Laboratory safety evaluation of lokivetmab, a canine anti-interleukin-31 monoclonal antibody, in dogs.

Lokivetmab (Cytopoint®, Zoetis) is a canine monoclonal antibody that specifically binds and neutralizes interleukin (IL)-31. Lokivetmab is approved for use in dogs for the treatment of atopic dermatitis (AD) and allergic dermatitis. The laboratory safety of lokivetmab was evaluated in 2 studies by adapting the science-based, case-by-case approach used for preclinical and early clinical safety evaluation of human biopharmaceuticals. The main objectives were to demonstrate the safety of lokivetmab in healthy laboratory Beagle dogs by using integrated clinical, morphologic, and functional evaluations. In Study 1, dogs were treated s.c. with saline or lokivetmab at 3.3 mg/kg (1X, label dose) or 10 mg/kg (3X intended dose) for 7 consecutive monthly doses, with terminal pathology and histology assessments. In Study 2, the functional immune response was demonstrated in naïve dogs using the T-cell dependent antibody response (TDAR) test with 2 different dose levels of unadjuvanted keyhole limpet hemocyanin (KLH) as the model immunogen. The primary endpoint was anti-KLH IgG antibody titer, and secondary endpoints were ex vivo IL-2 enzyme-linked immunospot (ELISpot) and peripheral blood mononuclear cell lymphoproliferation assays. Both studies included monitoring general health, periodic veterinary clinical evaluations, serial clinical pathology and toxicokinetics, and monitoring for anti-drug antibodies. In both studies, the health of dogs receiving lokivetmab was similar to controls, with no treatment-related changes uncovered. Extensive pathology evaluations of immune tissues (Study 1) revealed no lokivetmab-related morphologic changes, and in dogs treated at 10 mg/kg lokivetmab, immunization with the model antigen KLH did not impair the functional antibody or T-cell recall responses. There were no immunogenicity-related or hypersensitivity-related responses observed in either study. These studies in healthy laboratory dogs showed that lokivetmab was well-tolerated, did not produce any treatment-related effects, and had no effect on immune system morphology or its functional response. These studies also demonstrated the utility of a science-based case-by-case approach to the safety evaluation of a veterinary biopharmaceutical product.

Comparison of cellular assays for TLR activation and development of a species-specific reporter cell line for cattle.

PRRs are sentinels of the innate immune system, with TLRs being the most important. Assays for TLR ligand interactions have been used to gain insights into their function and signaling pathways. As significant differences exist between species with regard to ligand recognition, it is necessary to adapt these tools for TLRs of other species. In the present work, we describe a species-specific cell-based assay adapted for the analysis of single PRRs. Human embryonic kidney 293T cells were stably transfected with the NF-κB-inducible reporter gene secreted embryonic alkaline phosphatase (SEAP) together with bovine TLR2. We compared the SEAP response with an existing luciferase NF-κB reporter assay for correlation with IL-8 production. A dose-dependent response was detected upon stimulation using both methods with good correlation to IL-8 secretion. Lower stimulant concentrations were detected by SEAP assay than IL-8 secretion. The luciferase assay produced high non-specific background for all ligand concentrations. Of all assays tested, we found the bovine-specific SEAP reporter assay to be the most convenient and delivered results in the shortest time. The developed reporter cell line would lend well to rapid, high-throughput TLR ligand screening for cattle.

Characterization of a Broadly Reactive Anti-CD40 Agonistic Monoclonal Antibody for Potential Use as an Adjuvant.

Lack of safe and effective adjuvants is a major hindrance to the development of efficacious vaccines. Signaling via CD40 pathway leads to enhanced antigen processing and presentation, nitric oxide expression, pro-inflammatory cytokine expression by antigen presenting cells, and stimulation of B-cells to undergo somatic hypermutation, immunoglobulin class switching, and proliferation. Agonistic anti-CD40 antibodies have shown promising adjuvant qualities in human and mouse vaccine studies. An anti-CD40 monoclonal antibody (mAb), designated 2E4E4, was identified and shown to have strong agonistic effects on primary cells from multiple livestock species. The mAb recognize swine, bovine, caprine, and ovine CD40, and evoked 25-fold or greater proliferation of peripheral blood mononuclear cells (PBMCs) from these species relative to cells incubated with an isotype control (p<0.001). In addition, the mAb induced significant nitric oxide (p<0.0001) release by bovine macrophages. Furthermore, the mAb upregulated the expression of MHC-II by PBMCs, and stimulated significant (p<0.0001) IL-1α, IL6, IL-8, and TNF-α expression by PBMCs. These results suggest that the mAb 2E4E4 can target and stimulate cells from multiple livestock species and thus, it is a potential candidate for adjuvant development. This is the first study to report an anti-swine CD40 agonistic mAb that is also broadly reactive against multiple species.

