Calibration and Validation of a Mechanistic COVID-19 Model for Translational Quantitative Systems Pharmacology - A Proof-of-Concept Model Development for Remdesivir.

With the ongoing global pandemic of coronavirus disease 2019 (COVID-19), there is an urgent need to accelerate the traditional drug development process. Many studies identified potential COVID-19 therapies based on promising nonclinical data. However, the poor translatability from nonclinical to clinical settings has led to failures of many of these drug candidates in the clinical phase. In this study, we propose a mechanism-based, quantitative framework to translate nonclinical findings to clinical outcome. Adopting a modularized approach, this framework includes an in silico disease model for COVID-19 (virus infection and human immune responses) and a pharmacological component for COVID-19 therapies. The disease model was able to reproduce important longitudinal clinical data for patients with mild and severe COVID-19, including viral titer, key immunological cytokines, antibody responses, and time courses of lymphopenia. Using remdesivir as a proof-of-concept example of model development for the pharmacological component, we developed a pharmacological model that describes the conversion of intravenously administered remdesivir as a prodrug to its active metabolite nucleoside triphosphate through intracellular metabolism and connected it to the COVID-19 disease model. After being calibrated with the placebo arm data, our model was independently and quantitatively able to predict the primary endpoint (time to recovery) of the remdesivir clinical study, Adaptive Covid-19 Clinical Trial (ACTT). Our work demonstrates the possibility of quantitatively predicting clinical outcome based on nonclinical data and mechanistic understanding of the disease and provides a modularized framework to aid in candidate drug selection and clinical trial design for COVID-19 therapeutics.

New science, drug regulation, and emergent public health issues: The work of FDA's division of applied regulatory science.

The U.S. Food and Drug Administration (FDA) Division of Applied Regulatory Science (DARS) moves new science into the drug review process and addresses emergent regulatory and public health questions for the Agency. By forming interdisciplinary teams, DARS conducts mission-critical research to provide answers to scientific questions and solutions to regulatory challenges. Staffed by experts across the translational research spectrum, DARS forms synergies by pulling together scientists and experts from diverse backgrounds to collaborate in tackling some of the most complex challenges facing FDA. This includes (but is not limited to) assessing the systemic absorption of sunscreens, evaluating whether certain drugs can convert to carcinogens in people, studying drug interactions with opioids, optimizing opioid antagonist dosing in community settings, removing barriers to biosimilar and generic drug development, and advancing therapeutic development for rare diseases. FDA tasks DARS with wide ranging issues that encompass regulatory science; DARS, in turn, helps the Agency solve these challenges. The impact of DARS research is felt by patients, the pharmaceutical industry, and fellow regulators. This article reviews applied research projects and initiatives led by DARS and conducts a deeper dive into select examples illustrating the impactful work of the Division.

Innovations, challenges, and minimal information for standardization of humanized mice.

Mice xenotransplanted with human cells and/or expressing human gene products (also known as "humanized mice") recapitulate the human evolutionary specialization and diversity of genotypic and phenotypic traits. These models can provide a relevant in vivo context for understanding of human-specific physiology and pathologies. Humanized mice have advanced toward mainstream preclinical models and are now at the forefront of biomedical research. Here, we considered innovations and challenges regarding the reconstitution of human immunity and human tissues, modeling of human infections and cancer, and the use of humanized mice for testing drugs or regenerative therapy products. As the number of publications exploring different facets of humanized mouse models has steadily increased in past years, it is becoming evident that standardized reporting is needed in the field. Therefore, an international community-driven resource called "Minimal Information for Standardization of Humanized Mice" (MISHUM) has been created for the purpose of enhancing rigor and reproducibility of studies in the field. Within MISHUM, we propose comprehensive guidelines for reporting critical information generated using humanized mice.

The ELISA Detectability and Potency of Pegfilgrastim Decrease in Physiological Conditions: Key Roles for Aggregation and Individual Variability.

