An Aptamer-Based Proteomic Analysis of Plasma from Cats (Felis catus) with Clinical Feline Infectious Peritonitis.

Feline infectious peritonitis (FIP) is a systemic disease manifestation of feline coronavirus (FCoV) and is the most important cause of infectious disease-related deaths in domestic cats. FIP has a variable clinical manifestation but is most often characterized by widespread vasculitis with visceral involvement and/or neurological disease that is typically fatal in the absence of antiviral therapy. Using an aptamer-based proteomics assay, we analyzed the plasma protein profiles of cats who were naturally infected with FIP (n = 19) in comparison to the plasma protein profiles of cats who were clinically healthy and negative for FCoV (n = 17) and cats who were positive for the enteric form of FCoV (n = 9). We identified 442 proteins that were significantly differentiable; in total, 219 increased and 223 decreased in FIP plasma versus clinically healthy cat plasma. Pathway enrichment and associated analyses showed that differentiable proteins were related to immune system processes, including the innate immune response, cytokine signaling, and antigen presentation, as well as apoptosis and vascular integrity. The relevance of these findings is discussed in the context of previous studies. While these results have the potential to inform diagnostic, therapeutic, and preventative investigations, they represent only a first step, and will require further validation.

Expression of E. coli FimH Enhances Trafficking of an Orally Delivered Lactobacillus acidophilus Vaccine to Immune Inductive Sites via Antigen-Presenting Cells.

The development of lactic acid bacteria as mucosal vaccine vectors requires the identification of robust mucosal adjuvants to increase vaccine effectiveness. The E. coli type I fimbriae adhesion protein FimH is of interest as a mucosal adjuvant as it targets microfold (M) cells enhancing vaccine uptake into Peyer's patches and can activate the innate immune system via Toll-like receptor (TLR) 4 binding. Here, we displayed the N-terminal domain of FimH on the surface of a Lactobacillus acidophilus vaccine vector and evaluated its ability to increase uptake of L. acidophilus into Peyer's patches and activate innate immune responses. FimH was robustly displayed on the L. acidophilus surface but did not increase uptake into the Peyer's patches. FimH did increase trafficking of L. acidophilus to mesenteric lymph nodes by antigen-presenting cells including macrophages and dendritic cells. It also increased transcription of retinaldehyde dehydrogenase and decreased transcription of IL-21 in the Peyer's patches and mesenteric lymph nodes. The N-terminal domain of FimH did not activate TLR4 in vitro, indicating that FimH may stimulate innate immune responses through a not-yet-identified mechanism. These results indicate that E. coli FimH alters the innate immune response to L. acidophilus and should be further studied as an adjuvant for lactic acid bacterial vaccine platforms.

High-throughput viral microneutralization method for feline coronavirus using image cytometry.

Feline coronaviruses (FCoV) are members of the alphacoronavirus genus that are further characterized by serotype (types I and II) based on the antigenicity of the spike (S) protein and by pathotype based on the associated clinical conditions. Feline enteric coronaviruses (FECV) are associated with the vast majority of infections and are typically asymptomatic. Within individual animals, FECV can mutate and cause a severe and usually fatal disease called feline infectious peritonitis (FIP), the leading infectious cause of death in domestic cat populations. There are no approved antiviral drugs or recommended vaccines to treat or prevent FCoV infection. The plaque reduction neutralization test (PRNT) traditionally employed to assess immune responses and to screen therapeutic and vaccine candidates is time-consuming, low-throughput, and typically requires 2-3 days for the formation and manual counting of cytolytic plaques. Host cells are capable of carrying heavy viral burden in the absence of visible cytolytic effects, thereby reducing the sensitivity of the assay. In addition, operator-to-operator variation can generate uncertainty in the results and digital records are not automatically created. To address these challenges we developed a novel high-throughput viral microneutralization assay, with quantification of virus-infected cells performed in a plate-based image cytometer. Host cell seeding density, microplate surface coating, virus concentration and incubation time, wash buffer and fluorescent labeling were optimized. Subsequently, this FCoV viral neutralization assay was used to explore immune correlates of protection using plasma from naturally FECV-infected cats. We demonstrate that the high-throughput viral neutralization assay using the Celigo Image Cytometer provides a robust and efficient method for the rapid screening of therapeutic antibodies, antiviral compounds, and vaccines. This method can be applied to various viral infectious diseases to accelerate vaccine and antiviral drug discovery and development.