Induction of Robust Immune Responses in Swine by Using a Cocktail of Adenovirus-Vectored African Swine Fever Virus Antigens.

The African swine fever virus (ASFV) causes a fatal hemorrhagic disease in domestic swine, and at present no treatment or vaccine is available. Natural and gene-deleted, live attenuated strains protect against closely related virulent strains; however, they are yet to be deployed and evaluated in the field to rule out chronic persistence and a potential for reversion to virulence. Previous studies suggest that antibodies play a role in protection, but induction of cytotoxic T lymphocytes (CTLs) could be the key to complete protection. Hence, generation of an efficacious subunit vaccine depends on identification of CTL targets along with a suitable delivery method that will elicit effector CTLs capable of eliminating ASFV-infected host cells and confer long-term protection. To this end, we evaluated the safety and immunogenicity of an adenovirus-vectored ASFV (Ad-ASFV) multiantigen cocktail formulated in two different adjuvants and at two immunizing doses in swine. Immunization with the cocktail rapidly induced unprecedented ASFV antigen-specific antibody and cellular immune responses against all of the antigens. The robust antibody responses underwent rapid isotype switching within 1 week postpriming, steadily increased over a 2-month period, and underwent rapid recall upon boost. Importantly, the primed antibodies strongly recognized the parental ASFV (Georgia 2007/1) by indirect fluorescence antibody (IFA) assay and Western blotting. Significant antigen-specific gamma interferon-positive (IFN-γ+) responses were detected postpriming and postboosting. Furthermore, this study is the first to demonstrate induction of ASFV antigen-specific CTL responses in commercial swine using Ad-ASFV multiantigens. The relevance of the induced immune responses in regard to protection needs to be evaluated in a challenge study.

Protection against henipaviruses in swine requires both, cell-mediated and humoral immune response.

Hendra virus (HeV) and Nipah virus (NiV) are members of the genus Henipavirus, within the family Paramyxoviridae. Nipah virus has caused outbreaks of human disease in Bangladesh, Malaysia, Singapore, India and Philippines, in addition to a large outbreak in swine in Malaysia in 1998/1999. Recently, NiV was suspected to be a causative agent of an outbreak in horses in 2014 in the Philippines, while HeV has caused multiple human and equine outbreaks in Australia since 1994. A swine vaccine able to prevent shedding of infectious virus is of veterinary and human health importance, and correlates of protection against henipavirus infection in swine need to be better understood. In the present study, three groups of animals were employed. Pigs vaccinated with adjuvanted recombinant soluble HeV G protein (sGHEV) and challenged with HeV, developed antibody levels considered to be protective prior to the challenge (titers of 320). However, activation of the cell-mediated immune response was not detected, and the animals were only partially protected against challenge with 5×10(5) PFU of HeV per animal. In the second group, cross-neutralizing antibody levels against NiV in the sGHEV vaccinated animals did not reach protective levels, and with no activation of cellular immune memory, these animals were not protected against NiV. Only pigs orally infected with 5×10(4) PFU of NiV per animal were protected against nasal challenge with 5×10(5) PFU of NiV per animal. This group of pigs developed protective antibody levels, as well as cell-mediated immune memory. Peripheral blood mononuclear cells restimulated with UV-inactivated NiV upregulated IFN-gamma, IL-10 and the CD25 activation marker on CD4(+)CD8(+) T memory helper cells and to lesser extent on CD4(-)CD8(+) T cells. In conclusion, both humoral and cellular immune responses were required for protection of swine against henipaviruses.

Replacement of two aminoacids in the bovine Toll-like receptor 5 TIR domain with their human counterparts partially restores functional response to flagellin.

