Laboratory Diagnosis of Toscana Virus Infection by Enzyme Immunoassay with Recombinant Viral Nucleoprotein

A recombinant enzyme immunoassay (rEIA) to detect serum immunoglobulin M (IgM) and IgG to Toscana virus (TOSV) was developed with the aim of establishing a simple and easily available assay for diagnosing acute and/or previous infections. The rEIA, based on the recombinant nucleoprotein of TOSV expressed in Escherichia coli, was evaluated with 97 serum samples collected in an area where TOSV is endemic and compared to an analogous assay based on cell-derived TOSV. Discordant results were resolved by immunoblotting (IB). Twenty-two of these samples, obtained from subjects hospitalized during the summer season with meningitis of suspected TOSV etiology, were further characterized by indirect immunofluorescence and IB, and detection of specific TOSV RNA sequences in the cerebrospinal fluid of these patients was attempted by nested PCR. The results indicated that rEIA was able to diagnose acute TOSV infection by detection of specific serum IgM in all of the subjects with TOSV meningitis confirmed by nested PCR or serology. The overall sensitivity and specificity of rEIA were both 100% for IgM detection and 100 and 96.6%, respectively, for IgG detection. Thus, rEIA appears to be a simple and reliable laboratory test for the diagnosis of acute TOSV infection and for the assessment of immune status.     

Th17 Cells Facilitate the Humoral Immune Response in Patients with Acute Viral Myocarditis

Background  

Recently, the Th17 cell, a newly determined CD4+Th subset, was reported to participate in the inflammation of myocarditis combined with Th1 cells, and this study aimed to explore whether it was involved in the Th2 cell-mediated humoral immunity in viral myocarditis.     

Diagnostic Potential of Toscana Virus N Protein Expressed in Escherichia coli

The nucleocapsid (N) protein of the Toscana (TOS) virus was expressed in Escherichia coli by using a pET15b vector. The recombinant protein was purified by affinity chromatography and was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and enzyme immunoassay (EIA). The recombinant antigen was reactive with positive human sera, and the reactivity correlated very well (r = 0.9) with that of a whole-virus antigen when tested by EIA with 30 TOS virus-positive and 30 TOS virus-negative serum samples. The results demonstrate that the recombinant N protein can be easily produced in a procaryotic system and used for diagnostic assays for TOS virus immunity.     

Toscana Virus Encephalitis in a Traveler Returning to the United States

In Italy, Toscana virus is the most common cause of meningitis from May to October. Though only a few cases have been reported in U.S. travelers returning from Europe, most cases are likely unrecognized due to lack of familiarity with the disease. Here, we describe the case of an 82-year-old man presenting with fever, profound weakness, and hearing loss after returning to the United States following a 2-week summertime vacation in southern Italy who was ultimately diagnosed with Toscana virus encephalitis. This case should alert clinicians to the possibility of Toscana virus infection in returning travelers and provides information on how to obtain testing if Toscana virus is suspected.     

Vaccinia Virus Pathogenicity in Atopic Dermatitis is Caused by Allergen-Induced Immune Response that Prevents the Antiviral Cellular and Humoral Immunity

Atopic dermatitis (AD) serves as a contraindication for the immunization of AD patients with a live vaccinia virus (VV) vaccine. The antiallergen IgE interacts with the Fc receptors (FcRI) on dendritic cell (DC) membranes and with allergen molecules. The immunological events that lead to AD disease, the activation of the T-helper 2 (Th2) immune response, the synthesis of the cytokines IL-4, IL-5, IL-13, and the inhibition of the T-helper 1 (Th1) damage the capacity of the host to develop anti-VV cytotoxic cells (CTLs). In the presence of Th2-derived cytokine IL-4 in the AD skin and the synthesis of VV proteins that interfere recruitment of DCs by host cytokines, the VV can cause a generalized infection.Conceptually, new VV recombinants may be needed for human immunization. Such VV recombinants should lack the genes that interfere with the host immune system and express a mutated human IL-4 cytokine gene that will prevent negative regulatory mechanisms. Such improved VV recombinants may be used to express genes from pathogenic viruses.     

