Specific Cell-mediated Immune Responses Research Articles

APOE GENE POLYMORPHISM: THE IMPACT OF APOE4 ALLELE ON SYSTEMIC INFLAMMATION AND ITS ROLE IN THE PATHOGENESIS OF ALZHEIMER’S DISEASE

ApoE is a member of lipoprotein family. It is the most common lipoprotein in the central nervous system (CNS), secreted by astrocytes, microglia, neurons and immunocompetent cells, including lymphocytes, monocytes and macrophages. According to recent data, it has endotheliotropic and immunomodulatory functions, regulating inflammatory activation of mononuclear phagocytes and antigen-induced lymphocyte proliferation. APOE4 allele is a major genetic risk factor of Alzheimer’s disease, with prevalence 3-12 times higher in those who have this allele. Mechanisms that predispose carriers of the allele to earlier clinical presentation of neurodegeneration include changes in lipid metabolism in the CNS, in the buildup of neurotoxic amyloid-beta oligomers, in the clearance of amyloid-beta peptides from the CNS and in regulation of immune response. In this review the functions of ApoE protein in central nervous and immune system and changes in functional activity of the protein in APOE4 carriers are discussed. The impact of APOE4 allele on monocyte phenotype and inflammatory activation of monocytes, on specific cell-mediated immune response to amyloid-beta antigens and on effectiveness of immunomodulatory therapy in patients with Alzheimer’s disease summarized, as well as the possible role of changes in the immune response characteristic for APOE4 carriers in the increased risk of Alzheimer’s disease.

Therapeutic HIV-1 vaccine: time for immunomodulation and combinatorial strategies.

The purpose is to recall some of the key immunological elements that are at the crossroad and need to be combined for developing a potent therapeutic HIV-1 vaccine. Therapeutic vaccines and cytokines have been commonly used to enhance and/or recall preexisting HIV-1 specific cell-mediated immune responses aiming to suppress virus replication. While the vaccine is important to stimulate HIV-1 specific T-cell responses, the cytokine may support the expansion of the stimulated virus-specific T cells. Moreover, the current success of immune checkpoint blockers in cancer therapy render them very attractive to use in HIV-1 infected individuals, with the objective to preserve the function of HIV-specific T cells from exhaustion and target directly HIV-1 cell reservoir. More recently, the development of passive immunotherapy using broad neutralizing HIV antibodies (bNAbs) and their potential capacity to elicit innate or adaptive HIV-cellular responses, beyond their neutralizing activity, offers a new opportunity to improve the efficiency of therapeutic vaccine. These major advances provide the scientific basis for developing potent combinatorial interventions in HIV-1 infected patients. Major advances in our immunological understanding resulting from basic science and clinical trials studies have paved the way and established a solid platform to jump over the stumbling blocks that prevent the field from developing a therapeutic HIV-1 vaccine. It is time for immuno-modulation and combinatorial strategies towards HIV-1 eradication.

Virus like particles as a platform for cancer vaccine development.

Cancers have killed millions of people in human history and are still posing a serious health problem worldwide. Therefore, there is an urgent need for developing preventive and therapeutic cancer vaccines. Among various cancer vaccine development platforms, virus-like particles (VLPs) offer several advantages. VLPs are multimeric nanostructures with morphology resembling that of native viruses and are mainly composed of surface structural proteins of viruses but are devoid of viral genetic materials rendering them neither infective nor replicative. In addition, they can be engineered to display multiple, highly ordered heterologous epitopes or peptides in order to optimize the antigenicity and immunogenicity of the displayed entities. Like native viruses, specific epitopes displayed on VLPs can be taken up, processed, and presented by antigen-presenting cells to elicit potent specific humoral and cell-mediated immune responses. Several studies also indicated that VLPs could overcome the immunosuppressive state of the tumor microenvironment and break self-tolerance to elicit strong cytotoxic lymphocyte activity, which is crucial for both virus clearance and destruction of cancerous cells. Collectively, these unique characteristics of VLPs make them optimal cancer vaccine candidates. This review discusses current progress in the development of VLP-based cancer vaccines and some potential drawbacks of VLPs in cancer vaccine development. Extracellular vesicles with close resembling to viral particles are also discussed and compared with VLPs as a platform in cancer vaccine developments.