Impact of oral probiotic Lactobacillus acidophilus vaccine strains on the immune response and gut microbiome of mice.

The potential role of probiotic bacteria as adjuvants in vaccine trials led to their use as nonparenteral live mucosal vaccine vectors. Yet, interactions between these vectors, the host and the microbiome are poorly understood. This study evaluates impact of three probiotic, Lactobacillus acidophilus, vector strains, and their interactions with the host's immune response, on the gut microbiome. One strain expressed the membrane proximal external region from HIV-1 (MPER). The other two expressed MPER and either secreted interleukin-1ß (IL-1ß) or expressed the surface flagellin subunit C (FliC) as adjuvants. We also used MPER with rice bran as prebiotic supplement. We observed a strain dependent, differential effect suggesting that MPER and IL-1ß induced a shift of the microbiome while FliC had minimal impact. Joint probiotic and prebiotic use resulted in a compound effect, highlighting a potential synbiotic approach to impact efficacy of vaccination. Careful consideration of constitutive adjuvants and use of prebiotics is needed depending on whether or not to target microbiome modulation to improve vaccine efficacy. No clear associations were observed between total or MPER-specific IgA and the microbiome suggesting a role for other immune mechanisms or a need to focus on IgA-bound, resident microbiota, most affected by an immune response.

Effects of Regulatory T Cell Depletion on NK Cell Responses against Listeria monocytogenes in Feline Immunodeficiency Virus Infected Cats.

Regulatory T cells (Treg) are key players in the maintenance of peripheral tolerance, preventing autoimmune diseases and restraining chronic inflammatory diseases. Evidence suggests Treg cells and NK cells have important roles in feline immunodeficiency virus (FIV) pathogenesis; however, in vivo studies investigating the interplay between these two cell populations are lacking. We previously described innate immune defects in FIV-infected cats characterized by cytokine deficits and impaired natural killer cell (NK) and NK T cell (NKT) functions. In this study, we investigated whether in vivo Treg depletion by treatment with an anti-feline CD25 monoclonal antibody would improve the innate immune response against subcutaneous challenge with Listeria monocytogenes (Lm). Treg depletion resulted in an increased overall number of cells in Lm-draining lymph nodes and increased proliferation of NK and NKT cells in FIV-infected cats. Treg depletion did not normalize expression of perforin or granzyme A by NK and NKT cells, nor did Treg depletion result in improved clearance of Lm. Thus, despite the quantitative improvements in the NK and NKT cell responses to Lm, there was no functional improvement in the early control of Lm. CD1a+ dendritic cell percentages in the lymph nodes of FIV-infected cats were lower than in specific-pathogen-free control cats and failed to upregulate CD80 even when Treg were depleted. Taken together, Treg depletion failed to improve the innate immune response of FIV-infected cats against Lm and this may be due to dendritic cell dysfunction.

Mucosal Immune Response to Feline Enteric Coronavirus Infection.

