Onset and duration of immunity to equine influenza virus resulting from canarypox-vectored (ALVAC) vaccination.

Equine influenza virus remains an important problem in horses despite extensive use of vaccination. Efficacy of equine influenza vaccination depends on the onset and duration of protective immunity, and appropriate strain specificity of the immune response. This study was designed to test the protective immunity resulting from vaccination with the North American commercial ALVAC equine influenza vaccine (RECOMBITEK Influenza, Merial, USA)(1) against challenge with American lineage influenza viruses. In experiment 1, 12 ponies were vaccinated twice, at a 35 day interval, using the ALVAC-influenza vaccine expressing the HA genes of influenza A/eq/Newmarket/2/93 and A/eq/Kentucky/94 (H3N8), and 11 ponies served as unvaccinated controls. Six months after the second vaccination, all ponies were challenged with A/eq/Kentucky/91. In experiment 2, 10 ponies received one dose of the ALVAC-influenza vaccine, 10 ponies served as unvaccinated controls, and all ponies were challenge infected with A/equine/Ohio/03, 14 days after vaccination. Parameters studied included serological responses, and clinical disease and nasal viral shedding following challenge infection. In experiment 1, following the two-dose regimen, vaccinated ponies generated high titered anti-influenza virus IgGa and IgGb antibody responses to vaccination and demonstrated statistically significant clinical and virological protection to challenge infection compared to controls. Infection with A/eq/Kentucky/91 produced unusually severe signs in ponies in the control group, requiring therapy with NSAID's and antibiotics, and leading to the euthanasia of one pony. In experiment 2 following the one-dose regimen, vaccinates generated IgGa responses pre-challenge, and anamnestic IgGa and IgGb responses after challenge. Vaccinates demonstrated statistically significant clinical and virological protection to challenge infection compared to controls. The results of this study clearly demonstrate the early onset, and 6-month duration of protective immunity resulting from ALVAC-influenza vaccination against challenge with American lineage equine influenza viruses.

Detection and quantification of equine herpesvirus-1 viremia and nasal shedding by real-time polymerase chain reaction.

Equine herpesvirus-1 (EHV-1) infection is common in young horses throughout the world, resulting in respiratory disease, epidemic abortion, sporadic myelitis, or latent infections. To improve on conventional diagnostic tests for EHV-1, a real-time polymerase chain reaction (PCR) technique was developed, using primers and probes specific for the EHV-1 gB gene. Amplification efficiencies of 100% +/- 5% were obtained for DNA isolated from a plasmid, infected peripheral blood mononuclear cells (PBMCs), and nasal secretions from infected ponies. The dynamic range of the assay was 8 log10 dilutions, and the lower limit of detection was 6 DNA copies. Fifteen ponies, seronegative for EHV-1, were experimentally infected with EHV-1, and nasal samples were used to quantify shedding of virus by both virus isolation and real-time PCR analysis. Virus isolation identified nasal shedding of EHV-1 in 12/15 ponies on a total of 25 days; real-time PCR detected viral shedding in 15/15 ponies on 75 days. Viremia was quantified using PBMC DNA, subsequent to challenge infection in 3 additional ponies. Viremia was identified in 1/3 ponies on a single day by virus isolation; real-time PCR detected viremia in 3/3 ponies on 17 days. When real-time PCR was used to analyze PBMC DNA from 11 latently infected ponies (documented by nested PCR), EHV-1 was not detected. We conclude that real-time PCR is a sensitive and quantitative test for EHV-1 nasal shedding and viremia and provides a valuable tool for EHV-1 surveillance, diagnosis of clinical disease, and investigation of vaccine efficacy.

Effect of oestradiol on PAMP-mediated CCL20/MIP-3 alpha production by mouse uterine epithelial cells in culture.

The present study was undertaken to establish whether mouse uterine epithelial cells produce CCL20/macrophage inflammatory protein 3 alpha (CCL20/MIP-3 alpha) and to determine whether secretion is under hormonal control and influenced by pathogen-associated molecular patterns (PAMPs). In the absence of PAMPs, polarized uterine epithelial cells grown to confluence on cell culture inserts constitutively secreted CCL20/MIP-3 alpha with preferential accumulation into the apical compartment. When epithelial cells were treated with the Toll-like receptor (TLR) agonists Pam3Cys (TLR2/1), peptidoglycan (TLR2/6) or lipopolysaccharide (LPS; TLR4), CCL20/MIP-3 alpha increased rapidly (4 hr) in both apical and basolateral secretions. Time-course studies indicated that responses to PAMPs added to the apical surface persisted for 12-72 hr. Stimulation with loxoribin (TLR7) and DNA CpG motif (TLR9) increased basolateral but not apical secretion of CCL20/MIP-3 alpha. In contrast, the viral agonist Poly(I:C) (TLR3) had no effect on either apical or basolateral secretion. In other studies, we found that oestradiol added to the culture media decreased the constitutive release of CCL20/MIP-3 alpha. Moreover, when added to the culture media along with LPS, oestradiol inhibited LPS-induced increases in CCL20/MIP-3 alpha secretion into both the apical and basolateral compartments. In summary, these results indicate that CCL20/MIP-3 alpha is produced in response to PAMPs. Since CCL20/MIP-3 alpha is chemotactic for immature dendritic cells, B cells and memory T cells and has antimicrobial properties, these studies suggest that CCL20/MIP-3 alpha production by epithelial cells, an important part of the innate immune defence in the female reproductive tract, is under hormonal control and is responsive to microbial challenge.

