Review: The PI3K-AKT-mTOR signal transduction pathway in canine cancer.

Tumors in dogs and humans share many similar molecular and genetic features, incentivizing a better understanding of canine neoplasms not only for the purpose of treating companion animals, but also to facilitate research of spontaneously developing tumors with similar biologic behavior and treatment approaches in an immunologically competent animal model. Multiple tumor types of both species have similar dysregulation of signal transduction through phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB; AKT), and mechanistic target of rapamycin (mTOR), collectively known as the PI3K-AKT-mTOR pathway. This review aims to delineate the pertinent aspects of the PI3K-AKT-mTOR signaling pathway in health and in tumor development. It will then present a synopsis of current understanding of PI3K-AKT-mTOR signaling in important canine cancers and advancements in targeted inhibitors of this pathway.

Host Functional Response to a Prototypic Orally Delivered Self-Replicating Vaccine Platform.

The development of mucosal vaccines has been limited and could be aided by a systems vaccinology approach to identify platforms and adjuvant strategies that induce protective immune responses. The induction of local immune responses by mucosal-delivered vaccines has been difficult to evaluate from peripheral samples, as systemic responses often do not correlate with the mucosal response. Here, we utilized transcriptomics in combination with Gene Set Enrichment Analysis (GSEA) to assess innate immune activation by an oral probiotic Lactobacillus acidophilus-based vaccine platform in mice. The goal was to explore the earliest immune responses elicited after oral immunization at the Peyer's patch. Twenty-four hours after oral delivery of the L. acidophilus vaccine platform, we found an abundance of L. acidophilus at Peyer's patches and detected expression of the vaccine viral proteins and adjuvants, confirming in vivo vaccine delivery. Compared to mice orally dosed with buffer or wild-type L. acidophilus, we identified enhanced responses in immune pathways related to cytokine and gene signaling, T and B cell activation, phagocytosis, and humoral responses. While more work is needed to correlate these pathways with protection from infection and/or disease, they indicate this method's potential to evaluate and aid in the iterative development of next-generation mucosal vaccines.

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.

Lactobacillus acidophilus Expressing Murine Rotavirus VP8 and Mucosal Adjuvants Induce Virus-Specific Immune Responses.

Rotavirus diarrhea-associated illness remains a major cause of global death in children under five, attributable in part to discrepancies in vaccine performance between high- and low-middle-income countries. Next-generation probiotic vaccines could help bridge this efficacy gap. We developed a novel recombinant Lactobacillus acidophilus (rLA) vaccine expressing rotavirus antigens of the VP8* domain from the rotavirus EDIM VP4 capsid protein along with the adjuvants FimH and FliC. The upp-based counterselective gene-replacement system was used to chromosomally integrate FimH, VP8Pep (10 amino acid epitope), and VP8-1 (206 amino acid protein) into the L. acidophilus genome, with FliC expressed from a plasmid. VP8 antigen and adjuvant expression were confirmed by flow cytometry and Western blot. Rotavirus naïve adult BALB/cJ mice were orally immunized followed by murine rotavirus strain ECWT viral challenge. Antirotavirus serum IgG and antigen-specific antibody-secreting cell responses were detected in rLA-vaccinated mice. A day after the oral rotavirus challenge, fecal antigen shedding was significantly decreased in the rLA group. These results indicate that novel rLA constructs expressing VP8 can be successfully constructed and used to generate modest homotypic protection from rotavirus challenge in an adult murine model, indicating the potential for a probiotic next-generation vaccine construct against human rotavirus.

Oral Vaccination Using a Probiotic Vaccine Platform Combined with Prebiotics Impacts Immune Response and the Microbiome.

Unique to mucosal vaccination is the reciprocal influence of the microbiome and mucosal immune responses, where the immune system is constantly balancing between the clearance of pathogens and the tolerance of self-antigen, food, and the microbiota. Secretory IgA plays a major role in maintaining the homeostasis of a healthy gut microbiome. Natural polyreactive IgA often coats members of the commensal microbiota to aid in their colonization, while high-affinity specific IgA binds to pathogens resulting in their clearance. We developed a probiotic-based mucosal vaccination platform using the bacterium Lactobacillus acidophilus (rLA) with the potential to influence this balance in the IgA coating. In this study, we sought to determine whether repeated administration of rLA alters the host intestinal microbial community due to the immune response against the rLA vaccine. To address this, IgA-seq was employed to characterize shifts in IgA-bound bacterial populations. Additionally, we determined whether using rice bran as a prebiotic would influence the immunogenicity of the vaccine and/or IgA-bound bacterial populations. Our results show that the prebiotic influenced the kinetics of rLA antibody induction and that the rLA platform did not cause lasting disturbances to the microbiome.

