by other proinflammatory cytokines. Elevated TNF-α, IL-1β, IL-6, and IL-10 were detected in feces and in gut segments of infected animals. Bacteria were present inside epithelial cells and within colonic lamina propria. In contrast, an isogenic strain lacking Shiga toxin, induced similar but milder symptoms, with moderate mucosal damage and lower cytokine levels.
We conclude that piglets are highly susceptible to shigellosis providing a useful tool to compare vaccine candidates for immunogenicity, reactogenicity and response to challenge, investigate the role of virulence factors and test the efficacy of microbial agents.
PMCID: PMC2826551  PMID: 20136414
7.  An Oral versus Intranasal Prime/Boost Regimen Using Attenuated Human Rotavirus or VP2 and VP6 Virus-Like Particles with Immunostimulating Complexes Influences Protection and Antibody-Secreting Cell Responses to Rotavirus in a Neonatal Gnotobiotic Pig Model ▿ 
We determined the impact of mucosal prime/boost regimens and vaccine type (attenuated Wa human rotavirus [AttHRV] or nonreplicating Wa 2/6 rotavirus-like particles [VLP]) on protection and antibody-secreting cell (ASC) responses to HRV in a neonatal gnotobiotic pig disease model. Comparisons of delivery routes for AttHRV and evaluation of nonreplicating VLP vaccines are important as alternative vaccine approaches to overcome risks associated with live oral vaccines. Groups of neonatal gnotobiotic pigs were vaccinated using combinations of oral (PO) and intranasal (IN) inoculation routes as follows: (i) 3 oral doses of AttHRV (AttHRV3×PO); (ii) AttHRV3×IN; (iii) AttHRVPO, then 2/6VLP2×IN; (iv) AttHRVIN, then 2/6VLP2×IN; and (v) mock-inoculated controls. Subsets of pigs from each group were challenged with virulent Wa HRV [P1A(8) G1] (4 weeks post-primary inoculation) to assess protection. The AttHRVPO+2/6VLP2×IN pigs had the highest protection rates against virus shedding and diarrhea (71% each); however, these rates did not differ statistically among the vaccine groups, except for the AttHRVIN+2/6VLPIN group, which had a significantly lower protection rate (17%) against diarrhea. The isotype, magnitude, and tissue distribution of ASCs were analyzed by enzyme-linked immunospot assay. The highest mean numbers of virus-specific IgG and IgA ASCs were observed pre- and postchallenge in both intestinal and systemic lymphoid tissues of the AttHRVPO+2/6VLPIN group. Thus, the AttHRVPO+2/6VLPIN vaccine regimen using immunostimulating complexes (ISCOM) and multiple mucosal inductive sites, followed by AttHRV3×PO or IN regimens, were the most effective vaccine regimens, suggesting that either AttHRVPO+2/6VLPIN or AttHRV3×IN may be an alternative approach to AttHRV3×PO for inducing protective immunity against rotavirus diarrhea.
PMCID: PMC2837955  PMID: 20107005
8.  High Titers of Circulating Maternal Antibodies Suppress Effector and Memory B-Cell Responses Induced by an Attenuated Rotavirus Priming and Rotavirus-Like Particle-Immunostimulating Complex Boosting Vaccine Regimen 
Clinical and Vaccine Immunology  2006;13(4):475-485.
We investigated maternal antibody (MatAb) effects on protection and immune responses to rotavirus vaccines. Gnotobiotic pigs were injected intraperitoneally at birth with pooled serum from sows hyperimmunized with human rotavirus (HRV); control pigs received no sow serum. Pigs with or without MatAbs received either sequential attenuated HRV (AttHRV) oral priming and intranasal boosting with VP2/VP6 virus-like particle (VLP)-immunostimulating complex (ISCOM) (AttHRV/VLP) or intranasal VLP-ISCOM prime/boost (VLP) vaccines at 3 to 5 days of age. Subsets of pigs were challenged at 28 or 42 days postinoculation with virulent Wa HRV to assess protection. Isotype-specific antibody-secreting cell (ASC) responses to HRV were quantitated by enzyme-linked immunospot assay to measure effector and memory B-cell responses in intestinal and systemic lymphoid tissues pre- and/or postchallenge. Protection rates against HRV challenge (contributed by active immunity and passive circulating MatAbs) were consistently (but not significantly) lower in the MatAb-AttHRV/VLP groups than in the corresponding groups without MatAbs. Intestinal B-cell responses in the MatAb-AttHRV/VLP group were most suppressed with significantly reduced or no intestinal immunoglobulin A (IgA) and IgG effector and memory B-cell responses or antibody titers pre- and postchallenge. This suppression was not alleviated but was enhanced after extending vaccination/challenge from 28 to 42 days. In pigs vaccinated with nonreplicating VLP alone that failed to induce protection, MatAb effects differed, with intestinal and systemic IgG ASCs and prechallenge memory B cells suppressed but the low intestinal IgA and IgM ASC responses unaffected. Thus, we demonstrate that MatAbs differentially affect both replicating and nonreplicating HRV vaccines and suggest mechanisms of MatAb interference. This information should facilitate vaccine design to overcome MatAb suppression.
PMCID: PMC1459641  PMID: 16603615

Results 1-8 (8)