PEGylated recombinant human granulocyte colony stimulating factor (pegfilgrastim) is used clinically to accelerate immune reconstitution following chemotherapy and is being pursued for biosimilar development. One challenge to overcome in pegfilgrastim biosimilar development is establishing pharmacokinetic (PK) similarity, which is partly due to the degree of PK variability. We herein report that commercially available G-CSF and PEG ELISA detection kits have different capacities to detect pegfilgrastim aggregates that rapidly form in vitro in physiological conditions. These aggregates can be observed using SDS-PAGE, size-exclusion chromatography, dynamic light scattering, and real-time NMR analysis and are associated with decreased bioactivity as reflected by reduced drug-induced cellular proliferation and STAT3 phosphorylation. Furthermore, individual variability in the stability and detectability of pegfilgrastim in human sera is also observed. Pegfilgrastim levels display marked subject variability in sera from healthy donors incubated at 37 °C. The stability patterns of pegfilgrastim closely match the stability patterns of filgrastim, consistent with a key role for pegfilgrastim's G-CSF moiety in driving formation of inactive aggregates. Taken together, our results indicate that individual variability and ELISA specificity for inactive aggregates are key factors to consider when designing and interpreting studies involving the measurement of serum pegfilgrastim concentrations.

Bone marrow-liver-thymus (BLT) immune humanized mice as a model to predict cytokine release syndrome.

Cytokine release syndrome (CRS) is a serious and potentially life-threatening complication that can be associated with biological drug products. In vitro assays or in vivo tests using nonhuman primates may fail to predict CRS due to species differences and the complexity of immune system. Therefore, model species that have human-specific immune components may improve the ability to identify CRS and enhance product safety. In this study we used bone marrow-liver-thymus (BLT) humanized mice to test muromonab (OKT3), an anti-CD3 antibody with a black box warning for CRS. Initially, we completed pilot and dose escalation studies with muromonab and showed that when the dose was increased sufficiently, BLT-humanized mice experienced serious adverse outcomes including moribundity. Full studies compared muromonab treatment with adalimumab, saline, and a group pretreated with methylprednisolone prior to muromonab. We evaluated immune cell activation using flow cytometry and cytokine expression using a custom 10-plex cytokine assay to assess levels of human TNF-α, IFN-γ, IL-2, IL-6, IL-8, IL-10, IL-13, IL-17A, IL12/23p40, and GM-CSF. Muromonab treated mice had significant increases in all cytokines tested with T-cell depletion and T-cell activation noted. Adalimumab (active) and saline (inactive) control groups did not demonstrate cytokine expression changes or alterations in T-cell numbers or activation. Further, pretreatment with methylprednisolone blunted or abrogated cytokine increases. This study demonstrates that BLT-humanized mice are capable of experiencing CRS, and could be used to screen biologics for this adverse event to enhance patient safety.

BLT-Immune Humanized Mice as a Model for Nivolumab-Induced Immune-Mediated Adverse Events: Comparison of the NOG and NOG-EXL Strains.

Checkpoint inhibitors represent a new class of therapeutics in the treatment of cancer that has demonstrated remarkable clinical effectiveness. However, some patients have experienced serious immune-mediated adverse effects including pneumonitis, hepatitis, colitis, nephritis, dermatitis, encephalitis, and adrenal or pituitary insufficiency. These adverse events were not predicted by nonclinical studies. To determine if bone marrow-liver-thymus (BLT) immune humanized mice could demonstrate these adverse effects, we studied the effect of nivolumab on 2 strains of BLT-humanized mice, NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac (NOG) and NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(SV40/HTLV-IL3, CSF2)10-7Jic/JicTac (NOG-EXL). Mice were treated with 2.5, 5.0, or 10.0 mg/kg nivolumab or saline twice weekly for 28 days. BLT-NOG mice had significantly reduced survival compared with BLT-NOG-EXL mice. In spite of the difference in survival, both BLT-humanized strains showed adverse reactions similar to those reported in humans, including pneumonitis and hepatitis, with nephritis, dermatitis and adrenalitis also noted in some individuals. Additional histopathologic findings included pancreatic atrophy, myositis, and osteomyelitis in some animals. T-cell activation increased with concomitant loss of PD-1 detection. These findings show that BLT immune humanized mice can demonstrate immune-mediated adverse effects of antiPD1 therapy, and may represent a model that can be used to better understand toxicity of this class of drugs.

Evaluation of a TGN1412 analogue using in vitro assays and two immune humanized mouse models.