Feline infectious peritonitis is a devastating, fatal disease of domestic cats caused by a pathogenic mutant virus derived from the ubiquitous feline enteric coronavirus (FECV). Infection by FECV is generally subclinical, and little is known about the mucosal immune response that controls and eliminates the virus. We investigated the mucosal immune response against FECV in an endemically infected breeding colony over a seven-month period. Thirty-three cats were grouped according to FECV seropositivity and fecal virus shedding into naïve/immunologically quiescent, convalescent and actively infected groups. Blood, fecal samples and colon biopsies were collected to assess the mucosal and systemic immunologic and virologic profile. Results showed that cats with active FECV infections have strong systemic IgG and mucosal IgA responses that wane after virus clearance. Significant FECV-specific mucosal T cell IFNγ responses were not detected in any of the three groups. A shift toward an inflammatory state in the mucosa was suggested by increased IL17:FoxP3 expression. However, no histologic abnormalities were observed, and no shifts in lymphocyte subpopulation phenotype or proliferation were noted. Together, the results suggest that control of FECV is mediated by humoral mucosal and systemic responses and that perturbations in the primary reservoir organ (colon) are minimal.

Nod2 is required for antigen-specific humoral responses against antigens orally delivered using a recombinant Lactobacillus vaccine platform.

Safe and efficacious orally-delivered mucosal vaccine platforms are desperately needed to combat the plethora of mucosally transmitted pathogens. Lactobacillus spp. have emerged as attractive candidates to meet this need and are known to activate the host innate immune response in a species- and strain-specific manner. For selected bacterial isolates and mutants, we investigated the role of key innate immune pathways required for induction of innate and subsequent adaptive immune responses. Co-culture of murine macrophages with L. gasseri (strain NCK1785), L. acidophilus (strain NCFM), or NCFM-derived mutants-NCK2025 and NCK2031-elicited an M2b-like phenotype associated with TH2 skewing and immune regulatory function. For NCFM, this M2b phenotype was dependent on expression of lipoteichoic acid and S layer proteins. Through the use of macrophage genetic knockouts, we identified Toll-like receptor 2 (TLR2), the cytosolic nucleotide-binding oligomerization domain containing 2 (NOD2) receptor, and the inflammasome-associated caspase-1 as contributors to macrophage activation, with NOD2 cooperating with caspase-1 to induce inflammasome derived interleukin (IL)-1ß in a pyroptosis-independent fashion. Finally, utilizing an NCFM-based mucosal vaccine platform with surface expression of human immunodeficiency virus type 1 (HIV-1) Gag or membrane proximal external region (MPER), we demonstrated that NOD2 signaling is required for antigen-specific mucosal and systemic humoral responses. We show that lactobacilli differentially utilize innate immune pathways and highlight NOD2 as a key mediator of macrophage function and antigen-specific humoral responses to a Lactobacillus acidophilus mucosal vaccine platform.

Mucosal Immunogenicity of Genetically Modified Lactobacillus acidophilus Expressing an HIV-1 Epitope within the Surface Layer Protein.

Surface layer proteins of probiotic lactobacilli are theoretically efficient epitope-displaying scaffolds for oral vaccine delivery due to their high expression levels and surface localization. In this study, we constructed genetically modified Lactobacillus acidophilus strains expressing the membrane proximal external region (MPER) from human immunodeficiency virus type 1 (HIV-1) within the context of the major S-layer protein, SlpA. Intragastric immunization of mice with the recombinants induced MPER-specific and S-layer protein-specific antibodies in serum and mucosal secretions. Moreover, analysis of systemic SlpA-specific cytokines revealed that the responses appeared to be Th1 and Th17 dominant. These findings demonstrated the potential use of the Lactobacillus S-layer protein for development of oral vaccines targeting specific peptides.

Construction and immunological evaluation of dual cell surface display of HIV-1 gag and Salmonella enterica serovar Typhimurium FliC in Lactobacillus acidophilus for vaccine delivery.