Flagellin potently induces inflammatory responses in mammalian cells by activating Toll-like receptor (TLR) 5. Recently, we were able to show that stimulation of bovine TLR5 resulted in neither NFκB signalling nor CXCL8 production. Like other TLRs, TLR5 recruits signalling molecules to its intracellular TIR domain, leading to inflammatory responses. Analysis of available TLR5 sequences revealed substitutions in all artiodactyl sequences at amo acid (AA) position 798 and 799. Interestingly, a putative binding site for PI3K was identified at tyrosine 798 in the human TLR5 TIR domain, analogous to the PI3K recruitment domain in the IL-1 receptor. Mutation of the artiodactyl residues at position 798, 799 or both with their corresponding human counterparts partially restored the response of bovine (bo)TLR5 to flagellin as well as phosphorylation of PI3K. Together, our results suggest a potential lack of phosphorylation of F798 and H799 in boTLR5 partially explains the lack in observed response.

Hydrocortisone inhibits IFN-γ production in equine, ovine, and bovine PBMCs.

Hydrocortisone is widely accepted as an anti-inflammatory agent and there are many products available containing hydrocortisone as an active ingredient. Surprisingly, there is little data available specifically on the immunological effects of hydrocortisone in large animals. Glucocorticoids are well-characterized for their ability to repress inflammation via a wide variety of mechanisms including suppression of cytokine production. In this study the effects of hydrocortisone on IFN-γ production by equine, bovine, and ovine PBMCs were assessed using flow cytometric or ELISpot analysis. Hydrocortisone suppressed mitogen-driven IFN-γ production by PBMCs from all three species of animals, confirming that this agent mediates anti-inflammatory effects in large animals. Although the results from this study were expected based on the precedence set in murine and human systems, it is important to understand the effects of administration of a compound or product in the species of interest as species-specific indications are not always available.

Two Theileria parva CD8 T cell antigen genes are more variable in buffalo than cattle parasites, but differ in pattern of sequence diversity.

BACKGROUND: Theileria parva causes an acute fatal disease in cattle, but infections are asymptomatic in the African buffalo (Syncerus caffer). Cattle can be immunized against the parasite by infection and treatment, but immunity is partially strain specific. Available data indicate that CD8(+) T lymphocyte responses mediate protection and, recently, several parasite antigens recognised by CD8(+) T cells have been identified. This study set out to determine the nature and extent of polymorphism in two of these antigens, Tp1 and Tp2, which contain defined CD8(+) T-cell epitopes, and to analyse the sequences for evidence of selection. METHODOLOGY/PRINCIPAL FINDINGS: Partial sequencing of the Tp1 gene and the full-length Tp2 gene from 82 T. parva isolates revealed extensive polymorphism in both antigens, including the epitope-containing regions. Single nucleotide polymorphisms were detected at 51 positions (∼12%) in Tp1 and in 320 positions (∼61%) in Tp2. Together with two short indels in Tp1, these resulted in 30 and 42 protein variants of Tp1 and Tp2, respectively. Although evidence of positive selection was found for multiple amino acid residues, there was no preferential involvement of T cell epitope residues. Overall, the extent of diversity was much greater in T. parva isolates originating from buffalo than in isolates known to be transmissible among cattle. CONCLUSIONS/SIGNIFICANCE: The results indicate that T. parva parasites maintained in cattle represent a subset of the overall T. parva population, which has become adapted for tick transmission between cattle. The absence of obvious enrichment for positively selected amino acid residues within defined epitopes indicates either that diversity is not predominantly driven by selection exerted by host T cells, or that such selection is not detectable by the methods employed due to unidentified epitopes elsewhere in the antigens. Further functional studies are required to address this latter point.

Treatment of cattle with DNA-encoded Flt3L and GM-CSF prior to immunization with Theileria parva candidate vaccine antigens induces CD4 and CD8 T cell IFN-γ responses but not CTL responses.

Theileria parva antigens recognized by cytotoxic T lymphocytes (CTLs) are prime vaccine candidates against East Coast fever in cattle. A strategy for enhancing induction of parasite-specific T cell responses by increasing recruitment and activation of dendritic cells (DCs) at the immunization site by administration of bovine Flt3L and GM-CSF prior to inoculation with DNA vaccine constructs and MVA boost was evaluated. Analysis of immune responses showed induction of significant T. parva-specific proliferation, and IFN-γ-secreting CD4(+) and CD8(+) T cell responses in immunized cattle. However, antigen-specific CTLs were not detected. Following lethal challenge, 5/12 immunized cattle survived by day 21, whereas all the negative controls had to be euthanized due to severe disease, indicating a protective effect of the vaccine (p<0.05). The study demonstrated the potential of this technology to elicit significant MHC class II and class I restricted IFN-γ-secreting CD4(+) and CD8(+) T cells to defined vaccine candidate antigens in a natural host, but also underscores the need to improve strategies for eliciting protective CTL responses.

