Nicholas Svitek

Receptor (SLAM [CD150]) recognition and the V protein sustain swift lymphocyte-based invasion of mucosal tissue and lymphatic organs by a morbillivirus.

ABSTRACT Experimental infections of ferrets with canine distemper virus (CDV) recapitulate many hallmarks of measles: rash, high fever, viremia, depression of delayed-type hypersensitivity responses, lowered leukocyte counts, and reduced lymphocyte proliferation activity. To understand how a morbillivirus invades the host and causes immunosuppression, we generated CDV either unable to recognize one of the receptors or incapable of expressing either one or both of the candidate interferon antagonist proteins V and C. Variants of these viruses expressing green fluorescent protein were also generated. Striking similarities between CDV infection of ferrets and human immunodeficiency virus host invasion were documented: first, massive early replication in the gut-associated lymphatic tissue, including intestinal Peyers patches, followed by extensive infection of lymphatic organs, including thymus and circulating lymphocytes. Moreover, T cells were selectively depleted. Thus, CDV takes advantage of mucosal surfaces for host invasion and lymphocytes for swift dissemination. A CDV unable to recognize the signaling lymphocytic activation molecule (SLAM [CD150]) that is expressed in lymphocytes and other immune cells did not spread. A V-defective CDV multiplied with reduced efficiency in lymphocytes and did not inhibit the interferon and cytokine responses. Protein C affected the severity of rash and digestive symptoms elicited by V-defective CDV, but it was dispensable for the invasion of the lymphatic organs. These findings prove formally that SLAM recognition is necessary for morbillivirus virulence. They also reveal how two viral proteins affect pathogenesis: V sustains the swift lymphocyte-based invasion of mucosal tissue and lymphatic organs, whereas C sustains subsequent infection phases.

Severe seasonal influenza in ferrets correlates with reduced interferon and increased IL-6 induction.

Even though ferrets are one of the principal animal models for influenza pathogenesis, the lack of suitable immunological reagents has so far limited their use in host response studies. Using recently established real-time PCR assays for a panel of ferret cytokines, we analyzed the local ferret immune response to human influenza isolates of the H1N1 and H3N2 subtypes that varied in their virulence. We observed that the severity of clinical signs correlated with gross- and histopathological changes in the lungs and was subtype-independent. Strains causing a mild disease were associated with a strong and rapid innate response and upregulation of IL-8, while severe infections were characterized by a lesser induction of type I and II interferons and strong IL-6 upregulation. These findings suggest that more virulent strains may interfere more efficiently with the host response at early disease stages.

Early cytokine mRNA expression profiles predict Morbillivirus disease outcome in ferrets

Abstract Severe immunosuppression is a hallmark of Morbillivirus infections. To study the underlying mechanisms, we have developed a ferret model of canine distemper virus infection. The model reproduces all clinical signs of measles, but the lack of ferret-specific reagents has limited the characterization of the cellular immune response. Towards this, we cloned ferret cytokines and established semi-quantitative real-time PCR assays. To demonstrate the utility of these assays we compared the cytokine profiles elicited by lethal and non-lethal strains during the prodromal phase. We observed a general lack of cytokine induction in animals that later succumbed to the disease, whereas survivors mounted a robust and sustained response. The newly developed cytokine assays strengthen and expand the ferret model not only for Morbillivirus pathogenesis studies but also for several other human respiratory viruses including influenza and SARS.

Local Innate Immune Responses and Influenza Virus Transmission and Virulence in Ferrets

Host innate immunity is the first line of defense against invading pathogens, including influenza viruses. Ferrets are well recognized as the best model of influenza virus pathogenesis and transmission, but little is known about the innate immune response of ferrets after infection with this virus. The goal of this study was to investigate the contribution of localized host responses to influenza virus pathogenicity and transmissibility in this model by measuring the level of messenger RNA expression of 12 cytokines and chemokines in the upper and lower respiratory tracts of ferrets infected with H5N1, H1N1, or H3N2 influenza viruses that exhibit diverse virulence and transmissibility in ferrets. We found a strong temporal correlation between inflammatory mediators and the kinetics and frequency of transmission, clinical signs associated with transmission, peak virus shedding, and virulence. Our findings point to a link between localized innate immunity and influenza virus transmission and disease progression.