Oral vaccines that elicit a mucosal immune response may be effective against human immunodeficiency virus type 1 (HIV-1) because its transmission occurs mainly at the mucosa. The aim of this study was to construct recombinant Lactobacillus for oral delivery of oral vaccines against HIV-1 and to evaluate their immunogenicity. A recombinant Lactobacillus acidophilus strain expressing the HIV-1 Gag on the bacterial cell surface was established by fusion with the signal peptide and anchor motif of a mucus binding protein (Mub) from L. acidophilus with or without coexpression of Salmonella enterica serovar Typhimurium flagellin (FliC) fused to a different Mub signal peptide and anchor. Using HEK293 cells engineered to express Toll-like receptor 5 (TLR5), the biological activity of FliC on the bacterial cell surfaces was determined. The surface-exposed flagellin retained its TLR5-stimulating activity, suggesting that the recombinant strain with Gag and FliC dual display might provide a different immunopotency than the strain expressing only Gag. The immunological properties of the recombinant strains were assessed by coculture with human myeloid dendritic cells (DCs). The heterologous antigens on the cell surface affected maturation and cytokine responses of DCs. Acquired immune responses were also investigated by intragastric immunization of mice. The enzyme-linked immunosorbent spot assay showed induction of gamma interferon-producing cells at local mucosa after immunization of mice with the Gag-producing strain. Meanwhile, the immunization with L. acidophilus displaying both Gag and FliC resulted in an increase of Gag-specific IgA-secreting cells. These results suggested that the Gag-displaying L. acidophilus elicited specific immune responses and the coexistence of FliC conferred an adjuvant effect on local IgA production.

Assessment of Lactobacillus gasseri as a candidate oral vaccine vector.

Lactobacillus species are commensal bacteria that have long been recognized as probiotic microbes and are generally regarded as safe (GRAS) for human consumption. We have investigated the use of L. gasseri as a vaccine vector for oral immunization against mucosal pathogens. Recent research has shown that the immune response to different lactobacilli can vary widely depending on the species or subspecies of Lactobacillus being studied. While some lactobacilli seem to induce oral tolerance, others induce an adaptive immune response. This study characterized the systemic and mucosal immune response to wild-type and genetically modified L. gasseri. L. gasseri primarily activates TLR2/6, with additional activation through the TLR2 homodimer. To expand the Toll-like receptor (TLR) activation profile of L. gasseri and the immunogenicity of the vector, a plasmid containing fliC, the gene encoding bacterial flagellin, was introduced which resulted in the strong activation of TLR5. The treatment of human myeloid dendritic cells with recombinant lactobacilli expressing flagellin triggered phenotypic maturation and the release of proinflammatory cytokines. In contrast, bacterial treatment also resulted in a statistically significant increase in IL-10 production. In vivo studies established that treatment with L. gasseri led to a diversification of B-cell populations in the lamina propria of the murine colon. Furthermore, treatment with genetically modified L. gasseri led to a significant decrease in the percentage of FoxP3(+) colonic lymphocytes. Taken together, these data clarify the interaction of L. gasseri with the host immune system and support further investigation of the in vivo immunogenicity of L. gasseri expressing both flagellin and candidate vaccine antigens.

Dissimilar properties of two recombinant Lactobacillus acidophilus strains displaying Salmonella FliC with different anchoring motifs.

Display of heterologous antigens on the cell surface is considered a useful technique for vaccine delivery by recombinant lactobacilli. In this study, two recombinant Lactobacillus acidophilus derivatives displaying Salmonella flagellin (FliC) were constructed using different anchor motifs. In one instance, the FliC protein was fused to the C-terminal region of a cell envelope proteinase (PrtP) and was bound to the cell wall by electrostatic bonds. In the other case, the same antigen was conjugated to the anchor region of mucus binding protein (Mub) and was covalently associated with the cell wall by an LPXTG motif. These two recombinant L. acidophilus cell surface displays resulted in dissimilar maturation and cytokine production by human myeloid dendritic cells. The surface-associated antigen was highly sensitive to simulated gastric and small intestinal juices. By supplementation with bicarbonate buffer and soybean trypsin inhibitor, the cell surface antigen was protected from proteolytic enzymes during gastric challenge in vitro. The protective reagents also increased the viability of the L. acidophilus cells upon challenge with simulated digestive juices. These results demonstrate the importance of protecting cells and their surface-associated antigens during oral immunization.