Characterization of the fine specificity of bovine CD8 T-cell responses to defined antigens from the protozoan parasite Theileria parva.

Immunity against the bovine intracellular protozoan parasite Theileria parva has been shown to be mediated by CD8 T cells. Six antigens targeted by CD8 T cells from T. parva-immune cattle of different major histocompatibility complex (MHC) genotypes have been identified, raising the prospect of developing a subunit vaccine. To facilitate further dissection of the specificity of protective CD8 T-cell responses and to assist in the assessment of responses to vaccination, we set out to identify the epitopes recognized in these T. parva antigens and their MHC restriction elements. Nine epitopes in six T. parva antigens, together with their respective MHC restriction elements, were successfully identified. Five of the cytotoxic-T-lymphocyte epitopes were found to be restricted by products of previously described alleles, and four were restricted by four novel restriction elements. Analyses of CD8 T-cell responses to five of the epitopes in groups of cattle carrying the defined restriction elements and immunized with live parasites demonstrated that, with one exception, the epitopes were consistently recognized by animals of the respective genotypes. The analysis of responses was extended to animals immunized with multiple antigens delivered in separate vaccine constructs. Specific CD8 T-cell responses were detected in 19 of 24 immunized cattle. All responder cattle mounted responses specific for antigens for which they carried an identified restriction element. By contrast, only 8 of 19 responder cattle displayed a response to antigens for which they did not carry an identified restriction element. These data demonstrate that the identified antigens are inherently dominant in animals with the corresponding MHC genotypes.

Evaluation of the recognition of Theileria parva vaccine candidate antigens by cytotoxic T lymphocytes from Zebu cattle.

East Coast fever (ECF) is a highly fatal lymphoproliferative disease of cattle caused by Theileria parva, a tick-borne intracellular apicomplexan parasite. Parasite antigens that are targets of protective cytotoxic T lymphocyte (CTL) responses are required to formulate a sub-unit vaccine against ECF. A number of CTL target antigens have recently been identified and initial evaluation has shown their vaccine potential. This study aimed to evaluate whether these antigens were recognised by CTL obtained from six genetically diverse Zebu cattle immunized with a cocktail of T. parva stocks. T. parva Muguga specific polyclonal CD8(+) CTL lines were generated and confirmed to specifically lyse autologous infected cells. CTL recognition of autologous skin fibroblasts (iSF) transduced with recombinant modified vaccinia virus Ankara strain (MVA) expressing previously identified T. parva Muguga vaccine candidate antigens was evaluated using an IFN-gamma ELISpot assay. CTL lines from one of the four calves, BY120, responded specifically to cells infected with MVA expressing the antigen Tp2 and synthetic peptides were employed to map a new CTL epitope on this antigen. Immunoscreening of the T. parva genome with these CTL lines should identify novel antigens that will constitute valuable additions to the vaccine candidates currently being evaluated.

A novel strategy for the identification of antigens that are recognised by bovine MHC class I restricted cytotoxic T cells in a protozoan infection using reverse vaccinology.

BACKGROUND: Immunity against the bovine protozoan parasite Theileria parva has previously been shown to be mediated through lysis of parasite-infected cells by MHC class I restricted CD8+ cytotoxic T lymphocytes. It is hypothesized that identification of CTL target schizont antigens will aid the development of a sub-unit vaccine. We exploited the availability of the complete genome sequence data and bioinformatics tools to identify genes encoding secreted or membrane anchored proteins that may be processed and presented by the MHC class I molecules of infected cells to CTL. RESULTS: Of the 986 predicted open reading frames (ORFs) encoded by chromosome 1 of the T. parva genome, 55 were selected based on the presence of a signal peptide and/or a transmembrane helix domain. Thirty six selected ORFs were successfully cloned into a eukaryotic expression vector, transiently transfected into immortalized bovine skin fibroblasts and screened in vitro using T. parva-specific CTL. Recognition of gene products by CTL was assessed using an IFN-gamma ELISpot assay. A 525 base pair ORF encoding a 174 amino acid protein, designated Tp2, was identified by T. parva-specific CTL from 4 animals. These CTL recognized and lysed Tp2 transfected skin fibroblasts and recognized 4 distinct epitopes. Significantly, Tp2 specific CD8+ T cell responses were observed during the protective immune response against sporozoite challenge. CONCLUSION: The identification of an antigen containing multiple CTL epitopes and its apparent immunodominance during a protective anti-parasite response makes Tp2 an attractive candidate for evaluation of its vaccine potential.