Generation of populations of antigen-specific cytotoxic T cells using DCs transfected with DNA construct encoding HER2/neu tumor antigen epitopes

BackgroundRecent fundamental and clinical studies have confirmed the effectiveness of utilizing the potential of the immune system to remove tumor cells disseminated in a patient’s body. Cytotoxic T lymphocytes (CTLs) are considered the main effectors in cell-mediated antitumor immunity. Approaches based on antigen presentation to CTLs by dendritic cells (DCs) are currently being intensively studied, because DCs are more efficient in tumor antigen presentation to T cells through their initiation of strong specific antitumor immune responses than other types of antigen-presenting cells. Today, it has become possible to isolate CTLs specific for certain antigenic determinants from heterogeneous populations of mononuclear cells. This enables direct and specific cell-mediated immune responses against cells carrying certain antigens. The aim of the present study was to develop an optimized protocol for generating CTL populations specific for epitopes of tumor-associated antigen HER2/neu, and to assess their cytotoxic effects against the HER2/neu-expressing MCF-7 tumor cell line.MethodsThe developed protocol included sequential stages of obtaining mature DCs from PBMCs from HLA A*02-positive healthy donors, magnet-assisted transfection of mature DCs with the pMax plasmid encoding immunogenic peptides HER2 p369–377 (E75 peptide) and HER2 p689–697 (E88 peptide), coculture of antigen-activated DCs with autologous lymphocytes, magnetic-activated sorting of CTLs specific to HER2 epitopes, and stimulation of isolated CTLs with cytokines (IL-2, IL-7, and IL-15).ResultsThe resulting CTL populations were characterized by high contents of CD8+ cells (71.5% in cultures of E88-specific T cells and 90.2% in cultures of E75-specific T cells) and displayed strong cytotoxic effects against the MCF-7 cell line (percentages of damaged tumor cells in samples under investigation were 60.2 and 65.7% for E88- and E75-specific T cells, respectively; level of spontaneous death of target cells was 17.9%).ConclusionsThe developed protocol improves the efficiency of obtaining HER2/neu-specific CTLs and can be further used to obtain cell-based vaccines for eradicating targeted tumor cells to prevent tumor recurrence after the major tumor burden has been eliminated and preventing metastasis in patients with HER2-overexpressing tumors.

Field performance of six Mycobacterium avium subsp. paratuberculosis antigens in a 20 h interferon gamma release assay in Belgium

Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (Map), is a chronic granulomatous enteritis which primarily affects domestic and wild ruminants, resulting in serious economic losses for dairy and beef industry around the world. There is no satisfactory cure or vaccine, and actual diagnostic tests need improvement, particularly for the initial stages of the disease. Map specific cell-mediated immune responses may allow early detection of the infection at subclinical stages. In this study, over a period of 39 months, we collected 548 blood samples in two culture-confirmed Map-infected herds, 95 blood samples in five dairy herds that scored negative during 3 consecutive years of Map serology testing and 79 samples in three culture-confirmed M. bovis infected herds. Based on criteria of bacteriology, serology and ratio of IFN-γ induced with bovine and avian purified protein derivative of tuberculin (PPD-B/PPD-A), we classified the samples in four groups: 415 samples as Map-exposed/infected (MAP), 58 samples as aspecific reactors (AR), 179 samples as non-responders (NI) and 70 samples as M. bovis infected (TB). Age of the animals influenced the IFN-γ response in the MAP group, with PPD specific IFN-γ levels (but not PPD-B/PPD-A IFN-γ ratio) being significantly higher in animals <18 months of age. Map specific antibodies were detected by IDEXX ELISA in 13/415 (3%) sera of the MAP group, whereas fecal culture was positive for only 7/405 (1.7%) samples. Animals in the MAP group could therefore be considered being at the very early stage of Map infection. Six purified, recombinant Map antigens (Ag5, Ag6, MAP1637c, MAP0388, MAP3547c and MAP0586c), previously identified using combined advanced proteomic or reverse genomic approaches, were tested for their diagnostic potential in a 20h IFN-γ release assay. In the age group >18 months old, Ag5 and MAP0388 were recognized by only 10.1% and 7.7% of the animals in the MAP group, whereas a total of 38.6.%, 29.4%, 25.6% and 39.0% of the animals in the MAP group reacted to Ag6, MAP1637c, MAP3547c and MAP0586c respectively. None of the animals in the TB group reacted to Ag6, MAP1637c or MAP586c. Except for MAP0388, the % of reactors in the MAP group was significantly higher in animals <18 months old: 28.0%, 24.0%, 45.5%, 47.1%, 49.8% and 47.4% respectively. Further studies of these candidates and their combination are needed to confirm their diagnostic potential for the detection of early Map infection.

Abstract A30: AR20.5-based novel immunotherapy for pancreatic cancer

Abstract Pancreatic cancer is a fourth leading cause of cancer death in United States. Recent advances in tumor immunology have provided evidence for immune surveillance against pancreatic cancer. Previous studies have documented the presence of specific antibodies and cytotoxic T cell activities to Mucin 1 (MUC1), an antigen that is expressed in most pancreatic cancers. Hence, MUC1 remains a candidate for immunotherapeutic strategies for pancreatic cancer. Numerous attempts have been made to target this mucin glycoprotein; however, these efforts have shown only moderate success in part due to the complexity of the pancreatic tumor microenvironment. Previously, administration of murine monoclonal antibody BrevaRexAb-AR20.5 alone produced MUC1 specific immune response in a phase I study of advanced cancer patients, including increased human anti-MUC1 antibody levels and MUC1 specific T cell response in few patients. Though this was a phase I study, modest changes in tumor marker (CA15.3) but no anecdotal anti-tumor activity was observed in this small group of patients. Moving forward it is obvious that future immunization strategies should include other immune-modulators to amplify MUC1 specific immune responses of mAbAR20.5 and enhance other factors that contribute to tumor rejection. To that end, we investigated the anti-tumor effect of mAbAR20.5 in combination with anti-PD-L1 and poly (I:C) in murine models of pancreatic adenocarcinoma using human MUC1 expressing transgenic (hMUC1.Tg) mice, which are immunologically tolerant to MUC1. The therapeutic combination of mAb-AR20.5+anti-PD-L1+Poly (I:C) induced rejection or significant inhibition of tumor growth for two different MUC1 expressing pancreatic tumor cell lines, which was accompanied by persistent MUC1 specific memory immune response, which could be adoptively transferred to other mice and shown to protect against subsequent tumor challenge. We show that the anti-tumor response was effected by CD8 T cells, as their abrogation attenuated the anti-tumor response. Flow cytometric analysis of immunized mice demonstrated progressive increases in activated CD8 T cells in the peripheral circulation of combination treated mice. Together, these data support the hypothesis that targeting checkpoint induced immunosuppression (anti-PD-L1) together with the use of toll-like receptor 3 agonist as an adjuvant (poly (I:C) ) enhances the capacity of mAbAR20.5 to induce specific cell mediated immune responses to MUC1, which in turn provide long lasting anti-tumor response against pancreatic tumors. Our study supports the rapid translation of this strategy into clinical trials for pancreatic cancer patients. Citation Format: Kamiya Mehla, James A. Grunkemeyer, Kelly A. O'Connell, Maria M. Steele, Thomas Caffrey, Ragupathy Madiyalakan, Christopher F. Nicodemus, Michael A. Hollingsworth. AR20.5-based novel immunotherapy for pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A30.

