A Gag peptide encompassing B- and T-cell epitopes of the caprine arthritis encephalitis virus functions as modular carrier peptide.

Short synthetic peptides are important tools in biomedical research permitting to generate hapten specific polyclonal sera for analytical purposes or functional studies. In this paper we provide proof of principle that a peptide located in a highly conserved portion of the Gag protein of the caprine arthritis encephalitis virus and carrying an immunodominant T helper cell epitope functions as an efficient carrier peptide, mediating a strong antibody response to a peptidic hapten encompassing a well-characterized B cell epitope of Env. The carrier and hapten peptides were collinearly synthesized permutating their molecular arrangement. While the antibody response to the hapten was similar for both constructs, the antibody response to a B cell epitope overlapping the T helper cell epitope of the Gag carrier peptide was considerably different. This permits a modular use of the carrier peptide to generate antibody directed exclusively to the hapten peptide or a strong humoral response to both carrier- and hapten-peptide. Finally, we have mapped the epitopes involved in this polarized antibody response and discussed the potential immunological implications.

The viral RNase E(rns) prevents IFN type-I triggering by pestiviral single- and double-stranded RNAs.

Interferon (IFN) type-I is of utmost importance in the innate antiviral defence of eukaryotic cells. The cells express intra- and extracellular receptors that monitor their surroundings for the presence of viral genomes. Bovine viral diarrhoea virus (BVDV), a Pestivirus of the family Flaviviridae, is able to prevent IFN synthesis induced by poly(IC), a synthetic dsRNA. The evasion of innate immunity might be a decisive ability of BVDV to establish persistent infection in its host. We report that ds- as well as ssRNA fragments of viral origin are able to trigger IFN synthesis, and that the viral envelope glycoprotein E(rns), that is also secreted from infected cells, is able to inhibit IFN expression induced by these extracellular viral RNAs. The RNase activity of E(rns) is required for this inhibition, and E(rns) degrades ds- and ssRNA at neutral pH. In addition, cells infected with a cytopathogenic strain of BVDV contain more dsRNA than cells infected with the homologous non-cytopathogenic strain, and the intracellular viral RNA was able to excite the IFN system in a 5'-triphosphate-, i.e. RIG-I-, independent manner. Functionally, E(rns) might represent a decoy receptor that binds and enzymatically degrades viral RNA that otherwise might activate the IFN defence by binding to Toll-like receptors of uninfected cells. Thus, the pestiviral RNase efficiently manipulates the host's self-nonself discrimination to successfully establish and maintain persistence and immunotolerance.

RNase-dependent inhibition of extracellular, but not intracellular, dsRNA-induced interferon synthesis by Erns of pestiviruses.

Recombinant pestivirus envelope glycoprotein E(rns) has been shown to interfere with dsRNA-induced interferon (IFN-alpha/beta) synthesis. This study demonstrated that authentic, enzymically active E(rns) produced in mammalian cells prevented a dsRNA-induced IFN response when present in the supernatant of bovine cells. Strikingly, IFN synthesis of cells expressing E(rns) was eliminated after extracellular addition, but not transfection, of dsRNA. Importantly, the same applied to cells infected with bovine viral diarrhea virus (BVDV) expressing E(rns) but lacking the N-terminal protease N(pro). Free E(rns) concentrations circulating in the blood of animals persistently infected with BVDV were determined to be approximately 50 ng ml(-1), i.e. at a similar order of magnitude as that displaying an effect on dsRNA-induced IFN expression in vitro. Whilst N(pro) blocks interferon regulatory factor-3-dependent IFN induction in infected cells, E(rns) may prevent constant IFN induction in uninfected cells by dsRNA that could originate from pestivirus-infected cells. This probably contributes to the survival of persistently BVDV-infected animals and maintains viral persistence in the host population.

Vaccination with a T-cell-priming Gag peptide of caprine arthritis encephalitis virus enhances virus replication transiently in vivo.

CD4+ T cells are involved in several immune response pathways used to control viral infections. In this study, a group of genetically defined goats was immunized with a synthetic peptide known to encompass an immunodominant helper T-cell epitope of caprine arthritis encephalitis virus (CAEV). Fifty-five days after challenge with the molecularly cloned CAEV strain CO, the vaccinated animals had a higher proviral load than the controls. The measurement of gamma interferon and interleukin-4 gene expression showed that these cytokines were reliable markers of an ongoing immune response but their balance did not account for more or less efficient control of CAEV replication. In contrast, granulocyte-macrophage colony-stimulating factor appeared to be a key cytokine that might support virus replication in the early phase of infection. The observation of a potential T-cell-mediated enhancement of virus replication supports other recent findings showing that lentivirus-specific T cells can be detrimental to the host, suggesting caution in designing vaccine candidates.