Cloning of feline FOXP3 and detection of expression in CD4+CD25+ regulatory T cells.

Regulatory T cells (Treg) are increased and directly infected by feline immunodeficiency virus (FIV) and likely play a role in other feline autoimmune, neoplastic, and infectious diseases. Phenotypically, Treg are best characterized by surface expression of CD4 and CD25 and intranuclear expression of the forkhead transcription factor Foxp3. Our objective was to clone and sequence feline FOXP3 for the purpose of developing assays to enhance studies of feline Treg. We determined the feline FOXP3 is 1293 nucleotides in length and codes for a protein that shares high homology to other species. A splice variant devoid of exon 2 was also identified. A real-time PCR assay was developed and used to show Foxp3 mRNA expression occurs primarily in CD4+CD25+ T cells. Two cross-reacting antibodies were identified by immunocytochemical staining of HEK293 cells transfected with feline FOXP3. The antibody labeling confirmed the nuclear localization of the protein. A flow cytometric assay was also validated and used to correlate the phenotypic and functional characteristics of feline Treg induced by treatment of lymph node lymphocytes with flagellin or LPS in combination with mitogen or IL2. Together, these studies provide useful tools to further investigate Foxp3 and Tregs in cats.

Cytokine modulation of the innate immune response in feline immunodeficiency virus-infected cats.

BACKGROUND: In vitro data suggest that innate immune function in human immunodeficiency virus type 1-infected patients is compromised; however, in vivo studies are lacking. Feline immunodeficiency virus (FIV) infection in cats provides an excellent model to explore innate immune function in vivo. The innate response against Listeria monocytogenes is well understood, making it a useful immune probe. METHODS: Recombinant L. monocytogenes carrying eukaryotic expression plasmids for feline tumor necrosis factor (TNF)- alpha , interleukin (IL)-10, interferon (IFN)- gamma , and IL-15 were created to determine whether specific cytokines would modulate innate immune function. L. monocytogenes was delivered subcutaneously, and local lymph nodes were evaluated for size, cell subpopulations, and L. monocytogenes burden. Two months later, memory responses were evaluated by IFN- gamma enzyme-linked immunospot assay. RESULTS: FIV-positive cats had significantly less lymph-node enlargement and a greater L. monocytogenes burden than FIV-negative control cats. TNF- alpha improved listericidal activity in FIV-negative control cats but not in FIV-positive cats, whereas IL-10 modestly reduced function in FIV-negative control cats. IFN- gamma improved memory responses but not clearance of L. monocytogenes. IL-15 improved innate function in FIV-positive cats and increased the percentage of natural killer cells. CONCLUSIONS: Lentivirus infection impairs innate immune function in vivo, and IL-15 can significantly restore function. We hypothesize that altered dendritic-cell function and increased regulatory T cell activity may underlie the innate immune defect in HIV infection.

Desmoglein-1 is a minor autoantigen in dogs with pemphigus foliaceus.

The majority of human patients with pemphigus foliaceus (PF) have circulating IgG autoantibodies that target conformational epitopes on the desmosomal cadherin desmoglein-1 (dsg1). Limited studies using immunoblot techniques suggested that the principal autoantigen in dogs with PF might also be dsg1. It was the objective of this study to test this hypothesis. A comprehensive survey of canine PF sera was conducted using a novel screening strategy that detects conformational epitopes. This method consists of the ectopic expression of canine dsg1 at the surface of human 293T epithelial kidney cells and their live screening, i.e. prior to fixation. Out of seven control human PF sera that bound to canine epidermis, three (57%) contained IgG autoantibodies that recognized ectopically expressed canine dsg1 with a membrane and punctate pattern. Out of 83 canine PF sera only five (6%) contained IgG that recognized canine dsg1. Consistent with findings for human PF sera obtained in this study, autoantibody binding was conformation- and glycosylation-dependent as demonstrated by calcium chelation with EDTA and tunicamycin or wheat germ agglutinin treatment, respectively. In conclusion, these studies establish canine dsg1 as a minor autoantigen for canine PF. Antigenic epitopes appear to be conformation- and glycosylation-dependent.

