William T. Golde

A rapid, simple, and humane method for submandibular bleeding of mice using a lancet

Methods for obtaining blood samples from mice tend to be difficult, inhumane, or both. The authors describe an inexpensive, disposable, single-use lancet for submandibular bleeding of mice that allows investigators to quickly draw 0.2–0.5 ml of blood without the use of anesthesia.

Induction of lymphopenia and inhibition of T cell function during acute infection of swine with foot and mouth disease virus (FMDV)

Foot and mouth disease virus (FMDV) is a picornavirus that causes an acute vesicular disease of cloven-hoofed animals. This virus continues to be a threat to livestock worldwide with outbreaks causing severe economic losses. The present study shows an analysis of immune system phenotype and function during the acute phase of FMDV infection in swine. In the first days of infection, a significant lymphopenia is observed that involves all T cell subsets, CD4(+), CD8(+), and CD4(+)/CD8(+). This marked lymphopenia is not a result of active infection of PBMC with the virus. Further, the response of residual peripheral blood T cells to the mitogen, Concanavalin A (ConA) is significantly reduced and occasionally eliminated. Animals usually resolve clinical signs of disease and develop antigen specific T cell responses to the virus and recover ConA reactivity. These characteristics of acute phase infection likely play an important role in viral pathogenesis, propagation and shedding of viral particles and may be targeted as a way of improving vaccine formulations.

A synthetic peptide containing the consensus sequence of the G-H loop region of foot-and-mouth disease virus type-O VP1 and a promiscuous T-helper epitope induces peptide-specific antibodies but fails to protect cattle against viral challenge

A pilot study was carried out in cattle to determine the immunogenicity of a synthetic consensus peptide comprising the G-H loop region of foot-and-mouth disease virus (FMDV) type-O VP1 and a non-VP1 T-helper (Th) epitope. Cattle vaccinated intramuscularly either once (n = 5) or twice (n = 4) with 50 microg of the peptide preparation at a 21-day interval developed antibodies to the peptide as determined by ELISA with the exception of one steer that received a single dose. However, neutralizing antibody titers against FMDV type-O were modest and all animals presented with clinical FMD signs upon challenge 21 days after the last vaccination. In contrast, four of the five animals inoculated with an inactivated FMD type-O commercially prepared vaccine developed neutralizing antibodies and were fully protected against clinical disease following virus challenge 21 days post-vaccination (dpv). Nucleotide sequence comparison of the VP1 region between the challenge virus and RT-PCR products recovered from a lesion of the peptide-vaccinated animal with the highest neutralizing antibody titer 5 days post-challenge (dpc) showed no evidence for selection of a neutralization-resistant mutant. We conclude that although the synthetic peptide induced an antibody response in cattle, it failed to confer protection against FMDV challenge.

Constitutive Expression of Alpha Interferon by Skin Dendritic Cells Confers Resistance to Infection by Foot-and-Mouth Disease Virus

ABSTRACT The role of dendritic cells (DC) in the initiation of immune responses against foot-and-mouth disease virus (FMDV) is poorly understood. We analyzed the innate response of freshly isolated swine skin DC to the virus and show a rapid induction of beta interferon (IFN-β) mRNA but not IFN-α mRNA. However, these DC secreted both IFN-α and IFN-β proteins in response to live virus but not killed virus. Furthermore, the surface expression of swine major histocompatibility complex class II (SLA II) or CD80/CD86 molecules and antigen processing functions were not affected by FMDV exposure. Given the demonstrated sensitivity of FMDV to IFN-α/β, there was no productive or nonproductive infection of these cells. Finally, freshly isolated skin DC constitutively expressed intracellular IFN-α protein in the absence of stimulation, with no detectable secretion of the cytokine until virus exposure. In situ analysis of these DC showed that these cells express and store IFN-α in uninfected animals. This is the first demonstration of the constitutive expression of IFN-α in resident, tissue-derived DC and indicates that skin DC can play an important role in the innate immune response of swine to viral infections.