Bioengineering a bacterial pathogen to assemble its own particulate vaccine capable of inducing cellular immunity

Many bacterial pathogens naturally form cellular inclusions. Here the immunogenicity of polyhydroxyalkanoate (PHA) inclusions and their use as particulate vaccines delivering a range of host derived antigens was assessed. Our study showed that PHA inclusions of pathogenic Pseudomonas aeruginosa are immunogenic mediating a specific cell-mediated immune response. Protein engineering of the PHA inclusion forming enzyme by translational fusion of epitopes from vaccine candidates outer membrane proteins OprI, OprF, and AlgE mediated self-assembly of PHA inclusions coated by these selected antigens. Mice vaccinated with isolated PHA inclusions produced a Th1 type immune response characterized by antigen-specific production of IFN-γ and IgG2c isotype antibodies. This cell-mediated immune response was found to be associated with the production of functional antibodies reacting with cells of various P. aeruginosa strains as well as facilitating opsonophagocytic killing. This study showed that cellular inclusions of pathogenic bacteria are immunogenic and can be engineered to display selected antigens suitable to serve as particulate subunit vaccines against infectious diseases.

Immunogenicity of modified vaccinia virus Ankara expressing the hemagglutinin stalk domain of pandemic (H1N1) 2009 influenza virus

Background: Vaccination offers protection against influenza, although current vaccines need to be reformulated each year. The development of a broadly protective influenza vaccine would guarantee the induction of heterosubtypic immunity also against emerging influenza viruses of a novel subtype. Vaccine candidates based on the stalk region of the hemagglutinin (HA) have the potential to induce broad and persistent protection against diverse influenza A viruses.Methods: Modified vaccinia virus Ankara (MVA) expressing a headless HA (hlHA) of A/California/4/09 (CA/09) virus was used as a vaccine to immunize C57BL/6 mice. Specific antibody and cell-mediated immune responses were determined, and challenge experiments were performed by infecting vaccinated mice with CA/09 virus.Results: Immunization of mice with CA/09-derived hlHA, vectored by MVA, was able to elicit influenza-specific broad cross-reactive antibodies and cell-mediated immune responses, but failed to induce neutralizing antibodies and did not protect mice against virus challenge.Conclusion: Although highly immunogenic, our vaccine was unable to induce a protective immunity against influenza. A misfolded and unstable conformation of the hlHA molecule may have affected its capacity of inducing neutralizing antiviral, conformational antibodies. Design of stable hlHA-based immunogens and their delivery by recombinant MVA-based vectors has the potential of improving this promising approach for a universal influenza vaccine.

Elucidating Unusual Modes of Action and Resistance of Antibacterial Peptides.

The emergence of bacterial resistance to conventional antibiotics has been become a major issue in the health system. Currently, studies are being performed to develop new drugs that can replace conventional antibiotics. These new drugs include the antimicrobial peptides (AMPs), a heterogeneous group of molecules produced in a variety of bacterial, invertebrate, plant and animal species, which can serve as a nonspecific defense system that complements the highly specific cell-mediated immune response. AMPs have antimicrobial activities against a wide range of microorganisms, including bacteria, fungi, and viruses, and they are effective against pathogenic organisms that are resistant to common drugs. The most common mechanism of action of antibacterial peptides on microbial cells has been characterized as an alteration in the cellular membrane permeability. In contrast, there are AMPs with unusual strategies associated with cell wall and protein synthesis inhibition, nucleic acid binding or inactivation of toxins that enable microbial infection. Many pathogens can develop resistance strategies, inactivating or repelling AMPs through modification of the surface expression of pumps or secretion of proteases. In summary, this review focus on AMPs with non-conventional modes of actions and the microbial counter measures to resist these peptides.