Feline cytokine ELISPOT: issues in assay development.

The enzyme-linked immunospot (ELISPOT) assay is a sensitive and relatively simple assay for detecting secreted cellular products such as cytokines and has become an invaluable immunological tool. The ELISPOT has been used extensively in human and murine research but has only recently been used to assess the feline immune system. For researchers studying feline disease or using the cat as a model of human disease, the quantification of cytokine-producing cells by ELISPOT is an invaluable technique for investigations of disease immunopathogenesis and vaccine efficacy. For example, use of the interferon (IFN)-gamma ELISPOT to measure the frequency of antigen-specific T-cells during feline immunodeficiency virus (FIV) infection or after immunization with candidate FIV vaccines is of particular interest. This application of the ELISPOT may serve to expand the utility of FIV as a model for human immunodeficiency virus. Broader applications of the ELISPOT should further our understanding of feline diseases and be useful in the rational development of more efficacious vaccines and therapeutic modalities for the enhancement of feline health. This chapter discusses important parameters of ELISPOT design that will enable researchers to develop and analyze the feline-specific assays within their own laboratory.

Pre-existing immunity to pathogenic Listeria monocytogenes does not prevent induction of immune responses to feline immunodeficiency virus by a novel recombinant Listeria monocytogenes vaccine.

Listeria monocytogenes is an attractive biologic vaccine vector against HIV because it induces a strong cell mediated immune response, can be delivered by mucosal routes, can be readily manipulated to express viral antigens, and is easy and inexpensive to produce. Proof of concept studies have been performed using HIV Gag expressing recombinant L. monocytogenes in the mouse. Here we report the development and validation of recombinant L. monocytogenes to be evaluated in the FIV/cat model of HIV. Using a simplified approach to introduce individual and polyprotein FIV gag genes, we show that recombinant L. monocytogenes containing the entire gag expresses the full-length Gag polyprotein in a soluble secreted form. A DNA vaccine plasmid (pND14-Lc-env) that replicates in Gram positive bacteria and contains the FIV SU (gp100) and the ectodomain of TM (gp40) in a eukaryotic expression cassette was transfected into LM-gag to create LM-gag/pND14-Lc-env. After infection of target cells with LM-gag/pND14-Lc-env in vitro, both FIV Gag and Env proteins were detected in soluble cell lysates. Whether previous exposure to L. monocytogenes affects the immunogenicity of LM-gag/pND14-Lc-env was determined in cats infected with wild-type L. monocytogenes orally and/or subcutaneously. After a single oral dose of LM-gag/pND14-Lc-env, cats with existing anti-L. monocytogenes immune responses developed anti-FIV Gag IgA titers in vaginal secretions, saliva, and feces. Similarly, FIV Gag and Env specific IFN-gamma ELISPOT responses were measurable in spleen and lymph node but at a statistically higher frequency in cats exposed to a single subcutaneous dose of wild-type L. monocytogenes versus cats exposed both subcutaneously and orally. The FIV/cat model will provide a useful challenge system to determine whether recombinant L. monocytogenes can protect against a lentivirus in its natural host after challenge by the routes common to HIV transmission.

Cloning and expression of feline interleukin 15.

A cDNA encoding feline interleukin 15 (IL15) was cloned from the lymph node of a cat infected with feline infectious peritonitis virus. The cDNA is 486 bp in length and encodes a protein of 162 amino acids. Recombinant protein was readily expressed as a GST fusion in Escherichia coli and purified by glutathione affinity chromatography. Expression of recombinant protein in mammalian cells was only accomplished by eliminating the 5' and 3' UTR, replacing the IL15 signal peptide with the tissue plasminogen activator signal peptide, and adding 3' sequence to disrupt presumptive secondary structure of the mRNA. Biologically active feline IL15 was expressed in HEK293T cells and was shown to sustain primary feline lymphocytes, a feline T cell line, and mouse CTLL-2 cells. Proliferation of CTLL-2 cells was induced by the recombinant protein in a dose-dependent manner. Monoclonal and polyclonal antibodies against human IL15 recognized feline IL15 in immunofluorescence and Western blot assays. Additionally, feline IL15 was detectable using a commercially available human IL15 ELISA kit.