Analysis of the antibody response to an immunodominant epitope of the envelope glycoprotein of a lentivirus and its diagnostic potential.

The envelope glycoprotein of small ruminant lentiviruses (SRLV) is a major target of the humoral immune response and contains several linear B-cell epitopes. We amplified and sequenced the genomic segment encoding the SU5 antigenic site of the envelope glycoprotein of several SRLV field isolates. With synthetic peptides based on the deduced amino acid sequences of SU5 in an enzyme-linked immunosorbent assay (ELISA), we have (i) proved the immunodominance of this region regardless of its high variability, (ii) defined the epitopes encompassed by SU5, (iii) illustrated the rapid and peculiar kinetics of seroconversion to this antigenic site, and (iv) shown the rapid and strong maturation of the avidity of the anti-SU5 antibody. Finally, we demonstrated the modular diagnostic potential of SU5 peptides. Under Swiss field conditions, the SU5 ELISA was shown to detect the majority of infected animals and, when applied in a molecular epidemiological context, to permit rapid phylogenetic classification of the infecting virus.

The MHC-haplotype influences primary, but not memory, immune responses to an immunodominant peptide containing T- and B-cell epitopes of the caprine arthritis encephalitis virus Gag protein.

In this report, we describe a short peptide, containing a T helper- and a B-cell epitope, located in the Gag protein of the caprine arthritis encephalitis virus (CAEV). This T-cell epitope is capable of inducing a robust T-cell proliferative response in vaccinated goats with different genetic backgrounds and to provide help for a strong antibody response to the B-cell epitope, indicating that it may function as a universal antigen-carrier for goat vaccines. The primary immune response of goats homozygous for MHC class I and II genes showed an MHC-dependent partitioning in rapid-high and slow-low responses, whereas the memory immune response was strong in both groups, demonstrating that a vaccine based on this immunodominant T helper epitope is capable to overcome genetic differences.

Evidence for dissociation of TLR mRNA expression and TLR agonist-mediated functions in bovine macrophages.

Toll-like receptors are of key importance in the recognition of and response to infectious agents by cells of the innate immune system. TLR mRNA expression and TLR-mediated functions were determined in bovine macrophages (MPhi) infected with bovine viral diarrhea virus (BVDV) or stimulated with interferon-gamma (IFN-gamma) in order to see whether they are correlated under these conditions. As parameters quantitative real time RT-PCR (QRT-PCR) for TLR2, TLR3 and TLR4, NO and TNF production were measured. Triggering of bovine MPhi with bona fide TLR2 and TLR4 agonists (lipopolysaccharide, lipoteichoic acid, peptidoglycan, lipopetide) led to NO and TNF production but neither TLR3 nor TLR9 agonists (double-stranded RNA, CpG DNA) showed this effect. The mRNA expression of TLR2, TLR3 and TLR4 was neither influenced by MPhi costimulation with IFN-gamma nor by MPhi preinfection with BVDV nor by the ligands themselves. However, NO production induced by TLR2 or TLR4 agonists was strongly modulated either by IFN-gamma costimulation or BVDV preinfection. Thus costimulation of MPhi with IFN-gamma resulted in an increase of both NO synthesis and TNF expression by cells stimulated simultaneously by TLR2 or TLR4 agonists. Preinfection of bovine MPhi by BVDV resulted in upregulation of TLR2- and TLR4-mediated NO synthesis. Collectively, these data show that TLR-mediated functions may be modulated by viral infection or activation via IFN-gamma of MPhi whereas the mRNA concentrations of relevant TLR members were not significantly influenced. Thus, the amount of TLR2, TLR3 and TLR4 mRNA transcripts is stable at least under the conditions tested. More importantly, modulation of TLR-mediated responses was dissociated from mRNA expression of TLR members.

Innate immune responses of calves during transient infection with a noncytopathic strain of bovine viral diarrhea virus.

In this study, six immunocompetent calves were experimentally infected with a noncytopathic strain of bovine viral diarrhea virus (BVDV), and the effects of the viral infection on parameters of the innate immune response of the host were analyzed. Clinical and virological data were compared with the temporal activation of the alpha/beta interferon-regulated Mx gene in white blood cells (WBC) and skin as well as the upregulation of the acute-phase serum proteins haptoglobin (Hp) and serum amyloid A (SAA). The viral strain used did provoke transient health impairment, namely, fever and leukopenia that were associated with viremia, viral shedding with nasal secretions, and antiviral seroconversion. Complete recovery was observed within 3 weeks. Elevated levels of SAA and Hp were apparent from days 4 to 13 and 8 to 11, respectively. In WBC, the levels of Mx mRNA and Mx protein were elevated from days 2 to 15. In the context of this study with BVDV, the level of Mx protein expression in WBC provided the most telling diagnostic window to monitor the host's ongoing innate immune response.