Theileria parva candidate vaccine antigens recognized by immune bovine cytotoxic T lymphocytes.

East Coast fever, caused by the tick-borne intracellular apicomplexan parasite Theileria parva, is a highly fatal lymphoproliferative disease of cattle. The pathogenic schizont-induced lymphocyte transformation is a unique cancer-like condition that is reversible with parasite removal. Schizont-infected cell-directed CD8(+) cytotoxic T lymphocytes (CTL) constitute the dominant protective bovine immune response after a single exposure to infection. However, the schizont antigens targeted by T. parva-specific CTL are undefined. Here we show the identification of five candidate vaccine antigens that are the targets of MHC class I-restricted CD8(+) CTL from immune cattle. CD8(+) T cell responses to these antigens were boosted in T. parva-immune cattle resolving a challenge infection and, when used to immunize naïve cattle, induced CTL responses that significantly correlated with survival from a lethal parasite challenge. These data provide a basis for developing a CTL-targeted anti-East Coast fever subunit vaccine. In addition, orthologs of these antigens may be vaccine targets for other apicomplexan parasites.

Local skin reaction (chancre) induced following inoculation of metacyclic trypanosomes in cattle by tsetse flies is dependent on CD4 T lymphocytes.

The first visible response in livestock to the bite of a trypanosome-infected tsetse fly is the formation of a localized skin reaction, also known as a chancre. This is an inflammatory response in the skin associated with swelling and an influx of cells. It is thought to be associated with an acquired immune response to the injected metacyclic trypanosomes. In this study, we examined the role of T lymphocytes in the development of the inflammatory response, by depleting cattle of T cell subpopulations and monitoring the development of chancres. Depletion of CD4 cells, but not CD8 cells, resulted in a significant reduction in chancre formation, confirming that an acquired response mediates the inflammatory response. In addition, it was established that the CD4 T cells mediate the generation of memory for immunity to a homologueous re-challenge. The inflammatory response in the skin did not affect further progress of the infection.

Immunisation of cattle against heartwater by infection with Cowdria ruminantium elicits T lymphocytes that recognise major antigenic proteins 1 and 2 of the agent.

There is growing evidence that immunity of cattle to Cowdria ruminantium infection is mediated by T lymphocytes. C. ruminantium antigens that stimulate these responses are therefore of considerable importance to the development of a sub-unit vaccine against the disease. We have examined T cell responses against recombinant analogues of the surface-exposed C. ruminantium major antigen 1 (MAP1) a 28.8 kDa protein and MAP2 (21 kDa) antigen in cattle immunised by infection and treatment. Vigorous and sustained proliferative responses to both antigens were observed in peripheral blood mononuclear cells from immune cattle. MAP1-specific responses were predominantly restricted to cluster of differentiation four antigen positive T cells (CD4+ T cells). Reverse transcription polymerase chain reaction (RT-PCR) analysis of cytokine expression by T cell lines derived from this population revealed strong expression of interferon gamma (IFN-gamma), interferon alpha (IFN-alpha), tumour necrosis factor alpha (TNF-alpha), tumour necrosis factor beta (TNF-beta), interleukin-2 receptor alpha (IL-2Ralpha) transcripts, and weak expression of IL-2 and IL-4. Supernatants from these T cell cultures contained IFN-gamma protein. CD4+ T cell clones specific for MAP1 were generated. Two of these clones proliferated in the presence of autologous infected endothelial cells. In contrast, the response to MAP2 was characterised largely by proliferation of gamma delta (gammadelta) T cells. RT-PCR analysis of cytokine expression by T cell lines which were dominated by gammadelta T cells revealed expression of IFN-gamma, TNF-alpha, TNF-beta, IL-2Ralpha transcripts. Supernatants of these T cell cultures also contained IFN-gamma protein. Our findings indicate that immunisation of cattle by infection with C. ruminantium results in generation of MAP1- and MAP2-specific T cell responses that may play a role in protection against the pathogen.