Ferrets as a model for morbillivirus pathogenesis, complications, and vaccines.

The ferret is a standard laboratory animal that can be accommodated in most animal facilities. While not susceptible to measles, ferrets are a natural host of canine distemper virus (CDV), the closely related carnivore morbillivirus. CDV infection in ferrets reproduces all clinical signs associated with measles in humans, including the typical rash, fever, general immunosuppression, gastrointestinal and respiratory involvement, and neurological complications. Due to this similarity, experimental CDV infection of ferrets is frequently used to assess the efficacy of novel vaccines, and to characterize pathogenesis mechanisms. In addition, direct intracranial inoculation of measles isolates from subacute sclerosing panencephali-tis (SSPE) patients results in an SSPE-like disease in animals that survive the acute phase. Since the advent of reverse genetics systems that allow the targeted manipulation of viral genomes, the model has been used to evaluate the contribution of the accessory proteins C and V, and signalling lymphocyte activation molecule (SLAM)-binding to immunosuppression and overall pathogenesis. Similarly produced green fluorescent protein-expressing derivatives that maintain parental virulence have been instrumental in the direct visualization of systemic dissemination and neuroinvasion. As more immunological tools become available for this model, its contribution to our understanding of morbillivirus—host interactions is expected to increase.

The biology of Theileria parva and control of East Coast fever – Current status and future trends

Tremendous progress has been made over the last ten years on East Coast fever (ECF) research. Publication of a reference genome sequence of Theileria parva, the causative agent of ECF, has led to a more thorough characterization of the genotypic and antigenic diversity of the pathogen. It also facilitated identification of antigens that are targets of bovine major histocompatibility complex class I restricted cytotoxic T-lymphocytes (CTLs), induced by a live parasite-based infection and treatment method (ITM) vaccine. This has led to improved knowledge of epitope-specific T-cell responses to ITM that most likely contribute to the phenomenon of strain-specific immunity. The Muguga cocktail ITM vaccine, which provides broad-spectrum immunity to ECF is now a registered product in three countries in eastern Africa. Effort is directed at improving and scaling up the production process to make this vaccine more widely available on a commercial basis in the region. Meanwhile, research to develop a subunit vaccine based on parasite neutralizing antibodies and CTLs has been revived through convening of a research consortium to develop proof-of-concept for a next generation vaccine. Many new scientific and technical advances are facilitating this objective. Hence, the next decade promises even more progress toward an improved control of ECF.

A chimeric measles virus with canine distemper envelope protects ferrets from lethal distemper challenge.

CDV infects a broad range of carnivores, and over the past decades it has caused outbreaks in a variety of wild carnivore populations. Since the currently available live-attenuated vaccine is not sufficiently safe in these highly susceptible species, we produced a chimeric virus combining the replication complex of the measles Moraten vaccine strain with the envelope of a recent CDV wild type isolate. The resulting virus did not cause disease or immunosuppression in ferrets and conferred protection from challenge with a lethal wild type strain, demonstrating its potential value for wildlife conservation efforts.

Morbillivirus Control of the Interferon Response: Relevance of STAT2 and mda5 but Not STAT1 for Canine Distemper Virus Virulence in Ferrets