Serological Survey of Toscana Virus Infections in a High-Risk Population in Italy

Toscana virus is the most important agent responsible for meningitis in central Italy. We report a serosurveillance study, using an immunoenzymatic assay, of 360 serum samples harvested from a high-risk population occupationally exposed to Toscana virus in two regions of Italy, Tuscany and Piedmont. The results indicates a seroprevalence of Toscana virus of 77.2% in the forestry workers, particularly in the Tuscany region. This fact is strictly correlated with the ecological niches specific for the survival of Toscana virus arthropod vector.     

Serological Cross-Reactivities between Antibodies to Simian Virus 40, BK Virus, and JC Virus Assessed by Virus-Like-Particle-Based Enzyme Immunoassays

Enzyme immunoassays (EIAs) for detection of serum antibodies to simian virus 40 (SV40), BK virus (BKV), and JC virus (JCV) were developed by using virus-like-particles (VLPs) produced in insect cells from recombinant baculoviruses expressing the VP1 protein of the respective virus. Rhesus macaque sera with neutralizing antibodies to SV40 showed a high level of reactivity in the SV40 VLP-based EIA, and these sera also showed lower levels of reactivity in the BKV and JCV VLP-based EIAs. Rhesus macaque sera negative for neutralizing antibodies to SV40 were negative in all three EIAs. Competitive binding assays showed that SV40 VLPs inhibited BKV reactivity. In rhesus macaque sera, high optical density (OD) values for antibodies to SV40 VLPs were correlated with high OD values for antibodies to BKV but not with high OD values for antibodies to JCV VLPs. Human sera with neutralizing antibodies to SV40 were more reactive to SV40 VLPs than human sera without neutralizing antibodies to SV40. The greater SV40 reactivities of human sera were correlated with greater reactivities to BKV VLPs but not JCV VLPs. These data suggest that cross-reactivity with BKV antibodies may account for part of the low-level SV40 reactivity seen in human sera. With their greater versatility and their suitability for large-scale testing, the VLP-based EIAs for SV40, BKV, and JCV are likely to contribute to a better understanding of the biology of these viruses.     

Profiling the Native Specific Human Humoral Immune Response to Sudan Ebola Virus Strain Gulu by Chemiluminescence Enzyme-Linked Immunosorbent Assay

Ebolavirus, a member of the family Filoviridae, causes high lethality in humans and nonhuman primates. Research focused on protection and therapy for Ebola virus infection has investigated the potential role of antibodies. Recent evidence suggests that antibodies can be effective in protection from lethal challenge with Ebola virus in nonhuman primates. However, despite these encouraging results, studies have not yet determined the optimal antibodies and composition of an antibody cocktail, if required, which might serve as a highly effective and efficient prophylactic. To better understand optimal antibodies and their targets, which might be important for protection from Ebola virus infection, we sought to determine the profile of viral protein-specific antibodies generated during a natural cycle of infection in humans. To this end, we characterized the profile of antibodies against individual viral proteins of Sudan Ebola virus (Gulu) in human survivors and nonsurvivors of the outbreak in Gulu, Uganda, in 2000-2001. We developed a unique chemiluminescence enzyme-linked immunosorbent assay (ELISA) for this purpose based on the full-length recombinant viral proteins NP, VP30, and VP40 and two recombinant forms of the viral glycoprotein (GP_1-294 and GP_1-649) of Sudan Ebola virus (Gulu). Screening results revealed that the greatest immunoreactivity was directed to the viral proteins NP and GP_1-649, followed by VP40. Comparison of positive immunoreactivity between the viral proteins NP, GP_1-649, and VP40 demonstrated a high correlation of immunoreactivity between these viral proteins, which is also linked with survival. Overall, our studies of the profile of immunorecognition of antibodies against four viral proteins of Sudan Ebola virus in human survivors may facilitate development of effective monoclonal antibody cocktails in the future.     

Mechanism of Enhanced Humoral Response in Rodents by a Synthetic Polymer

Abstract

We previously reported that administration of a low molecular weight (MW=800) synthetic polymer, NED 137, significantly increases humoral and cellular immune responses in the rat. The effect of NED 137 on the murine humoral response to T-dependent (TD) and T-independent (TI) antigens was studied in C57 BL/6, CBA/J and Balb/c mice. The TD antigens (SRBC, DNP-DA with adjuvant) or TI antigen (DNP-Ficoll) were administered simultaneously with NED 137. The polymer significantly increased the direct PFC response to all antigens tested in normal mice. However, it could not restore the PFC response to SRBC in athymic (nu/nu) mice. The effect of NED 137 on accessory cells was studied by the assessment of the in vitro response to SRBC in normal and macrophage-depleted rat spleen cultures. The polymer stimulated both, the primary and secondary IgM response and its immunopotentiating activity was the greatest in macrophage-depleted spleen cell preparations. The lack of effect of NED 137 in systems devoid of functional T cells, dependency on and specificity for a sensitizing antigen and its ability to stimulate a secondary response suggest that this polymer does not act as a “conventional” B-cell polyclonal activator.     