Cytokine release syndrome (CRS) is a serious and potentially life-threatening complication typically associated with biological drug products. Pre-clinical testing in vitro and in vivo studies using non-human primates had failed to reliably predict CRS. To determine if bone marrow-thymus-liver (BLT) humanized mice with a fully engrafted human immune system or a CD34-humanized mouse model could predict CRS, we tested an anti-CD28 monoclonal antibody (mAb) similar to TGN1412. This TGN1412 analogue (TGN1412A) was initially tested in vitro and found to produce significant dose-dependent increases in cytokine production. For in vivo studies, adalimumab, an anti-tumor necrosis factor-alpha antibody known not to cause CRS, served as a negative control. We evaluated immune cell activation and cytokine expression in three independent experiments. In BLT humanized mice, significant increases in levels of human cytokines were identified in animals treated with anti-CD28 mAb. As expected, CD28+ cell detection was strongly reduced in the anti-CD28 treated group. Increased T cell activation was also observed. The control group did not show reductions in CD28+ T-cells and did not experience increased cytokine levels. Responses by CD34-humanized mice showed no significant differences between adalimumab and anti-CD28 treatment at doses used to test BLT-humanized mice. These results suggest that the TGN1412A produces similar results in vitro to the original TGN1412 monoclonal antibody. The BLT immune humanized mice but not the CD34 humanized mice produce both robust and specific cytokine responses and may represent a pre-clinical model to identify CRS.

Evaluation of the Ability of Immune Humanized Mice to Demonstrate CD20-Specific Cytotoxicity Induced by Ofatumumab.

CD20 monoclonal antibodies are well-established therapeutics for the treatment of B-cell malignancies. Several mechanisms of target cell killing occur from anti-CD20 therapy, including complement-dependent cytotoxicity (CDC) cell lysis and antibody-dependent cell-mediated cytotoxicity. Human Fc receptors (FcRs) are required to mediate these functions and are either not present or not cross-reactive in mice and most animal species. In contrast, some nonhuman primates have cross-reactive FcR; however, their cellular expression and function may differ from humans. Therefore, we tested bone marrow-liver-thymus (BLT) humanized mice to determine if they could recapitulate the pharmacokinetics (PKs), pharmacodynamics, and potential toxicities of ofatumumab, for which CDC is the predominant mechanism of action. Ofatumumab-treated BLT mice depleted B cells in a dose-dependent manner in all tissues sampled and recapitulated the PKs observed in humans, suggesting that BLT mice can mediate the CDC effector mechanism associated with biological drug products.

Outbreak of Opportunistic Ascending Pyelonephritis with Numerous Yeast after Experimental Humanization Surgery in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ and NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ Immunodeficient Mice.

Unexpected mortality occurred in a group of 12 NOD.Cg-NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) and 12 NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ (NRG) immunodeficient mice. At 10 d after routine bone marrow-liver-thymus humanization surgery, 9 mice were found dead without observation of initiating clinical signs; 1 d later (day 11), 3 additional mice showed signs of morbidity, including severe hunching, lateral recumbency, slow movement, shallow respiration, and decreased response to external stimulus. All remaining mice rapidly decompensated and were found dead or were euthanized within 4 d after the first death. Histopathology revealed severe ascending pyelonephritis with numerous yeast. Cultures in some mice were positive for Enterococcus faecalis or Staphylococcus xylosus, 2 bacteria considered commensals in rodents. In addition, Candida albicans was cultured from some animals. Further investigation revealed that a restraining device used for tail vein injections was the likely fomite harboring Candida organisms. These findings indicate that ascending pyelonephritis, with Candida as the etiologic agent, can cause significant mortality in NSG and NRG immunodeficient mice.

Evaluating the potential of gold, silver, and silica nanoparticles to saturate mononuclear phagocytic system tissues under repeat dosing conditions.

BACKGROUND: As nanoparticles (NPs) become more prevalent in the pharmaceutical industry, questions have arisen from both industry and regulatory stakeholders about the long term effects of these materials. This study was designed to evaluate whether gold (10 nm), silver (50 nm), or silica (10 nm) nanoparticles administered intravenously to mice for up to 8 weeks at doses known to be sub-toxic (non-toxic at single acute or repeat dosing levels) and clinically relevant could produce significant bioaccumulation in liver and spleen macrophages. RESULTS: Repeated dosing with gold, silver, and silica nanoparticles did not saturate bioaccumulation in liver or spleen macrophages. While no toxicity was observed with gold and silver nanoparticles throughout the 8 week experiment, some effects including histopathological and serum chemistry changes were observed with silica nanoparticles starting at week 3. No major changes in the splenocyte population were observed during the study for any of the nanoparticles tested. CONCLUSIONS: The clinical impact of these changes is unclear but suggests that the mononuclear phagocytic system is able to handle repeated doses of nanoparticles.