Detection of herpes simplex virus type 2 (HSV-2) -specific cell-mediated immune responses in guinea pigs during latent HSV-2 genital infection

Genital infections with herpes simplex virus type 2 (HSV-2) are a source of considerable morbidity and are a health concern for newborns exposed to virus during vaginal delivery. Additionally, HSV-2 infection diminishes the integrity of the vaginal epithelium resulting in increased susceptibility of individuals to infection with other sexually transmitted pathogens. Understanding immune protection against HSV-2 primary infection and immune modulation of virus shedding events following reactivation of the virus from latency is important for the development of effective prophylactic and therapeutic vaccines. Although the murine model of HSV-2 infection is useful for understanding immunity following immunization, it is limited by the lack of spontaneous reactivation of HSV-2 from latency. Genital infection of guinea pigs with HSV-2 accurately models the disease of humans including the spontaneous reactivation of HSV-2 from latency and provides a unique opportunity to examine virus-host interactions during latency. Although the guinea pig represents an accurate model of many human infections, relatively few reagents are available to study the immunological response to infection. To analyze the cell-mediated immune response of guinea pigs at extended periods of time after establishment of HSV-2 latency, we have modified flow-cytometry based proliferation assays and IFN-γ ELISPOT assays to detect and quantify HSV-specific cell-mediated responses during latent infection of guinea pigs. Here we demonstrate that a combination of proliferation and ELISPOT assays can be used to quantify and characterize effecter function of virus-specific immune memory responses during HSV-latency.

Humoral and Cell-Mediated Immune Responses to Alternate Booster Schedules of Anthrax Vaccine Adsorbed in Humans.

Protective antigen (PA)-specific antibody and cell-mediated immune (CMI) responses to annual and alternate booster schedules of anthrax vaccine adsorbed (AVA; BioThrax) were characterized in humans over 43 months. Study participants received 1 of 6 vaccination schedules: a 3-dose intramuscular (IM) priming series (0, 1, and 6 months) with a single booster at 42 months (4-IM); 3-dose IM priming with boosters at 18 and 42 months (5-IM); 3-dose IM priming with boosters at 12, 18, 30, and 42 months (7-IM); the 1970 licensed priming series of 6 doses (0, 0.5, 1, 6, 12, and 18 months) and two annual boosters (30 and 42 months) administered either subcutaneously (SQ) (8-SQ) or IM (8-IM); or saline placebo control at all eight time points. Antibody response profiles included serum anti-PA IgG levels, subclass distributions, avidity, and lethal toxin neutralization activity (TNA). CMI profiles included frequencies of gamma interferon (IFN-γ)- and interleukin 4 (IL-4)-secreting cells and memory B cells (MBCs), lymphocyte stimulation indices (SI), and induction of IFN-γ, IL-2, IL-4, IL-6, IL-1β, and tumor necrosis factor alpha (TNF-α) mRNA. All active schedules elicited high-avidity PA-specific IgG, TNA, MBCs, and T cell responses with a mixed Th1-Th2 profile and Th2 dominance. Anti-PA IgG and TNA were highly correlated (e.g., month 7,r(2)= 0.86,P< 0.0001, log10 transformed) and declined in the absence of boosters. Boosters administered IM generated the highest antibody responses. Increasing time intervals between boosters generated antibody responses that were faster than and superior to those obtained with the final month 42 vaccination. CMI responses to the 3-dose IM priming remained elevated up to 43 months. (This study has been registered at ClinicalTrials.gov under registration no. NCT00119067.).