Mechanism of Enhanced Humoral Response in Rodents by a Synthetic Polymer

We previously reported that administration of a low molecular weight (MW=800) synthetic polymer, NED 137, significantly increases humoral and cellular immune responses in the rat. The effect of NED 137 on the murine humoral response to T-dependent (TD) and T-independent (TI) antigens was studied in C57 BL/6, CBA/J and Balb/c mice. The TD antigens (SRBC, DNP-DA with adjuvant) or TI antigen (DNP-Ficoll) were administered simultaneously with NED 137. The polymer significantly increased the direct PFC response to all antigens tested in normal mice. However, it could not restore the PFC response to SRBC in athymic (nu/nu) mice. The effect of NED 137 on accessory cells was studied by the assessment of the in vitro response to SRBC in normal and macrophage-depleted rat spleen cultures. The polymer stimulated both, the primary and secondary IgM response and its immunopotentiating activity was the greatest in macrophage-depleted spleen cell preparations. The lack of effect of NED 137 in systems devoid of functional T cells, dependency on and specificity for a sensitizing antigen and its ability to stimulate a secondary response suggest that this polymer does not act as a “conventional” B-cell polyclonal activator.     

Outcomes and Predictors of Response in Steroid-Refractory Acute Graft-versus-Host Disease

The prognosis of steroid-refractory acute graft-versus-host disease (aGVHD) is poor, and predictors of response and survival are unclear. In an exploratory analysis of 203 steroid-refractory aGVHD patients with prospectively collected GVHD data who received antithymocyte globulin, etanercept, or mycophenolate mofetil (MMF) as second-line treatment, we determined the predictors of day 28 response, 2-year overall survival, and 2-year nonrelapse mortality (NRM). To minimize the risk of finding false-positive results, we used least absolute shrinkage and selection operator regression, aggressively eliminating variables that are unlikely to be associated with outcome. Day 28 response to second-line therapy was 38% (complete response, 23%), with a 2-year overall survival of 25% and a 2-year NRM of 62%. Factors associated with response were GVHD prophylaxis, organ involvement, and initial aGVHD to steroid-refractory aGVHD interval. Specifically, compared with cyclosporine/MMF as GVHD prophylaxis, the odds ratio (OR) for calcineurin inhibitor/methotrexate was .8 and for cyclosporine/prednisone .6. The OR for aGVHD to steroid-refractory aGVHD interval ≥ 14 versus <14 days was 1.3. The ORs for skin only involvement and gut or liver only involvement when compared with multiorgan involvement were 1.4 and 1.2, respectively. The only variable associated with worse survival was age, with a hazard ratio (HR) per decade of 1.04 for overall mortality. Similarly, age was the only variable associated with NRM (HR per decade, 1.02). When compared with complete response, no response at day 28 increased the risk of death (HR, 2.4; 95% confidence interval, 1.5 to 3.7). In conclusion, by means of an underused statistical technique in the field of transplantation, we identified predictors of response and survival in steroid-refractory aGVHD. Our results highlight the importance of developing novel treatment strategies because current treatments yield poor outcomes.     

Induction of Cross-reactive Humoral Immune Response by Immunization with Mimotopes of the Hypervariable Region 1 of the Hepatitis C Virus

Hepatitis C Virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, worldwide, and the development of an effective vaccine represents a high priority goal. The Hyper Variable Region 1 (HVRI) of the second Envelope protein (E2) of HCV contains a principal neutralizing determinant, but it is highly variable among different isolates and it is involved in the escape from host immune response. Thus, to be effective, a vaccine should elicit a cross-reacting humoral response against the majority of viral variants. We show that it is possible to achieve a broadly cross-reactive immune response in rabbits by immunization with mimotopes of the HVRI, selected from a specialized phage library using HCV patients' sera. At least some of the cross-reacting anti-mimotope antibodies, elicited in rabbits, recognize discontinuous epitopes in a manner similar to those induced by the virus in infected patients.     