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.

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.

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.

Altered bone marrow dendritic cell cytokine production to toll-like receptor and CD40 ligation during chronic feline immunodeficiency virus infection.

Impaired dendritic cell (DC) function is thought to be central to human immunodeficiency virus-associated immunodeficiency. In this study, we examined the effect of chronic feline immunodeficiency virus (FIV) infection on DC cytokine production in response to microbial and T-cell stimulation. Cytokine production after either Toll-like receptor (TLR) or CD40 ligation in bone marrow-derived DCs (BM-DCs) was measured in naïve and chronically FIV-infected cats. The BM-DCs were stimulated with ligands to TLR-2, -3, -4, -7 and -9 or cocultured with 3T3 cells expressing feline CD40 ligand. Ligation of TLR-4 and TLR-9 in BM-DCs from infected cats resulted in a significant decrease in the ratio of interleukin-12 (IL-12) to IL-10. Conversely, TLR-7 ligation produced a significant increase in the IL-12 : IL-10 ratio in BM-DCs from infected cats. No difference was noted for TLR-3 ligation. RNA expression levels of TLR-2, -3, -4, -7 and -9 were not significantly altered by FIV infection. CD40 ligation significantly elevated both IL-10 and IL-12 messenger RNA production but did not alter the IL-12 : IL-10 ratio. Chronic FIV infection alters the ratio of immunoregulatory cytokines produced by BM-DCs in response to certain pathogen-derived signals, which is probably relevant to the increased risk of opportunistic infections seen in lentiviral infection.

What is your diagnosis? Abdominal mass aspirate in a cat with an increased Na:K ratio.

A 13-year-old domestic shorthair cat was presented for evaluation of pollakiuria. Laboratory abnormalities included mild hypercholesterolemia, moderate hypertriglyceridemia, and a mild increase in the Na:K ratio (43, reference interval 32-41). Abdominal ultrasonography revealed urinary calculi and a soft tissue mass between the right caudate liver lobe and the right kidney. Surgery was done to remove the cystic calculi, and aspirates of the mass were obtained. Cytologic specimens contained a population of large, round to angular cells with round nuclei, coarse irregularly stippled chromatin, 1-2 prominent round to angular nucleoli, and abundant pale basophilic cytoplasm distended by numerous well-delineated vacuoles. Rare binucleated cells and micronuclei, and moderate anisocytosis, anisokaryosis, and anisonucleoleosis were noted. The cytologic interpretation was adrenal neoplasia, consistent with adrenal carcinoma. Approximately 4 months later, the cat developed vomiting, dehydration, weakness, and cervical ventroflexion. Serum biochemical alterations at that time included marked hypokalemia (2.4 mmol/L, reference interval 3.4-5.6 mmol/L) and a markedly increased Na:K ratio (65, reference interval 32-41). Mean systolic blood pressure was 205 mmHg. Surgical removal of the mass was accomplished via right adrenalectomy and a diagnosis of adrenal carcinoma was confirmed histologically. Plasma aldosterone concentration (measured preoperatively) was 1358 pmol/L (reference interval 194-388 pmol/L). Primary hyperaldosteronism caused by a functional adrenal carcinoma is an uncommon condition in cats.

Adjuvant Strategies for Lactic Acid Bacterial Mucosal Vaccines.

Lactic acid bacteria (LAB) are Gram-positive, acid-tolerant bacteria that have long been used in food fermentation and are generally recognized as safe (GRAS). LAB are a part of a normal microbiome and act as probiotics, improving the gastrointestinal microbiome and health when consumed. An increasing body of research has shown the importance of the microbiome on both mucosal immune heath and immune response to pathogens and oral vaccines. Currently, there are few approved mucosal vaccines, and most are attenuated viruses or bacteria, which necessitates cold chain, carries the risk of reversion to virulence, and can have limited efficacy in individuals with poor mucosal health. On account of these limitations, new types of mucosal vaccine vectors are necessary. There has been increasing interest and success in developing recombinant LAB as next generation mucosal vaccine vectors due to their natural acid and bile resistance, stability at room temperature, endogenous activation of innate and adaptive immune responses, and the development of molecular techniques that allow for manipulation of their genomes. To enhance the immunogenicity of these LAB vaccines, numerous adjuvant strategies have been successfully employed. Here, we review these adjuvant strategies and their mechanisms of action which include: Toll-like receptor ligands, secretion of bacterial toxins, secretion of cytokines, direct delivery to antigen presenting cells, and enterocyte targeting. The ability to increase the immune response to LAB vaccines gives them the potential to be powerful mucosal vaccine vectors against mucosal pathogens.