Induction of Foot-and-Mouth Disease Virus-Specific Cytotoxic T Cell Killing by Vaccination

ABSTRACT Foot-and-mouth disease (FMD) continues to be a significant threat to the health and economic value of livestock species. This acute infection is caused by the highly contagious FMD virus (FMDV), which infects cloven-hoofed animals, including large and small ruminants and swine. Current vaccine strategies are all directed toward the induction of neutralizing antibody responses. However, the role of cytotoxic T lymphocytes (CTLs) has not received a great deal of attention, in part because of the technical difficulties associated with establishing a reliable assay of cell killing for this highly cytopathic virus. Here, we have used recombinant human adenovirus vectors as a means of delivering FMDV antigens in a T cell-directed vaccine in pigs. We tested the hypothesis that impaired processing of the FMDV capsid would enhance cytolytic activity, presumably by targeting all proteins for degradation and effectively increasing the class I major histocompatibility complex (MHC)/FMDV peptide concentration for stimulation of a CTL response. We compared such a T cell-targeting vaccine with the parental vaccine, previously shown to effectively induce a neutralizing antibody response. Our results show induction of FMDV-specific CD8+ CTL killing of MHC-matched target cells in an antigen-specific manner. Further, we confirm these results by MHC tetramer staining. This work presents the first demonstration of FMDV-specific CTL killing and confirmation by MHC tetramer staining in response to vaccination against FMDV.

Characterization and functional analysis of skin-derived dendritic cells from swine without a requirement for in vitro propagation.

A low-density cell population was isolated from skin explants of pigs and characterized as a highly enriched dendritic cell (DC) population based on phenotypical and functional properties. The skin-derived DCs were identified by their characteristic ultrastructural properties as well as by consistent co-expression of the CD1 and SWC3a antigens that clearly differentiate them from other porcine leukocytes. These cells exhibit higher expression of porcine MHC class II (SLAII) and CD80/86 antigens as compared to macrophage/monocyte cells. They consistently expressed the S100 beta antigen at high levels and did not express the lymphoid markers CD3, CD4 or CD8. Within this population of skin-derived DCs there was variable expression of CD11c, CD14 and CD16. Functional characterization of this DC population revealed that they are efficient in uptake and processing of soluble protein antigens and in endocytosis of small (0.02 microm) but not large (2 microm) polystyrene beads. Further, these cells were efficient inducers of primary allogeneic responses and in stimulating antigen-specific and mitogen-induced proliferation and IFN gamma responses in autologous lymphocytes. This study provides important information to further characterize the cutaneous DCs and develop models to analyze the role of these cells in immune responses in vivo.

Interferon-α Production by Swine Dendritic Cells Is Inhibited During Acute Infection with Foot-and-Mouth Disease Virus

Viruses have evolved multiple mechanisms to evade the innate immune response, particularly the actions of interferons (IFNs). We have previously reported that exposure of dendritic cells (DCs) to foot-and-mouth disease virus (FMDV) in vitro yields no infection and induces a strong type I IFN (IFN-alpha and IFN-beta) response, indicating that DCs may play a critical role in the innate response to the virus. In vivo, FMDV induces lymphopenia and reduced T-cell proliferative responses to mitogen, viral effects that may contribute to evasion of early immune responses. In this study we analyzed the in vivo effects of FMDV infection on the IFN-alpha response of two populations of dendritic cells. During the acute phase of infection of swine, production of IFN-alpha from monocyte-derived DCs (MoDCs) and skin-derived DCs (skin DCs) is inhibited. This effect occurs concurrently with rising viral titers in the blood; however, these cells are not productively infected. Interestingly, there are no changes in the capability of these DCs to take up particles and process antigens, indicating that antigen-presenting cell function is normal. These data indicate that inhibition of the IFN-alpha response of dendritic cell populations from blood and skin by FMDV enhances viral pathogenesis in infected animals.