BVDV and innate immunity.

Infection with bovine viral diarrhea virus (BVDV) is prevalent in the cattle population worldwide. The virus exists in two biotypes, cytopathic and non-cytopathic, depending on the effect of the viruses on cultured cells. BVDV may cause transient and persistent infections which differ fundamentally in the host's antiviral immune response. Transient infection may be due to both cytopathic and non-cytopathic biotypes of BVDV and leads to a specific immune response. In contrast, only non-cytopathic BVD viruses can establish persistent infection as a result of infection of the embryo early in its development. Persistent infection is characterized by immunotolerance specific for the infecting viral strain. In this paper we discuss the role of innate immune responses in the two types of infection. In general, both transient and persistent infections are associated with an increased frequency of secondary infections. Associated with the increased risk of such infections are, among others, impaired bacteria killing and decreased chemotaxis. Interestingly, non-cytopathic BVDV fails to induce interferon type I in cultured bovine macrophages whereas cytopathic biotypes readily trigger this response. Cells infected with non-cytopathic BVDV are also resistant to induction of interferon by double stranded RNA, a potent interferon inducer signalling the presence of viral replication in the cell. Thus, non-cytopathic BVDV may dispose of a mechanism suppressing a key element of the antiviral defence of the innate immune system. Since interferon is also important in the activation of the adaptive immune response, suppression of this signal may be essential for the establishment of persistent infection and immunotolerance.

Production of monospecific antibody to immunodominant epitopes of the caprine arthritis encephalitis virus transmembrane glycoprotein and analysis of their activity in vitro.

Caprine arthritis encephalitis virus (CAEV)-infected goats develop high titers of nonneutralizing antibody to several immunodominant epitopes of the viral envelope glycoprotein. Antibodies directed to these structures, and especially the principal immunodominant domain (PID) of the transmembrane portion of the envelope glycoprotein, have been implicated in the immunopathological mechanisms leading to the development of arthritis. We previously mapped the PID and additional epitopes of CAEV gp38 and showed an association between the development of clinical arthritis in infected animals and the antibody response to these epitopes. The development of clinical arthritis is associated with a higher rate of viral expression, especially in the affected tissue, indicating that antibody may favorably modulate virus replication. To test this hypothesis, we immunized goats with synthetic peptides corresponding to the mapped epitopes. The immunized animals developed high titers of nonneutralizing antibody to the immunizing peptides. In radioimmunoprecipitation experiments these antibodies were shown to react to the envelope glycoproteins in extracts obtained under nondenaturing conditions. Finally, these sera were tested in cultures of primary macrophages infected at low multiplicity without showing any (either positive or negative) modulatory activity.

B-cell epitopes of the envelope glycoprotein of caprine arthritis-encephalitis virus and antibody response in infected goats.

Goats infected with caprine arthritis-encephalitis virus (CAEV) develop high titres of antibodies to Env. Not only is no consistent neutralizing response found but anti-Env antibodies have even been associated with disease in infected goats. To identify the continuous antigenic determinants involved in this atypical anti-Env response, we mapped CAEV-CO Env by screening an epitope expression library with infected goat sera. In addition to the four previously described epitopes, seven novel antigenic sites were identified, of which five were located on the surface (SU) and two in the transmembrane (TM) subunits of Env. The SU antibody-binding domains located in the variable regions of the C-terminal part of the molecule (SU3 to SU5) showed the strongest reactivity and induced a rapid seroconversion in six experimentally infected goats. However, the response to these immunodominant epitopes did not appear to be associated with any neutralizing activity. The pattern of serum reactivity of naturally infected goats with these epitopes was restricted, suggesting a type-specific reaction. Interestingly, the reactivity of peptides representing SU5 sequences derived from CAEV field isolates varied with the geographical and/or breeding origin of the animals. This suggests that peptides corresponding to the immunodominant SU epitopes may well be useful in the serotyping of CAEV isolates. Furthermore, the identification of the CAEV Env epitopes will permit us to functionally dissect the antibody response and to address the role of anti-Env antibodies either in the protection from or in the pathogenesis of CAEV infection.