Michael Bailey

A DNA Vaccine for Ebola Virus Is Safe and Immunogenic in a Phase I Clinical Trial

ABSTRACT Ebola viruses represent a class of filoviruses that causes severe hemorrhagic fever with high mortality. Recognized first in 1976 in the Democratic Republic of Congo, outbreaks continue to occur in equatorial Africa. A safe and effective Ebola virus vaccine is needed because of its continued emergence and its potential for use for biodefense. We report the safety and immunogenicity of an Ebola virus vaccine in its first phase I human study. A three-plasmid DNA vaccine encoding the envelope glycoproteins (GP) from the Zaire and Sudan/Gulu species as well as the nucleoprotein was evaluated in a randomized, placebo-controlled, double-blinded, dose escalation study. Healthy adults, ages 18 to 44 years, were randomized to receive three injections of vaccine at 2 mg (n = 5), 4 mg (n = 8), or 8 mg (n = 8) or placebo (n = 6). Immunogenicity was assessed by enzyme-linked immunosorbent assay (ELISA), immunoprecipitation-Western blotting, intracellular cytokine staining (ICS), and enzyme-linked immunospot assay. The vaccine was well-tolerated, with no significant adverse events or coagulation abnormalities. Specific antibody responses to at least one of the three antigens encoded by the vaccine as assessed by ELISA and CD4+ T-cell GP-specific responses as assessed by ICS were detected in 20/20 vaccinees. CD8+ T-cell GP-specific responses were detected by ICS assay in 6/20 vaccinees. This Ebola virus DNA vaccine was safe and immunogenic in humans. Further assessment of the DNA platform alone and in combination with replication-defective adenoviral vector vaccines, in concert with challenge and immune data from nonhuman primates, will facilitate evaluation and potential licensure of an Ebola virus vaccine under the Animal Rule.

A monoclonal antibody recognising an epitope associated with pig interleukin-2 receptors.

A monoclonal antibody is described which recognises an epitope associated with a receptor for interleukin-2 (IL-2) on pig lymphocytes. The monoclonal antibody inhibits high affinity binding of radiolabelled recombinant human IL-2 (rhIL-2) by pig lymphoblasts and also non-competitively inhibits both pig-TCGF and rhIL-2 maintained proliferation. By flow cytometry the antigen is apparently not present on freshly isolated blood lymphocytes but is detectable on small cells between 6 and 12 h after activation and on large cells by 24-h. These findings are comparable with those obtained using monoclonal antibodies recognising the 55 kDa alpha chain of the human and mouse IL-2 receptor (p55, TAC) expressed on activated cells in vivo and in vitro. However, the molecular weight of the porcine antigen is between 65 and 70 kDa.

Interleukin-6 is produced during both murine and avian Eimeria infections

The production of interleukin-6 (IL-6) during Eimeria infection was investigated in an attempt to gain a better understanding of the role of this multi-functional cytokine in resistance to this parasite. IL-6 production was measured in both chickens, in which the disease is of economic importance, and the better-characterised murine model system. Systemic and local IL-6 production in mice during E. vermiformis infection was investigated, in the relatively resistant BALB/c strain, and the relatively susceptible C57 BL/6 strain, using a murine IL-6 ELISA and the 7TD1 assay. Enhanced systemic production of IL-6 in serum was seen in infected BALB/c mice when compared to C57 BL/6 mice. This difference was also reflected in the draining lymph node of the site of infection, assessed by testing supernatants from stimulated mesenteric lymph node cells taken from infected mice at different times post-infection. Production of chicken IL-6-like factor activity was investigated using a murine IL-6 7TD1 bioassay. The presence of substantial quantities of IL-6-like factor activity was detected in serum taken from some chickens infected with E. tenella during the course of primary infection and, in a separate experiment, during the first few hours post-infection, a time when the pro-inflammatory capacity of IL-6 would influence the developing immune response. These results suggest that IL-6 is also important in the induction of immune effector responses to Eimeria infections in the chicken.

Direct experimental evidence that early-life farm environment influences regulation of immune responses

To cite this article: Lewis MC, Inman CF, Patel D, Schmidt B, Mulder I, Miller B, Gill BP, Pluske J, Kelly D, Stokes CR, Bailey M. Direct experimental evidence that early‐life farm environment influences regulation of immune responses. Pediatr Allergy Immunol 2012: 23: 265–269.

cDNA cloning of porcine interleukin 2 by polymerase chain reaction

Porcine interleukin 2 (IL-2) cDNA was cloned by polymerase chain reaction (PCR), using primers derived from the corresponding bovine sequence. The resulting porcine DNA sequence encodes a 154 residue IL-2 primary translation product. Comparison of the mature, secreted form of porcine IL-2 with those of other species was carried out in an attempt to identify differences that might contribute to the observed differing species specificities.