Nonclinical evaluation of the potential for mast cell activation by an erythropoietin analog.

The erythropoietin analog peginesatide was withdrawn from marketing due to unexpected severe anaphylactic reactions associated with administration of the multi-use formulation. The adverse events occurred rapidly following the first ever administration of the drug with most affected patients becoming symptomatic in less than 30min. This is most consistent with an anaphylactoid reaction due to direct activation of mast cells. Laboratory evaluation was undertaken using rat peritoneal mast cells as the model system. Initial studies showed that high concentrations of the formulated drug as well as formulated vehicle alone could cause mast cell degranulation as measured by histamine release. The purified active drug was not able to cause histamine release whereas the vehicle filtrate and lab created drug vehicle were equally potent at causing histamine release. Individual formulations of vehicle leaving one component out showed that histamine release was due to phenol. Dose response studies with phenol showed a very sharp dose response curve that was similar in three buffer systems. Cellular analysis by flow cytometry showed that the histamine release was not due to cell death, and that changes in light scatter parameters consistent with degranulation were rapidly observed. Limited testing with primary human mast cells showed a similar dose response of histamine release with exposure to phenol. To provide in vivo confirmation, rats were injected with vehicle formulated with various concentrations of phenol via a jugular vein cannula. Significant release of histamine was detected in blood samples taken 2min after dosing at the highest concentrations tested.

Development and validation of a liquid-chromatography tandem mass spectrometry method to determine in vitro and in vivo histamine release.

Histamine is an important biogenic amine involved in regulating numerous physiological and pathophysiological processes in humans and animals. To date, there have been very few studies focused on developing and validating sensitive liquid-chromatography-tandem mass spectrometric (LC-MS/MS) assays capable of quantitative trace level histamine analysis in biological matrices. In the present study, a rapid and sensitive LC-MS/MS assay, amenable to high throughput analysis was developed and validated to characterize in vitro and in vivo histamine release. The LC-MS/MS procedure incorporating deuterium labeled internal standards provides rapid resolution of histamine with excellent sensitivity, precision, and accuracy. Histamine eluted at 1.5 min and was well separated from endogenous plasma peaks. The total run time of the assay was 8.0 min. A linear (r(2) ≥ 0.99) instrument response over the entire concentration range of 1.0-1000 ng/mL was observed. Excellent accuracy (error ± 3.4%) and precision (CV ± 10%) of the assay was demonstrated, with the lower limit of quantitation (LLOQ) at 15.6 ng/mL. The validated LC-MS/MS assay was applied to determine histamine release in both in vitro and in vivo models. Peritoneal mast cells treated with prototypical degranulating agents (Compound 48/80 and Teicoplanin) showed that the two chemicals caused approximately 40% histamine release. In rats, using this assay, basal histamine plasma levels were typically under 100 ng/mL. Treatment with an agent suspected of causing anaphylactic type reactions resulted in plasma histamine levels to increase above 3000 ng/mL. The LC-MS/MS assay presented in this study can be applied to further characterize the physiological and pathophysiological role of histamine release in complex in vitro and in vivo models. Importantly, the LC-MS/MS assay may be useful in assessing active pharmaceutical ingredient-mediated degranulation and anaphylaxis as part of either a pre-market or a post-market assessment of drug products.

In vivo assessment of natural killer cell responses during chronic feline immunodeficiency virus infection.

