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Background: Limited options are available for the treatment of pain in cats. Monoclonal antibodies (mAbs) that neutralize nerve growth factor (NGF) have demonstrated analgesic capacity in rodent models, people with osteoarthritis, and dogs with degenerative joint disease., Hypothesis/objectives: This study describes the design and characterization of a fully felinized anti-NGF monoclonal antibody. In vitro potency, pharmacokinetics, and the ability of the antibody to treat pain in a self-resolving, acute inflammation model were investigated in cats., Animals: Thirty-eight cats at a research colony at Charles River Laboratories, Ireland., Methods: Felinized anti-NGF mAb, NV-02, was produced using a complementary DNA (cDNA)-based method (PETization). Purified NV-02 was tested for affinity, potency, and immunoreactivity in vitro, then for safety and plasma pharmacokinetic distribution in vivo, and analgesic efficacy in a model of kaolin-induced inflammatory pain., Results: Anti-NGF mAb, NV-02 neutralized NGF with high affinity and potency and did not bind complement. NV-02-administered SC had a plasma half-life of 7-15 days and was well tolerated at dosages up to 28 mg/kg. A dosage of 2 mg/kg NV-02 SC significantly decreased signs of lameness on day 2 (P = .0027), day 3 (P = .016), day 4, (P = .0063), day 5 (P = .0085), day 6 (P = .0014), and day 7 (P = .0034) after induction of inflammation., Conclusions and Clinical Importance: The high affinity, long plasma half-life, safety, and analgesic efficacy of felinized anti-NGF mAb (NV-02) support further investigation of the analgesic potential of this antibody in the cat., (Copyright © 2016 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Background: Monoclonal antibodies are a major class of biological therapies in human medicine but have not yet been successfully applied to veterinary species. We have developed a novel approach, PETisation, to rapidly convert antibodies for use in veterinary species. As an example, anti-nerve growth factor (anti-NGF) monoclonal antibodies (mAbs) which are effective in reducing acute and chronic pain in rodents and man are potentially useful for treating pain in dogs but a fully caninised mAb is required in order to avoid an immune response. The aim of this study was to determine the optimal properties of a caninised anti-NGF mAb for safe, repeated administration to dogs, to determine its pharmacokinetic properties and to evaluate its efficacy in a model of inflammatory pain in vivo., Results: Starting with a rat anti-NGF mAb, we used a novel algorithm based on expressed canine immunoglobulin sequences to design and characterise recombinant caninised anti-NGF mAbs. Construction with only 2 of the 4 canine IgG heavy chain isotypes (A and D) resulted in stable antibodies which bound and inhibited NGF with high-affinity and potency but did not bind complement C1q or the high-affinity Fc receptor gamma R1 (CD64). One of the mAbs (NV-01) was selected for scale-up manufacture, purification and pre-clinical evaluation. When administered to dogs, NV-01 was well tolerated, had a long serum half-life of 9 days, was not overtly immunogenic following repeated dosing in the dog and reduced signs of lameness in a kaolin model of inflammatory pain., Conclusions: The combination of stability, high affinity and potency, no effector activity and long half-life, combined with safety and activity in the model of inflammatory pain in vivo suggests that further development of the caninised anti-NGF mAb NV-01 as a therapeutic agent for the treatment of chronic pain in dogs is warranted.

Background: Hendra virus (HeV) is an Australian bat-borne zoonotic paramyxovirus that repeatedly spills-over to horses causing fatal disease. Human cases have all been associated with close contact with infected horses., Methods: A full-length antigenome clone of HeV was assembled, a reporter gene (GFP or luciferase) inserted between the P and M genes and transfected to 293T cells to generate infectious reporter gene-encoding recombinant viruses. These viruses were then assessed in vitro for expression of the reporter genes. The GFP expressing recombinant HeV was used to challenge ferrets to assess the virulence and tissue distribution by monitoring GFP expression in infected cells., Results: Three recombinant HeV constructs were successfully cloned and rescued; a wild-type virus, a GFP-expressing virus and a firefly luciferase-expressing virus. In vitro characterisation demonstrated expression of the reporter genes, with levels proportional to the initial inoculum levels. Challenge of ferrets with the GFP virus demonstrated maintenance of the fatal phenotype with disease progressing to death consistent with that observed previously with the parental wild-type isolate of HeV. GFP expression could be observed in infected tissues collected from animals at euthanasia., Conclusions: Here, we report on the first successful rescue of recombinant HeV, including wild-type virus and viruses expressing two different reporter genes encoded as an additional gene cassette inserted between the P and M genes. We further demonstrate that the GFP virus retained the ability to cause fatal disease in a well-characterized ferret model of henipavirus infection despite the genome being an extra 1290 nucleotides in length.