ABSTRACT The V proteins of paramyxoviruses control the innate immune response. In particular, the V protein of the genus Morbillivirus interferes with the signal transducer and activator of transcription 1 (STAT1), STAT2, and melanoma differentiation-associated protein 5 (mda5) signaling pathways. To characterize the contributions of these pathways to canine distemper virus (CDV) pathogenesis, we took advantage of the knowledge about the mechanisms of interaction between the measles virus V protein with these key regulators of innate immunity. We generated recombinant CDVs with V proteins unable to properly interact with STAT1, STAT2, or mda5. A virus with combined STAT2 and mda5 deficiencies was also generated, and available wild-type and V-protein-knockout viruses were used as controls. Ferrets infected with wild-type and STAT1-blind viruses developed severe leukopenia and loss of lymphocyte proliferation activity and succumbed to the disease within 14 days. In contrast, animals infected with viruses with STAT2 or mda5 defect or both STAT2 and mda5 defects developed a mild self-limiting disease similar to that associated with the V-knockout virus. This study demonstrates the importance of interference with STAT2 and mda5 signaling for CDV immune evasion and provides a starting point for the development of morbillivirus vectors with reduced immunosuppressive properties. IMPORTANCE The V proteins of paramyxoviruses interfere with the recognition of the virus by the immune system of the host. For morbilliviruses, the V protein is known to interact with the signal transducer and activator of transcription 1 (STAT1) and STAT2 and the melanoma differentiation-associated protein 5 (mda5), which are involved in interferon signaling. Here, we examined the contribution of each of these signaling pathways to the pathogenesis of the carnivore morbillivirus canine distemper virus. Using viruses selectively unable to interfere with the respective signaling pathway to infect ferrets, we found that inhibition of STAT2 and mda5 signaling was critical for lethal disease. Our findings provide new insights in the mechanisms of morbillivirus immune evasion and may lead to the development of new vaccines and oncolytic vectors.

Characterization of binding specificities of bovine leucocyte class I molecules: impacts for rational epitope discovery.

The binding of peptides to classical major histocompatibility complex (MHC) class I proteins is the single most selective step in antigen presentation. However, the peptide-binding specificity of cattle MHC (bovine leucocyte antigen, BoLA) class I (BoLA-I) molecules remains poorly characterized. Here, we demonstrate how a combination of high-throughput assays using positional scanning combinatorial peptide libraries, peptide dissociation, and peptide-binding affinity binding measurements can be combined with bioinformatics to effectively characterize the functionality of BoLA-I molecules. Using this strategy, we characterized eight BoLA-I molecules, and found the peptide specificity to resemble that of human MHC-I molecules with primary anchors most often at P2 and P9, and occasional auxiliary P1/P3/P5/P6 anchors. We analyzed nine reported CTL epitopes from Theileria parva, and in eight cases, stable and high affinity binding was confirmed. A set of peptides were tested for binding affinity to the eight BoLA proteins and used to refine the predictors of peptide–MHC binding NetMHC and NetMHCpan. The inclusion of BoLA-specific peptide-binding data led to a significant improvement in prediction accuracy for reported T. parva CTL epitopes. For reported CTL epitopes with weak or no predicted binding, these refined prediction methods suggested presence of nested minimal epitopes with high-predicted binding affinity. The enhanced affinity of the alternative peptides was in all cases confirmed experimentally. This study demonstrates how biochemical high-throughput assays combined with immunoinformatics can be used to characterize the peptide-binding motifs of BoLA-I molecules, boosting performance of MHC peptide-binding prediction methods, and empowering rational epitope discovery in cattle.

BoLA-6*01301 and BoLA-6*01302, two allelic variants of the A18 haplotype, present the same epitope from the Tp1 antigen of Theileria parva

We have recently shown that the BoLA-A18 variant haplotype (BoLA-6*01302) is more prevalent than the BoLA-A18 haplotype (BoLA-6*01301) in a sample of Holstein/Friesian cattle in Kenya. These MHC class I allelic variants differ by a single amino acid polymorphism (Glu97 to Leu97) in the peptide-binding groove. We have previously mapped an 11-mer peptide epitope from the Theileria parva antigen Tp1 (Tp1214-224) that is presented by BoLA-6*01301. Crystal structure data indicates that Glu97 in the MHC molecule plays a role in epitope binding through electro-static interaction with a lysine residue in position 5 of the epitope, which also functions as an additional anchor residue. In contrast to expectations, we demonstrate that the amino acid substitution in BoLA-6*01302 does not divert the CTL response away from Tp1214-224. The two MHC molecules exhibit similar affinity for the Tp1 epitope and can present the epitope to parasite-specific CTLs derived from either BoLA allelic variants. These data confirm that this BoLA polymorphism does not alter Tp1 epitope specificity and that both allelic variants can be used for Tp1 vaccine studies.