Accumulating evidence suggests that natural killer (NK) cells may have an important role in HIV-1 disease pathogenesis; however, in vivo studies are lacking. Feline immunodeficiency virus (FIV) infection of cats provides a valuable model to study NK cell function in vivo. The immune response against Listeria monocytogenes (Lm) is well characterized, allowing its use as an innate immune probe. We have previously shown that locally delivered IL-15 can improve Lm clearance in FIV-infected animals, and this correlated with an increase in NK cell number. In the present study, chronically FIV-infected and SPF-control cats were challenged with Lm by unilateral subcutaneous injection next to the footpad and then treated with 5-bromo-2'-deoxyuridine (BrdU). The Lm draining and contralateral control lymph nodes were evaluated for NK, NKT, CD4+ and CD8+ T cell number, proliferation, apoptosis, and NK cell function. Listeria monocytogenes burden was also assessed in both control and Lm draining lymph nodes. NK, NKT, CD4+ T and CD8+ T cells in the Lm-challenged lymph node of FIV-infected cats did not increase in number. In addition, after Lm challenge, NK cells from FIV-infected cats did not increase their proliferation rate, apoptosis was elevated, and perforin expression was not upregulated when compared to SPF-control cats. The failure of the NK cell response against Lm challenge in the draining lymph node of FIV-infected cats correlates with the delayed control and clearance of this opportunistic bacterial pathogen.

Acute mucosal pathogenesis of feline immunodeficiency virus is independent of viral dose in vaginally infected cats.

BACKGROUND: The mucosal pathogenesis of HIV has been shown to be an important feature of infection and disease progression. HIV-1 infection causes depletion of intestinal lamina propria CD4+ T cells (LPL), therefore, intestinal CD4+ T cell preservation may be a useful correlate of protection in evaluating vaccine candidates. Vaccine studies employing the cat/FIV and macaque/SIV models frequently use high doses of parenterally administered challenge virus to ensure high plasma viremia in control animals. However, it is unclear if loss of mucosal T cells would occur regardless of initial viral inoculum dose. The objective of this study was to determine the acute effect of viral dose on mucosal leukocytes and associated innate and adaptive immune responses. RESULTS: Cats were vaginally inoculated with a high, middle or low dose of cell-associated and cell-free FIV. PBMC, serum and plasma were assessed every two weeks with tissues assessed eight weeks following infection. We found that irrespective of mucosally administered viral dose, FIV infection was induced in all cats. However, viremia was present in only half of the cats, and viral dose was unrelated to the development of viremia. Importantly, regardless of viral dose, all cats experienced significant losses of intestinal CD4+ LPL and CD8+ intraepithelial lymphocytes (IEL). Innate immune responses by CD56+CD3- NK cells correlated with aviremia and apparent occult infection but did not protect mucosal T cells. CD4+ and CD8+ T cells in viremic cats were more likely to produce cytokines in response to Gag stimulation, whereas aviremic cats T cells tended to produce cytokines in response to Env stimulation. However, while cell-mediated immune responses in aviremic cats may have helped reduce viral replication, they could not be correlated to the levels of viremia. Robust production of anti-FIV antibodies was positively correlated with the magnitude of viremia. CONCLUSIONS: Our results indicate that mucosal immune pathogenesis could be used as a rapid indicator of vaccine success or failure when combined with a physiologically relevant low dose mucosal challenge. We also show that innate immune responses may play an important role in controlling viral replication following acute mucosal infection, which has not been previously identified.

Basal expression levels of IFNAR and Jak-STAT components are determinants of cell-type-specific differences in cardiac antiviral responses.

Viral myocarditis is an important human disease, and reovirus-induced murine myocarditis provides an excellent model system for study. Cardiac myocytes, like neurons in the central nervous system, are not replenished, yet there is no cardiac protective equivalent to the blood-brain barrier. Thus, cardiac myocytes may have evolved a unique antiviral response relative to readily replenished cell types, such as cardiac fibroblasts. Our previous comparisons of these two cell types revealed a conundrum: reovirus T3D induces more beta-interferon (IFN-beta) mRNA in cardiac myocytes, yet there is a greater induction of IFN-stimulated genes (ISGs) in cardiac fibroblasts. Here, we investigated possible underlying molecular determinants. We found that greater basal expression of IFN-beta in cardiac myocytes results in greater basal activated nuclear STAT1 and STAT2 and greater basal ISG mRNA expression and provides greater basal antiviral protection relative to cardiac fibroblasts. Conversely, cardiac fibroblasts express greater basal IFN-alpha/beta receptor 1 (IFNAR1) and greater basal cytoplasmic Jak1, Tyk2, STAT2, and IRF9, leading to a greater increase in reovirus T3D- or IFN-induced nuclear activated STAT1 and STAT2 and greater induction of ISGs for a greater IFN-induced antiviral protection relative to cardiac myocytes. Our results suggest that high basal IFN-beta expression in cardiac myocytes prearms this vulnerable, nonreplenishable cell type, while high basal expression of IFNAR1 and latent Jak-STAT components in adjacent cardiac fibroblasts renders these cells more responsive to IFN and prevents them from inadvertently serving as a reservoir for viral replication and spread to cardiac myocytes. These studies provide the first indication of an integrated network of cell-type-specific innate immune components for organ protection.