Modulation of the Humoral Immune Response by Placental Secretory Factors

PROBLEM: To investigate how the factors secreted by human placenta modify the quality and the quantity of the antibody produced by the hybridoma as well as its cellular proliferation. METHOD: Supernatants of cultures of human placenta (PS) were added to a mouse IgG1 hybridoma culture producing anti-DNP antibodies. The quantity of monoclonal antibody produced, the nature of these antibodies and the proliferation of the hybridoma cells were studied. RESULT: It was found that PS augmented by 40–50% the quantity of total antibody produced, increased the proportion of asymmetric (blocking) antibodies from 15% to 30%, and diminished the cellular proliferation, as measured by ³H-thymidine incorporation. CONCLUSION: These results, together with other similar observations already described in human, rat and mouse pregnancies, suggest that secretory factors produced by the placenta do modify the immune response of the mother against paternal antigens and participate in the mechanisms that make possible the survival of the allogenic fetus.     

Humoral Inhibition of Neutrophil Chemotaxis in Crohn''s Disease

In patients with Crohn's disease (CD) we investigated the C3 conversion of zymosan-activated serum (ZAS) and looked for the occurrence of chemotactic factor inactivation (CFI). We also studied the cell-directed inhibitory effect (CDI) of the CD patients' plasma and, in the same group, complement activation and complement-mediated deactivation. The mean value of ZAS C3 conversion in CD was no different from that of healthy controls, but in steroid-treated patients it was lower than in untreated CD. CFI occurred in 1 of the 23 CD sera tested, and CDI was observed in 6 out of the 22 patients tested. EDTA C3 conversion was present in 12 patients, and complement-mediated deactivation was associated with high values of EDTA C3 conversion. Our findings indicate that complement dysfunction and inhibitory factors of neutrophil chemotaxis are present in CD. These findings could explain the defective neutrophil migration into skin windows. Whether they are relevant to the pathogenesis of tissue injury or of infectious complications and are specific for CD, however, remains to be established.     

Natural Killer Cell Dysfunction during Acute Infection with Foot-and-Mouth Disease Virus