The Mucosal Immune Response to Laryngopharyngeal Reflux

RATIONALE Laryngopharyngeal reflux (LPR) affects up to 20% of Western populations. Although individual morbidity is usually moderate, treatment costs are high and there are associations with other diseases, including laryngeal cancer. To date, there have been no studies of the mucosal immune response to this common inflammatory disease. OBJECTIVES To determine the mucosal immune response to LPR. METHODS We performed a prospective immunologic study of laryngeal biopsies from patients with LPR and control subjects (n = 12 and 11, respectively), and of primary laryngeal epithelial cells in vitro. MEASUREMENTS AND MAIN RESULTS Quantitative multiple-color immunofluorescence, using antibodies for lymphocytes (CD4, CD8, CD3, CD79, CD161), granulocytes (CD68, EMBP), monocytic cells (CD68, major histocompatibility complex [MHC] class II), and classical and nonclassical MHC (I, II, beta(2)-microglobulin, CD1d). Univariate and multivariate analysis and colocalization measurements were applied. There was an increase in percentage area of mucosal CD8(+) cells in the epithelium (P < 0.005), whereas other leukocyte and granulocyte antigens were unchanged. Although epithelial MHC class I and II expression was unchanged by reflux, expression of the nonclassical MHC molecule CD1d increased (P < 0.05, luminal layers). In vitro, laryngeal epithelial cells constitutively expressed CD1d. CD1d and MHC I expression were inversely related in all subjects, in a pattern which appears to be unique to the upper airway. Colocalization of natural killer T (NKT) cells with CD1d increased in patients (P < 0.01). CONCLUSIONS These data indicate a role for the CD1d-NKT cell axis in response to LPR in humans. This represents a useful target for novel diagnostics and treatments in this common condition.

Regional and global changes in TCRαβ T cell repertoires in the gut are dependent upon the complexity of the enteric microflora.

The repertoire of gut associated T cells is shaped by exposure to microbes, including the natural enteric microflora. Previous studies compared the repertoire of gut associated T cell populations in germ free (GF) and conventional mammals often focussing on intra-epithelial lymphocyte compartments. Using GF, conventional and monocolonised (gnotobiotic) chickens and chicken TCRbeta-repertoire analysis techniques, we determined the influence of microbial status on global and regional enteric TCRbeta repertoires. The gut of conventionally reared chickens exhibited non-Gaussian distributions of CDR3-lengths with some shared over-represented peaks in neighbouring gut segments. Sequence analysis revealed local clonal over-representation. Germ-free chickens exhibited a polyclonal, non-selected population of T cells in the spleen and in the gut. In contrast, gnotobiotic chickens exhibited a biased repertoire with shared clones evident throughout the gut. These data indicate the dramatic influence of enteric microflora complexity on the profile of TCRbeta repertoire in the gut at local and global levels.

A defined intestinal colonization microbiota for gnotobiotic pigs.

Maximising the ability of piglets to survive exposure to pathogens is essential to reduce early piglet mortality, an important factor in efficient commercial pig production. Mortality rates can be influenced by many factors, including early colonization by microbial commensals. Here we describe the development of an intestinal microbiota, the Bristol microbiota, for use in gnotobiotic pigs and its influence on synthesis of systemic immunoglobulins. Such a microbiota will be of value in studies of the consequences of early microbial colonization on development of the intestinal immune system and subsequent susceptibility to disease. Gnotobiotic pig studies lack a well-established intestinal microbiota. The use of the Altered Schaedler Flora (ASF), a murine intestinal microbiota, to colonize the intestines of Caesarean-derived, gnotobiotic pigs prior to gut closure, resulted in unreliable colonization with most (but not all) strains of the ASF. Subsequently, a novel, simpler porcine microbiota was developed. The novel microbiota reliably colonized the length of the intestinal tract when administered to gnotobiotic piglets. No health problems were observed, and the novel microbiota induced a systemic increase in serum immunoglobulins, in particular IgA and IgM. The Bristol microbiota will be of value for highly controlled, reproducible experiments of the consequences of early microbial colonization on susceptibility to disease in neonatal piglets, and as a biomedical model for the impact of microbial colonization on development of the intestinal mucosa and immune system in neonates.

Ontogeny of Pig Discrete Peyer’s Patches: Distribution and Morphometric Analysis

We investigated the development of lymphoid and non-lymphoid cells in discrete Peyer’s patches (PP) of the pig using immuno-histology and image analysis. In newborn piglets discrete PP were mainly populated by CD2+, CD3+ T cells, and major histocompatibility complex class II+ cells, many of which were of macrophage and dendritic cell lineage. Four days after birth, cells were localized in defined regions: the follicle; the inter-follicular area and the dome region. Compartmentalization within the follicle started about 6 days after birth. The first signs of secondary follicles were seen from about 14 days. The pig discrete PP attained their mature structure at about 3 weeks after birth. Here we show that despite the demonstration at birth of the cell types that support antigen processing and presentation, PP did not fully differentiate morphologically until at least this time when antigen can be handled in an efficient manner.

Effects of infection with transmissible gastroenteritis virus on concomitant immune responses to dietary and injected antigens.

ABSTRACT Normal piglets weaned onto soy- or egg-based diets generated antibody responses to fed protein. Concurrent infection with transmissible gastroenteritis virus (TGEV) did not affect the responses to dietary antigens at weaning, nor did it affect the subsequent development of tolerance. However, TGEV infection did enhance the primary immunoglobulin M (IgM) and IgG1, but not IgG2, antibody responses to injected soy in comparison to those of uninfected animals. Paradoxically, TGEV-infected animals showed an enhanced primary IgG1 antibody response to injected soy at 4 weeks of age, but they subsequently showed a reduced secondary response after an intraperitoneal challenge at 9 weeks of age in comparison to uninfected animals. The results suggest that an enteric virus, either used as a vaccine vector or present as a subclinical infection, may not have significant effects on the development of dietary allergies but may have effects both on the primary response and on the subsequent recall response to systemic antigens to which the animal is exposed concurrently with virus antigens.