Henipaviruses encode several proteins from the P gene, of which V and W have been demonstrated by gene-based transfection studies to antagonize the innate immune response, blocking both type I interferon production and signaling. This study examines the effects of henipavirus infection on the innate immune response in human cell lines. We report that henipavirus infection does not result in interferon production, with the virus antagonizing this response. In contrast to published transfection studies, our study found that the interferon signaling pathways are only partially blocked by henipavirus infection of human cell lines.

Bats are known to harbor a number of emerging and re-emerging zoonotic viruses, many of which are highly pathogenic in other mammals but result in no clinical symptoms in bats. The ability of bats to coexist with viruses may be the result of rapid control of viral replication early in the immune response. IFNs provide the first line of defense against viral infection in vertebrates. Type III IFNs (IFN-λs) are a recently identified IFN family that share similar antiviral activities with type I IFNs. To our knowledge, we demonstrate the first functional analysis of type III IFNs from any species of bat, with the investigation of two IFN-λ genes from the pteropid bat, Pteropus alecto. Our results demonstrate that bat type III IFN has similar antiviral activity to type I and III IFNs from other mammals. In addition, the two bat type III IFNs are differentially induced relative to each other and to type I IFNs after treatment or transfection with synthetic dsRNA. Infection with the bat paramyxovirus, Tioman virus, resulted in no upregulation of type I IFN production in bat splenocytes but was capable of inducing a type III IFN response in three of the four bats tested. To our knowledge, this is the first report to describe the simultaneous suppression of type I IFN and induction of type III IFN after virus infection. These results may have important implications for the role of type III IFNs in the ability of bats to coexist with viruses.

Bats are natural reservoirs for a spectrum of infectious zoonotic diseases including the recently emerged henipaviruses (Hendra and Nipah viruses). Henipaviruses have been observed both naturally and experimentally to cause serious and often fatal disease in many different mammal species, including humans. Interestingly, infection of the flying fox with henipaviruses occurs in the absence of clinical disease. The extreme variation in the disease pattern between humans and bats has led to an investigation into the effects of henipavirus infection on the innate immune response in bat cell lines. We report that henipavirus infection does not result in the induction of interferon expression, and the viruses also inhibit interferon signaling. We also confirm that the interferon production and signaling block in bat cells is not due to differing viral protein expression levels between human and bat hosts. This information, in addition to the known lack of clinical signs in bats following henipavirus infection, suggests that bats control henipavirus infection by an as yet unidentified mechanism, not via the interferon response. This is the first report of henipavirus infection in bat cells specifically investigating aspects of the innate immune system.

Background: Bats are the suspected natural reservoir hosts for a number of new and emerging zoonotic viruses including Nipah virus, Hendra virus, severe acute respiratory syndrome coronavirus and Ebola virus. Since the discovery of SARS-like coronaviruses in Chinese horseshoe bats, attempts to isolate a SL-CoV from bats have failed and attempts to isolate other bat-borne viruses in various mammalian cell lines have been similarly unsuccessful. New stable bat cell lines are needed to help with these investigations and as tools to assist in the study of bat immunology and virus-host interactions., Methodology/findings: Black flying foxes (Pteropus alecto) were captured from the wild and transported live to the laboratory for primary cell culture preparation using a variety of different methods and culture media. Primary cells were successfully cultured from 20 different organs. Cell immortalisation can occur spontaneously, however we used a retroviral system to immortalise cells via the transfer and stable production of the Simian virus 40 Large T antigen and the human telomerase reverse transcriptase protein. Initial infection experiments with both cloned and uncloned cell lines using Hendra and Nipah viruses demonstrated varying degrees of infection efficiency between the different cell lines, although it was possible to infect cells in all tissue types., Conclusions/significance: The approaches developed and optimised in this study should be applicable to bats of other species. We are in the process of generating further cell lines from a number of different bat species using the methodology established in this study.

Prior to the emergence of Hendra virus in Australia in 1994, paramyxoviruses were considered to be a taxonomic group of ubiquitous pathogens, consisting primarily of Biosafety Level 2 agents, which possessed narrow host ranges and often caused only mild or preventable diseases in humans and animals. In recent years, a number of Paramyxoviridae members have emerged, including previously unrecognized human pathogens and highly pathogenic zoonoses. The recent emergence of paramyxoviruses in humans suggests that there is an increased incidence of zoonotic transmission between wildlife, livestock and human hosts. This article explores the current body of scientific knowledge, disease burden and knowledge of reservoirs of these emerging paramyxoviruses and provides a comparative review of both older and emerging viruses that have been shown to infect humans.