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Callaway J.B.,University of North Carolina at Chapel Hill | Smith S.A.,Vanderbilt Vaccine Center | Smith S.A.,Vanderbilt Medical Center | Widman D.G.,University of North Carolina at Chapel Hill | And 8 more authors.
PLoS ONE | Year: 2015

Dengue virus is a major global health threat and can lead to life-threatening hemorrhagic complications due to immune activation and cytokine production. Cross-reactive antibodies to an earlier dengue virus infection are a recognized risk factor for severe disease. These antibodies bind heterologous dengue serotypes and enhance infection into Fc-receptorbearing cells, a process known as antibody-dependent enhancement of infection. One crucial cytokine seen elevated in severe dengue patients is IL-1β, a potent inflammatory cytokine matured by the inflammasome. We used a highly-physiologic system by studying antibody-dependent enhancement of IL-1β in primary human monocytes with anti-dengue human monoclonal antibodies isolated from patients. Antibody-enhancement increased viral replication in primary human monocytes inoculated with supernatant harvested from Vero cells infected with dengue virus serotype 2 (DENV-2) 16681. Surprisingly, IL-1β secretion induced by infectious supernatant harvested from two independent Vero cell lines was not enhanced by antibody. Secretion of multiple other inflammatory cytokines was also independent of antibody signaling. However, IL-1β secretion did require NLRP3 and caspase- 1 activity. Immunodepletion of dengue virions from the infectious supernatant confirmed that virus was not the main IL-1β-inducing agent, suggesting that a supernatant component(s) not associated with the virion induced IL-1β production. We excluded RNA, DNA, contaminating LPS, viral NS1 protein, complement, and cytokines. In contrast, purified Vero-derived DENV-2 16681 exhibited antibody-enhancement of both infection and IL-1β induction. Furthermore, C6/36 mosquito cells did not produce such an inflammatory component, as crude supernatant harvested from insect cells infected with DENV-2 16681 induced antibody-dependent IL-1β secretion. This study indicates that Vero cells infected with DENV-2 16681 may produce inflammatory components during dengue virus propagation that mask the virus-specific immune response. Thus, the choice of host cell and viral purity should be carefully considered, while insect-derived virus represents a systemthat elicits antibody- dependent cytokine responses to dengue virus with fewer confounding issues. © 2015 Callaway et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Sapparapu G.,Vanderbilt Medical Center | Sapparapu G.,Vanderbilt Vaccine Center | Czako R.,Baylor College of Medicine | Alvarado G.,Vanderbilt Medical Center | And 6 more authors.
PLoS Pathogens | Year: 2016

Noroviruses (NoV) are the most common cause of non-bacterial acute gastroenteritis and cause local outbreaks of illness, especially in confined situations. Despite being identified four decades ago, the correlates of protection against norovirus gastroenteritis are still being elucidated. Recent studies have shown an association of protection with NoV-specific serum histo-blood group antigen-blocking antibody and with serum IgA in patients vaccinated with NoV VLPs. Here, we describe the isolation and characterization of human monoclonal IgG and IgA antibodies against a GI.I NoV, Norwalk virus (NV). A higher proportion of the IgA antibodies blocked NV VLP binding to glycans than did IgG antibodies. We generated isotype-switched variants of IgG and IgA antibodies to study the effects of the constant domain on blocking and binding activities. The IgA form of antibodies appears to be more potent than the IgG form in blocking norovirus binding to histo-blood group antigens. These studies suggest a unique role for IgA antibodies in protection from NoV infections by blocking attachment to cell receptors. © 2016 Sapparapu et al.

Callaway J.B.,University of North Carolina at Chapel Hill | Smith S.A.,Vanderbilt Vaccine Center | Smith S.A.,Vanderbilt Medical Center | McKinnon K.P.,University of North Carolina at Chapel Hill | And 4 more authors.
Journal of Biological Chemistry | Year: 2015