Rapid and Transient Activation of γδ T Cells to IFN-γ Production, NK Cell-Like Killing, and Antigen Processing during Acute Virus Infection

γδ T cells are the majority peripheral blood T cells in young cattle. The role of γδ T cells in innate responses against infection with foot-and-mouth disease virus was analyzed on consecutive 5 d following infection. Before infection, bovine WC1+ γδ T cells expressed a nonactivated phenotype relative to CD62L, CD45RO, and CD25 expression and did not produce IFN-γ ex vivo. Additionally, CD335 expression was lacking and no spontaneous target cell lysis could be detected in vitro, although perforin was detectable at a very low level. MHC class II and CD13 expression were also lacking. Following infection with foot-and-mouth disease virus, expression of CD62L and CD45RO was greatly reduced on WC1+ γδ T cells, and unexpectedly, CD45RO expression did not recover. A transient increase in expression of CD25 correlated with production of IFN-γ. Expression of CD335 and production of perforin were detected on a subset of γδ T cells, and this correlated with an increased spontaneous killing of xenogeneic target cells. Furthermore, increased MHC class II expression was detected on WC1+ γδ T cells, and these cells processed protein Ags. These activities are rapidly induced, within 3 d, and wane by 5 d following infection. All of these functions, NK-like killing, Ag processing, and IFN-γ production, have been demonstrated for these cells in various species. However, these results are unique in that all these functions are detected in the same samples of WC1+ γδ T cells, suggesting a pivotal role of these cells in controlling virus infection.

Loss of plasmacytoid dendritic cell function coincides with lymphopenia and viremia during foot-and-mouth disease virus infection.

Foot-and-mouth disease virus (FMDV) causes an acute, highly contagious disease of livestock. Though FMDV is very sensitive to interferon-alpha (IFN-alpha), IFN-beta, and IFN-gamma, the virus has evolved mechanisms to evade such innate responses. For instance, during acute infection, FMDV suppresses IFN-alpha production by skin and myeloid dendritic cells (DCs). We have previously reported that FMDV infection induces a transient lymphopenia and interruption of T-lymphocyte responses to mitogenic stimuli. To further understand the immunopathogenesis of FMD, we have now analyzed the serum IFN-alpha response in relation to lymphopenia, and the number and function of plasmacytoid DCs (pDCs) following infection of pigs with multiple serotypes of FMDV. Serum IFN-alpha peaked 2-3 d post-infection (PI), regardless of FMDV serotype. Lymphopenia coincided with peak viremia and the serum IFN-alpha response. Circulating pDC numbers and in-vitro pDC IFN-alpha secretion transiently declined by 48 h following infection. Infection of lymphocytes or pDCs was never detected regardless of the FMDV serotype inoculated or the age of the animal infected. These data indicate that, like other DC subsets, there is suppression of interferon production by pDCs, which abrogates this important innate response. Rapid induction of serum IFN-alpha, albeit short-lived, may contribute to the rapid resolution of FMDV viremia prior to induction of specific immunity.

Porcine major histocompatibility complex (MHC) class I molecules and analysis of their peptide-binding specificities

In all vertebrate animals, CD8+ cytotoxic T lymphocytes (CTLs) are controlled by major histocompatibility complex class I (MHC-I) molecules. These are highly polymorphic peptide receptors selecting and presenting endogenously derived epitopes to circulating CTLs. The polymorphism of the MHC effectively individualizes the immune response of each member of the species. We have recently developed efficient methods to generate recombinant human MHC-I (also known as human leukocyte antigen class I, HLA-I) molecules, accompanying peptide-binding assays and predictors, and HLA tetramers for specific CTL staining and manipulation. This has enabled a complete mapping of all HLA-I specificities (“the Human MHC Project”). Here, we demonstrate that these approaches can be applied to other species. We systematically transferred domains of the frequently expressed swine MHC-I molecule, SLA-1*0401, onto a HLA-I molecule (HLA-A*11:01), thereby generating recombinant human/swine chimeric MHC-I molecules as well as the intact SLA-1*0401 molecule. Biochemical peptide-binding assays and positional scanning combinatorial peptide libraries were used to analyze the peptide-binding motifs of these molecules. A pan-specific predictor of peptide–MHC-I binding, NetMHCpan, which was originally developed to cover the binding specificities of all known HLA-I molecules, was successfully used to predict the specificities of the SLA-1*0401 molecule as well as the porcine/human chimeric MHC-I molecules. These data indicate that it is possible to extend the biochemical and bioinformatics tools of the Human MHC Project to other vertebrate species.