Assessment of CD4+ and CD8+ IFN-gamma producing cells by ELISPOT in naïve and FIV-infected cats.

IFN-gamma is critical for the development of antiviral cell-mediated immunity in HIV infected humans and FIV infected cats. The ELISPOT has proven to be a technically straightforward assay to quantify the number of IFN-gamma producing cells and offers a reasonable alternative for the quantitative measurement of T-cell function in cats. We used a feline-specific ELISPOT to identify constitutive as well as Con A stimulated IFN-gamma production in T-cell subsets and determine if there were differences between purified (positively sorted) and negatively depleted populations from naïve and FIV infected cats. We found no difference in the total number of PBMC constitutively producing IFN-gamma in naïve and FIV+ cats. Con A exposure was associated with increased numbers of IFN-gamma producing PBMC in naïve, but not FIV+, cats. Equivalent numbers of CD4+ and CD8+ T cells constitutively expressed IFN-gamma in naïve cats. However, in FIV+ cats, the number of IFN-gamma producing CD8+ T-cells was approximately two-fold over that seen for CD4+ T-cells. We found minimal differences between purified (e.g. CD4+ or CD8+) and corresponding depleted (e.g. CD8- or CD4-) populations in samples from FIV+ cats. In contrast, depleted populations from naïve cats showed greater response to Con A than did purified populations. Thus, while determination of the number of IFN-gamma producing cells by feline-specific ELISPOT is a useful tool for the evaluation of the feline immune response, determination of the initial sample population and T-cell subset is critical for optimal interpretation of the IFN-gamma ELISPOT.

Peptide mapping of feline immunodeficiency virus by IFN-gamma ELISPOT.

Interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) has become an important tool in studying antigen-specific T lymphocyte responses. Soluble peptides can be used to map T-cell epitopes, providing information that is useful in the design and evaluation of vaccines as well as studies of immunopathogenesis. To date, this assay has not been widely utilized in feline immunodeficiency virus (FIV) research. We have developed a feline IFN-gamma ELISPOT assay and used it to determine FIV-specific T-cell epitopes recognized by infected cats. A panel of 331 peptides, 15 amino acids in length and overlapping by 10 residues was synthesized. The peptide library spanned the FIV structural (Gag), envelope (Env), reverse transcriptase (RT), and open-reading-frame A (OrfA) proteins. Initially, 34 pools, containing 7-10 peptides each were screened by IFN-gamma ELISPOT against peripheral blood mononuclear cells (PBMC) from eight cats chronically infected with the NCSU(1) molecular clone of FIV and four uninfected control cats. Individual peptides from pools recognized by FIV+ cats were then evaluated and optimal peptides were combined into pools representing Gag, Env, RT, and OrfA. A higher percentage of FIV infected cats were identified as responders against the peptide pools when using fresh PBMC as compared to cryopreserved PBMC. In vitro restimulation of cryopreserved PBMC with the peptide pools improved the sensitivity of the assay to similar levels as observed from fresh samples. Individual peptides used in the pools were generally found to stimulate CD8+ T-cells more efficiently than CD4+ T-cells. Comparison of the peptide sequences to representative FIV sequences from clades A-D showed conservation was high among Gag and RT peptides, variable among Env peptides and low for OrfA peptides. The IFN-gamma ELISPOT assay and FIV-specific peptide pools we describe here will be useful in assessing cell-mediated responses to experimental FIV vaccines.