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.

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.

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.

In vivo depletion of CD4+CD25+ regulatory T cells in cats.

To establish a characterized model of regulatory T cell (Treg) depletion in the cat we assessed the kinetics of depletion and rebound in peripheral and central lymphoid compartments after treatment with anti-CD25 antibody as determined by cell surface markers and FOXP3 mRNA expression. An 82% decrease in circulating CD4+CD25+ Tregs was observed by day 11 after treatment. CD4+CD25+ cells were also reduced in the thymus (69%), secondary lymphoid tissues (66%), and gut (67%). Although CD4+CD25+ cells rebound by day 35 post-treatment, FOXP3 levels remain depressed suggesting anti-CD25 antibody treatment has a sustainable diminutive effect on the Treg population. To determine whether CD25+ Treg depletion strategies also deplete activated CD25+ effector cells, cats were immunized with feline immunodeficiency virus (FIV) p24-GST recombinant protein, allowing them to develop a measurable memory response, prior to depletion with anti-CD25 antibody. Anti-FIV p24-GST effector cell activity in peripheral blood after depletion was sustained as determined by antigen-specific T cell proliferation and humoral responses against FIV p24-GST with an ELISA for antigen-specific feline IgG. Furthermore, development of an anti-mouse response in Treg-depleted cats was similar to control levels indicating the retained capacity to respond to a novel antigen. We conclude that despite alterations in CD25+ cell levels during depletion, the feline immune system remains functional. We demonstrate here a model for the study of disease pathogenesis in the context of reduced numbers of immunosuppressive CD4+CD25+ Tregs throughout the feline immune system.

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.

Lactobacillus Mucosal Vaccine Vectors: Immune Responses against Bacterial and Viral Antigens.

Lactic acid bacteria (LAB) have been utilized since the 1990s for therapeutic heterologous gene expression. The ability of LAB to elicit an immune response against expressed foreign antigens has led to their exploration as potential mucosal vaccine candidates. LAB vaccine vectors offer many attractive advantages: simple, noninvasive administration (usually oral or intranasal), the acceptance and stability of genetic modifications, relatively low cost, and the highest level of safety possible. Experimentation using LAB of the genus Lactobacillus has become popular in recent years due to their ability to elicit strong systemic and mucosal immune responses. This article reviews Lactobacillus vaccine constructs, including Lactobacillus species, antigen expression, model organisms, and in vivo immune responses, with a primary focus on viral and bacterial antigens.

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.

Failure of TLR4-driven NF-kappa B activation to stimulate virus replication in models of HIV type 1 activation.

The interaction of HIV-1 with Toll-like receptors (TLR) on host target cells is incompletely understood. Data from several in vivo and in vitro model systems suggest that TLR2, TLR4, and TLR9 remain functional and if stimulated, cause an upregulation of viral replication. In the present studies employing two different chronically HIV-1-infected cell lines and highly purified TLR agonists, we found ligation of TLR2 and TLR9, but not TLR4, resulted in significant upregulation of HIV-1 production. This result was not due to a lack of TLR4 expression or impaired NF-kappa B activation. Using HEK293 cells transfected with individual TLRs and an HIV-1 LTR reporter confirmed that TLR4 signaling does not directly activate the HIV-1 LTR. Finally, ultrapurified LPS did not enhance production of IL-1 beta or IL-6 in chronically infected U1 cells, whereas significant cytokine production was observed in uninfected U937 cells. These results confirm the biological activity of ultrapurified LPS and raise the possibility that TLR4 signaling pathways may be altered during chronic HIV-1 infection. Collectively, these studies suggest that although several TLR can upregulate NF-kappaB in HIV-1-infected cells, upregulation of NF-kappaB alone is insufficient to activate the viral LTR. Further dissection of the TLR signaling pathways is necessary to determine how TLR stimulation leads to LTR activation and whether HIV-1 infection can alter signaling through TLR4.