Mucosal challenge with cell-associated or cell-free feline immunodeficiency virus induces rapid and distinctly different patterns of phenotypic change in the mucosal and systemic immune systems.

The majority of human immunodeficiency virus type 1 (HIV-1) infections occur via mucosal transmission through contact with genital secretions containing cell-associated and cell-free virus. However, few studies have assessed whether exposure to cells, HIV-1 infected or uninfected, plays a role in the sexual transmission of HIV-1. This study examined phenotypic changes in mucosal and systemic lymphoid tissue 24 hr after vaginal exposure to in vitro equilibrated infectious doses of cell-associated or cell-free feline immunodeficiency virus, uninfected heterologous cells, or medium alone. We found that even at this early time-point, mucosal exposure to virus induced substantial alterations in the phenotype and distribution of leucocytes, particularly in the tissues of the mucosal immune system. Second, we found that the type of virus inoculum directly influenced the phenotypic changes seen. Vaginal exposure to cell-free virus tended to induce more generalized phenotypic changes, typically in the peripheral immune system (blood and systemic lymph nodes). In contrast, exposure to cell-associated virus was primarily associated with phenotypic shifts in the mucosal immune system (gut and mucosal/draining lymph nodes). In addition, we found that exposure to uninfected heterologous cells also induced alterations in the mucosal immune system. These data suggest that significant immune changes occur within the first 24 hr of virus exposure, well before substantial replication would be anticipated. As the mucosal immune system, and particularly the gut, is an early and persistent target for lentiviral replication, these findings have substantial implications for HIV-1 pathogenesis and vaccine development.

FIV infection induces unique changes in phenotype and cellularity in the medial iliac lymph node and intestinal IEL.

Studies of human immunodeficiency virus-1 (HIV-1)-infected patients and simian immunodeficiency virus (SIV)-infected macaques have identified profound depletion of CD4(+) T cells and expansion of CD8(+) T cells in the gastrointestinal lamina propria. Less attention has been given to CD8(+) intraepithelial lymphocytes (IEL), and no studies have concurrently examined inductive sites such as draining lymph nodes. Our preliminary data in the feline immunodeficiency virus (FIV) animal model suggested additional changes in IEL, and marked differences in the responses of lymph nodes draining different mucosal sites. To address this, we quantified the absolute leukocyte yield and examined the phenotype of cells from small intestinal IEL, mesenteric lymph node (MLN), and medial iliac lymph node (ILN) from chronically FIV-infected cats. The cellularity of the ILN was increased 530% in FIV-infected animals with an expansion of CD62L(+) cells, suggesting an increased population of naive T cells. The number of CD4(+), as well as CD8(+), T cells was increased in the ILN, resulting in a CD4:CD8 ratio greater than 1:1. In contrast, reduced cellularity, specific loss of CD4(+) T cells, and inversion of the CD4:CD8 ratio was observed in the MLN, which drains the intestine. In IEL, loss of CD8alpha, CD8beta, and CD4-expressing T cells was found in FIV-infected cats. Furthermore, expression intensity of CD8alpha and CD5, markers known to be important in T cell function, was markedly decreased on IEL. These findings expand the array of immune alterations induced by lentiviral infection and indicate that characterization of multiple mucosal sites will be necessary to fully understand the pathogenesis of HIV-1 infection.

Methodology for isolation and phenotypic characterization of feline small intestinal leukocytes.