Natural killer (NK) cells provide one of the initial barriers of cellular host defense against pathogens, in particular intracellular pathogens. The role of these cells in foot-and-mouth disease virus (FMDV) infection is unknown. Previously, we characterized the phenotype and function of NK cells from swine (F. N. Toka et al., J. Interferon Cytokine Res. 29:179-192, 2009). In the present study, we report the analysis of NK cells isolated from animals infected with FMDV and tested ex vivo and show that NK-dependent cytotoxic activity against tumor cells as targets was impaired. More relevantly to this infection, the killing of target cells infected with FMDV also was inhibited. Further, the proportion of NK cells capable of producing gamma interferon and storing perforin was reduced. Peripheral blood mononuclear cells isolated from infected animals are not productively infected, but virus exposure in vivo resulted in the significant induction of NKp30 and Toll-like receptor 3 expression and the moderate activation of SOCS3 and interleukin-15 receptor mRNA. However, there was little alteration of mRNA expression from a number of other receptor genes in these cells, including SH2D1B and NKG2A (inhibitory) as well as NKp80, NKp46, and NKG2D (activating). These data indicate that this virus infection influences the ability of NK cells to recognize and eliminate FMDV-infected cells. In addition, a reduction in NK cell cytotoxicity coincided with the increase in virus titers, indicating the virus blocking of NK cell-associated innate responses, albeit temporarily. These effects likely culminate in brief but effective viral immune evasion, allowing the virus to replicate and disseminate within the host.Innate immunity is a vital part of the overall host immune response to invasion by pathogens, particularly during virus infections. Natural killer (NK) cells occupy a critical position in the initial host responses against infection. Originally, NK cells were discovered on the basis of their capability to kill certain tumors without prior activation. Now the role of NK cells has been defined in virus infections such as human cytomegalovirus (8, 11, 55), murine cytomegalovirus virus (2, 32), influenza virus (28, 35), herpes simplex virus (44, 52), ectromelia virus (16, 41), and human immunodeficiency virus (HIV) (14, 50, 56). In two of these infections, a lack or deficiency in NK cell function leads to increased susceptibility to infection (6, 10).The initiation of NK cell responses is thought to originate from signals delivered by the professional pathogen-sensing system, which is comprised mainly of dendritic cells (DC) (21, 47, 57). Although the evidence is not yet definitive, the direct activation of NK cells also may occur through pathogen recognition receptors expressed by NK cells (49, 51). The cross-talk between NK cells and DC leads to the activation of NK cells, after which they operate in a manner that is dependent on the sensing expression of specific molecules induced on virus-infected cells through receptors present on the NK cell surface. In part, the recognition of an infected cell by NK cells relies on the detection of the missing self, i.e., the lack of major histocompatibility complex class I expression on the infected cell surface. Ultimately, the balance between signals from both inhibiting and activation receptors (34) on NK cells control NK cell function in response to infection. Subsequently, NK cells engage in cytokine secretion and, upon the encounter of a virus-infected cell, release cytotoxic granule contents or induce apoptosis. These mechanisms lead to the elimination of virus-infected cells. Whereas the discovery of activating or inhibitory receptors on NK cells has progressed tremendously, the identification of respective ligands on infected or transformed cells has been difficult (reviewed in reference 12).Although much is known about the function of NK cells in humans and mice, NK cell activity in swine or cattle remains preliminary, and their role in animal viral diseases still is obscure. The recent progress in these animal species has been reviewed by Boysen and Storset (9) and Gerner et al. (20). In pigs, NK cells may account for a total of 5 to 10% of circulating lymphocytes and currently are identified as belonging to a subset of cells that coexpress CD2 and CD8 molecules (17). Although mRNAs of many activating and inhibitory receptors have been detected, no studies have been conducted to define their role in the generic function of porcine NK cells. But it is known that porcine NK cells can secret gamma interferon (IFN-γ), store perforin, and kill in vitro targets (54). Their function can be modulated by direct stimulation with cytokines such as interleukin-2 (IL-2), IL-12, IL-15, IL-18 (42, 54), or IFN-α, or Toll-like receptor (TLR) agonists such as polyinosinic:poly(C) (pI:C), CpG, imiquimod, and resiquimod in humans (23).In this study, we examine NK cell responses during infection with foot-and-mouth disease virus (FMDV). FMDV is a contagious disease of cloven-hoofed animals caused by a picornavirus (25). Infection with FMDV presents as an acute disease characterized by fever, short-lived viremia, and the occurrence of lesions on feet and tongue (reviewed in reference 25). The control of FMDV in certain regions of the world depends on the use of inactivated vaccines. The use of these as emergency vaccines, however, is compromised by the time from vaccination to protection (7 days in cattle) (22) and the difficulty in distinguishing infected animals from vaccinated animals. In FMDV-free countries vaccination is not practiced, leading to outbreak responses relying mostly on the elimination of all susceptible animals within the areas of outbreaks (15).The immune response to FMDV infection in pigs has not been fully dissected and remains an area of speculation. Whereas B-cell responses are activated early and neutralizing antibody titers correlate with protection (36), there is no clear picture regarding the induction of functional T-cell immunity in infected animals. Bautista et al. (4) found the inhibition of T-cell responses to mitogens during the infection of pigs with FMDV. Some groups have reported the induction of antigen-specific T cells reactive with nonstructural proteins of the virus (7). More recently, antigen-specific, major histocompatibility complex-restricted CD8⁺ T-cell responses were detected following infection with FMDV (26).The importance of innate immunity in FMDV infection is perhaps the most overlooked systemic response to FMDV in susceptible species and remains largely undefined. To date, there is no comprehensive study addressing the role of NK cells during infection with FMDV in either swine or cattle. A more sophisticated understanding of the innate response mechanisms involved in infection with FMDV is pertinent to the design of potent emergency vaccines that can protect against infection.In these studies, we characterized the response of NK cells derived from swine peripheral blood following infection with the O1 Campos strain of FMDV. Results demonstrate that these cells are compromised in the killing of either tumor cells or FMDV-infected target cells ex vivo. Moreover, this dysfunction includes a reduction in the proportion of IFN-γ-producing and perforin-storing cells and an alteration in the expression pattern of activating and inhibitory receptor genes. These data suggest a profound effect of FMDV infection on porcine NK cell function. Taken together, this abnormal functional status likely contributes to the acute nature of FMDV infection in this species and toward making it one of the most contagious viral infections of swine.