Approximately 500,000 people are hospitalized with severe dengue illness annually. Antibody-dependent enhancement (ADE) of dengue virus (DENV) infection is believed to contribute to the pathogenic cytokine storm described in severe dengue patients, but the precise signaling pathways contributing to elevated cytokine production are not elucidated. IL-1β is a potent inflammatory cytokine that is frequently elevated during severe dengue, and the unique dual regulation of IL-1β provides an informative model to study ADE-induced cytokines. This work utilizes patient-derived anti-DENV mAbs and primary human monocytes to study ADE-induced IL-1β and other cytokines. ADE of DENV serotype 2 (DENV-2) elevates mature IL-1βsecretion by monocytes independent of DENV replication by 4 h postinoculation (hpi). Prior to this, DENV immune complexes activate spleen tyrosine kinase (Syk) within 1 hpi. Syk induces elevated IL1B, TNF, and IL6 mRNA by 2 hpi. Syk mediates elevated IL-1β secretion by activating ERK1/2, and both Syk and ERK1/2 inhibitors ablated ADE-induced IL-1β secretion. Maturation of pro-IL-1β during ADE requires caspase-1 and NLRP3, but caspase-1 is suboptimally increased by ADE and can be significantly enhanced by a typical inflammasome agonist, ATP. Importantly, this inflammatory Syk-ERK signaling axis requires DENV immune complexes, because DENV-2 in the presence of serotype-matched anti-DENV-2 mAb, but not anti- DENV-1 mAb, activates Syk, ERK, and IL-1β secretion. This study provides evidence that DENV-2 immune complexes activate Syk to mediate elevated expression of inflammatory cytokines. Syk and ERK may serve as new therapeutic targets for interfering with ADE-induced cytokine expression during severe dengue. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Goins W.P.,Baylor College of Medicine | Edwards K.M.,Vanderbilt Vaccine Research Program | Rock M.T.,Vanderbilt Vaccine Center | Schaffner W.,Vanderbilt University | Talbot T.R.,Vanderbilt University
Clinical Infectious Diseases | Year: 2012

Background. Antibiotic postexposure prophylaxis (PEP) following pertussis exposure is recommended but has never been evaluated in healthcare personnel (HCP) vaccinated with acellular pertussis vaccine (Tdap). Methods. Tdap-vaccinated HCP were randomized to receive azithromycin PEP or no PEP following pertussis exposure. Acute and convalescent nasopharyngeal swabs and sera were obtained for pertussis testing by polymerase chain reaction (PCR) and anti-pertussis toxin (PT) immunoglobulin G, respectively. A nasopharyngeal aspirate was also collected for PCR and culture from subjects who reported respiratory symptoms within 21 days following identification of the exposure. Pertussis infection was defined as a positive culture or PCR, a 2-fold rise in anti-PT titer, or a single anti-PT titer of ≥94 enzyme-linked immunosorbent assay units/mL. Daily symptom monitoring without PEP was considered noninferior to PEP after pertussis exposure if the lower limit of the 1-sided 95% confidence interval (CI) for the reduction in pertussis was greater than-7%.Results.During 30 months of study, 86 subjects were randomized following a pertussis exposure. Using the predefined definition of infection, pertussis infection did not develop in 41 (97.6%) of 42 subjects who received azithromycin PEP and 38 (86.4%) of 44 subjects who did not receive PEP (absolute risk difference,-11.3%; lower bound of the 1-sided 95% CI,-20.6%; P =. 81). However, no subject developed symptomatic pertussis confirmed with culture or a specific PCR assay, and possibly no subject developed subclinical pertussis infection based upon additional serologic testing. Conclusions. Using the predefined definition of pertussis infection, noninferiority for preventing pertussis following exposure was not demonstrated for daily symptom monitoring of Tdap-vaccinated HCP without PEP when compared with antibiotic PEP. However, the small number of exposed HCP warrants further study of this approach. Clinical Trial Registration. NCT00469274. © 2011 The Author.

Keitel W.A.,Baylor College of Medicine | Jackson L.A.,Group Health Research Institute | Edupuganti S.,Hope Clinic of the Emory Vaccine Center | Edupuganti S.,Emory University | And 15 more authors.
Journal of Infectious Diseases | Year: 2015

Background. Variant influenza A(H3N2) viruses (H3N2v) have transmitted recently from pigs to humans in the United States. Vaccines strategies are needed. Methods. Healthy adults received 2 doses of subvirion H3N2v vaccine (15 μg of hemagglutinin/dose) 21 days apart in this open-label trial. Serum hemagglutination inhibition (HAI) and neutralizing (Neut) antibody (Ab) titers were measured before and 8 and 21 days after each dose. Memory B-cell (MBC) responses were assessed. Results. Vaccine was well tolerated. A total of 40% of subjects had an HAI Ab titer of ≥40 before vaccination. Eight-seven percent (95% confidence interval [CI], 79%-93%) and 73% (95% CI, 63%-81%) of subjects 18-64 years old (98 subjects) and ≥65 years old (90 subjects), respectively, had an HAI titer of ≥40 21 days after dose 1 (P =. 01); 51% (95% CI, 41%-61%) and 52% (95% CI, 41%-62%) of younger and older subjects, respectively, developed ≥4-fold rises in titer (P = not significant). Neut Ab response patterns were similar. Geometric mean titers were higher in younger subjects. Dose 2 provided no significant enhancement in responses. Cross-reactive MBCs were detected before vaccination and expanded after vaccination. Preexisting H3N2v-specific MBCs positively correlated with early increases in vaccine-induced Ab. Conclusions. In most healthy adults, one 15-μg dose of vaccine elicited levels of HAI Abs associated with protection. Studies in children and elderly individuals are indicated to define the immunization needs of these groups. © 2015 The Author.

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