Effect of toll-like receptor (TLR) agonists on TLR and microbicide expression in uterine and vaginal tissues of the mouse.

PROBLEM: Epithelial cells lining the uterine lumen are the first line of defense against pathogenic microbes. The objective of this study was to examine the expression of Toll-like receptors (TLRs), defensins and secretory leukocyte protease inhibitor (SLPI) in the mouse uterus and vagina and in primary uterine epithelial cells and to determine whether TLR agonists induce TLR and defensin expression. METHOD OF STUDY: The mRNA expression of alpha- and beta-defensins (AD1, 2 and 5 and BD1, 2 and 4) and SLPI was examined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) along with the secretion of macrophage chemotactic protein-1 (MCP-1), measured by enzyme-linked immunosorbent assay. RESULTS: Expression of TLR1-9 as well as beta-defensins 1, 2 and 4 and SLPI by uterine and vaginal tissues was demonstrated by RT-PCR. beta-Defensins and SLPI expression was greater in the vagina than in the uterus. Comparison of fresh and polarized uterine epithelial cells indicated that TLR2-6 expression was unaffected by culture. Incubation of polarized epithelial cells with TLR agonists [lipopolysaccharide (LPS), Pam3Cys, Poly (I:C) or PGN] induced TLR5 and TLR9 expression but had no effect on TLR4, defensins or SLPI. Furthermore, exposure to LPS, Pam3Cys, Poly (I:C) or PGN, induced MCP-1 secretion by polarized epithelial cells in culture. CONCLUSION: These results indicate that the uterus and vagina as well as uterine epithelial cells are responsive to bacterial and viral pathogens. Not only do epithelial cells respond to TLR agonists by releasing MCP-1, which mediates inflammatory responses, but they also influence the expression of selected TLR genes to further enhance innate immune protection.

Expression of Toll-like receptors (TLR) and responsiveness to TLR agonists by polarized mouse uterine epithelial cells in culture.

The objective of the present study was to examine the expression of Toll-like receptors (TLRs) by mouse uterine epithelial cells and to determine if stimulation of the expressed TLR induces changes in cytokine and/or chemokine secretion. Using RT-PCR, the expression of TLRs 1-6 by mouse uterine epithelial cells was demonstrated, with TLRs 7-9 expressed only periodically. In the absence of pathogen-associated molecular patterns, polarized uterine epithelial cells constitutively secrete interleukin (IL) 1A, cysteine-cysteine ligand (CCL) 2, IL6, granulocyte-macrophage colony-stimulating factor 2 (CSF2), tumor necrosis factor A (TNFA), CSF3, and IL8 in vitro, with levels of cytokines/chemokines secreted into the apical compartment being significantly greater than those released into the basolateral compartment. When added to the apical surface for 48 h before analysis, the TLR2-agonist Pam3Cys-Ser-(Lys)4 and TLR1/6-agonist peptidoglycan increased epithelial cell apical secretion of IL1A, CCL2, and IL6 and apical/basolateral bidirectional secretion of CSF2, TNFA, CSF3, and IL8 when compared to controls. The TLR3-agonist poly (I:C) significantly increased bidirectional secretion of CCL2, IL6, TNFA, and CSF2 and basolateral secretion of CSF3. Lastly, the TLR4-agonist lipopolysaccharide increased bidirectional secretion CCL2, CSF2, TNFA, CSF3, and IL8 and apical secretion of IL6. These results indicate that mRNAs for Tlr1 through Tlr6 are expressed by uterine epithelial cells and that treatment with specific TLR agonists alters the expression of key chemokines and proinflammatory cytokines that contribute to the defense of the uterus against potential pathogens.

Identification of equine herpesvirus-1 antigens recognized by cytotoxic T lymphocytes.

Equine herpesvirus-1 (EHV-1) causes serious disease in horses throughout the world, despite the frequent use of vaccines. CTLs are thought to be critical for protection from primary and reactivating latent EHV-1 infections. However, the antigen-specificity of EHV-1-specific CTLs is unknown. The aim of this study was to identify EHV-1 genes that encode proteins containing CTL epitopes and to determine their MHC I (or ELA-A in the horse) restriction. Equine dendritic cells, transfected with a series of EHV-1 genes, were used to stimulate autologous CTL precursor populations derived from previously infected horses. Cytotoxicity was subsequently measured against EHV-1-infected PWM lymphoblast targets. Dendritic cells were infected with EHV-1 (positive control) or transfected with plasmids encoding the gB, gC, gD, gE, gH, gI, gL, immediate-early (IE) or early protein of EHV-1 using the PowderJect XR-1 research device. Dendritic cells transfected with the IE gene induced CTL responses in four of six ponies. All four of these ponies shared a common ELA-A3.1 haplotype. Dendritic cells transfected with gC, gD, gI and gL glycoproteins induced CTLs in individual ponies. The cytotoxic activity was ELA-A-restricted, as heterologous targets from ELA-A mismatched ponies were not killed and an MHC I blocking antibody reduced EHV-1-specific killing. This is the first identification of an EHV-1 protein containing ELA-A-restricted CTL epitopes. This assay can now be used to study CTL specificity for EHV-1 proteins in horses with a broad range of ELA-A haplotypes, with the goal of developing a multi-epitope EHV-1 vaccine.