Critical assessment of intestinal immune responses requires the ability to characterize leukocytes from different anatomic locations as leukocytes from inductive sites such as Peyer's patches and lymphoid follicles vary significantly from their effector counterparts, intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL). This study describes (1) methods developed to isolate specific intestinal leukocyte populations with high yield and purity, (2) difficulties encountered in establishing a panel of monoclonal antibodies to assess phenotype, and (3) the phenotypic characterization of effector and inductive sites in the feline small intestine. We found that the phenotypic distribution of feline intestinal leukocytes was similar to that found in other species such as humans, macaques and mice. The majority of IEL were CD5(+) T-cells with less than 7% B-cells. CD8(+) T-cells comprised approximately 60% of the IEL with roughly half displaying CD8alphaalpha homodimers. Approximately 10% of IEL were CD4(+) T-cells. In the LPL, CD4(+) T-cells predominated at 42%, with 33% CD8(+) T-cells and 10% B-cells. As would be expected, B-cells predominated in Peyer's patches with 40% B-cells, 28% CD4(+) T-cells and 20% CD8(+) T-cells. Increased MHCII expression was found in the Peyer's patches as compared to the IEL and LPL. B7.1 expression was significantly higher in mucosal leukocyte populations as compared to organized lymphoid tissue in the periphery with expression detected on 65% of IEL and 53% of LPL. Plasma cells were found in all regions of small intestine examined with greater numbers in lamina propria and Peyer's patches. Lymphoblasts were only identified in inductive tissue. In general, no differences were found between the phenotype of mucosal leukocyte populations from specific pathogen free or random source cats. However, the percentage of CD4(+) CD25(+) T-cells was significantly greater in both IEL and LPL from random source animals. This study provides techniques and a baseline from which future studies of the feline intestinal immune system can be conducted.

Toll-like receptor expression in feline lymphoid tissues.

Toll-like receptors (TLRs) are germline-encoded pattern recognition receptors (PRRs) that activate the innate immune system. While it is clear that TLRs are important in the immune response against pathogens, they may also be exploited by some pathogens. Our objective is to determine whether feline immunodeficiency virus (FIV) infection affects TLR expression or function thereby resulting in innate immune dysfunction. To this end, we cloned partial sequences for feline TLRs 1--3, 5--8, and developed real-time PCR assays to quantify feline TLRs 1--9. TLR expression was quantified in normal cat lymphoid tissues, purified lymphocyte subsets, and FIV-infected cell lines. Different expression patterns of TLRs were found in spleen, mesenteric lymph node, retropharyngeal lymph node, thymus, intestinal intraepithelial lymphocytes, and lamina propria lymphocytes. B lymphocytes, CD4+ T cells, and CD8+ T cells all expressed TLRs 2--5, 7--9; however, the relative levels of expression varied among lymphocyte phenotypes. Infection of cell lines with FIV resulted in altered TLR expression levels that differed depending on cell type. These results demonstrate that tissue distribution of TLRs is associated with the immunological role of a particular tissue, that lymphocytes may also express these 'innate immune' receptors, and that FIV infection can alter TLR expression.

Oral immunization with recombinant listeria monocytogenes controls virus load after vaginal challenge with feline immunodeficiency virus.

Recombinant Listeria monocytogenes has many attractive characteristics as a vaccine vector against human immunodeficiency virus (HIV). Wild-type and attenuated Listeria strains expressing HIV Gag have been shown to induce long-lived mucosal and systemic T-cell responses in mice. Using the feline immunodeficiency virus (FIV) model of HIV we evaluated recombinant L. monocytogenes in a challenge system. Five cats were immunized with recombinant L. monocytogenes that expresses the FIV Gag and delivers an FIV Env-expressing DNA vaccine (LMgag/pND14-Lc-env). Control cats were either sham immunized or immunized with wild-type L. monocytogenes (LM-wt). At 1 year after vaginal challenge, provirus could not be detected in any of the nine tissues evaluated from cats immunized with the recombinant bacteria but was detected in at least one tissue in 8 of 10 control animals. Virus was isolated from bone marrow of four of five LMgag/pND14-Lc-env-immunized cats by use of a stringent coculture system but required CD8(+) T-cell depletion, indicating CD8(+) T-cell suppression of virus replication. Control animals had an inverted CD4:CD8 ratio in mesenteric lymph node and were depleted of both CD4(+) and CD8(+) intestinal epithelial T cells, while LMgag/pND14-Lc-env-immunized animals showed no such abnormalities. Vaginal FIV-specific immunoglobulin A was present at high titer in three LMgag/pND14-Lc-env-immunized cats before challenge and in all five at 1 year postchallenge. This study demonstrates that recombinant L. monocytogenes conferred some control of viral load after vaginal challenge with FIV.