Pertussis specific cell-mediated immune responses ten years after acellular pertussis booster vaccination in young adults

BackgroundOne of the reasons for pertussis resurgence is waning immunity. Both humoral and cell mediated immunity (CMI) are essential for protection. The aim of this study was to evaluate CMI responses after acellular pertussis vaccination in young adults. MethodsFifty-seven young adults were followed for ten years after a diphtheria-tetanus acellular pertussis (dTpa) booster vaccination. A second booster was administrated at year 10. CMI was determined from peripheral blood mononuclear cells (PBMC) stimulated with vaccine antigens pertussis toxin (PT), filamentous hemagglutinin (FHA) and pertactin (PRN) before and one month after the second vaccination, using proliferation and IFN-γ and IL-17 ELISpot. In addition, the response to ten selected cytokines was measured from 14 subjects. ResultsBefore the booster dose, positive proliferation was recognized in 51%, 53% and 89% of the subjects against PT, PRN and FHA, respectively. One month after, the positivity rate increased to 81%, 81% and 96%. Although the number of IFN-γ and IL-17 secreting cells was increased, the expression of most of the tested cytokines was found to be downregulated. After PT stimulation, only one (7.1%) subject had increased production in all cytokines, whereas six (42.9%) had decreased production of all cytokines. Ten subjects (71.4%) had decreased concentration of IFN-γ, the cytokine important for pertussis protection. ConclusionsCMI persists even when antibodies have decayed, and acellular pertussis vaccine enhances the CMI response. Further studies are needed to illustrate what factors cause the low production of some important cytokines.

A novel lipid nanoparticle adjuvant significantly enhances B cell and T cell responses to sub-unit vaccine antigens

Sub-unit vaccines are primarily designed to include antigens required to elicit protective immune responses and to be safer than whole-inactivated or live-attenuated vaccines. But their purity and inability to self-adjuvant often result in weaker immunogenicity. Emerging evidence suggests that bio-engineered nanoparticles can be used as immunomodulatory adjuvants. Therefore, in this study we explored the potential of novel Merck-proprietary lipid nanoparticle (LNP) formulations to enhance immune responses to sub-unit viral antigens. Immunization of BALB/c and C57BL/6 mice revealed that LNPs alone or in combination with a synthetic TLR9 agonist, immune-modulatory oligonucleotides, IMO-2125 (IMO), significantly enhanced immune responses to hepatitis B virus surface antigen (HBsAg) and ovalbumin (OVA). LNPs enhanced total B-cell responses to both antigens tested, to levels comparable to known vaccine adjuvants including aluminum based adjuvant, IMO alone and a TLR4 agonist, 3-O-deactytaled monophosphoryl lipid A (MPL). Investigation of the quality of B-cell responses demonstrated that the combination of LNP with IMO agonist elicited a stronger Th1-type response (based on the IgG2a:IgG1 ratio) than levels achieved with IMO alone. Furthermore, the LNP adjuvant significantly enhanced antigen specific cell-mediated immune responses. In ELISPOT assays, depletion of specific subsets of T cells revealed that the LNPs elicited potent antigen-specific CD4+ and CD8+T cell responses. Intracellular FACS analyses revealed that LNP and LNP+IMO formulated antigens led to higher frequency of antigen-specific IFNγ+TNFα+IL-2+, multi-functional CD8+T cell responses, than unadjuvanted vaccine or vaccine with IMO only. Overall, our results demonstrate that lipid nanoparticles can serve as future sub-unit vaccine adjuvants to boost both B-cell and T-cell responses in vivo, and that addition of IMO can be used to manipulate the quality of immune responses.

A transgenic ginseng vaccine for bovine viral diarrhea.

BackgroundBovine viral diarrhea virus (BVDV) infections are endemic in cattle populations worldwide and cause major economic losses. Thus, an effective vaccine is needed against the transmission of BVDV. The glycoprotein Erns is one of the envelope proteins of this virus and shows BVDV-related immunogenicity. Here, we report the use of Panax ginseng as an alternative production platform for the expression of glycoprotein Erns via Agrobacterium-mediated transformation.ResultPolymerase chain reaction (PCR) and reverse transcription (RT)-PCR analyses showed that pBI121-Erns was stably integrated into the chromosome of transformants. ELISA assay and Western blot analysis confirmed the antigenicity of plant-derived Erns glycoprotein. Immunogenicity was evaluated subcutaneously in deer using a soluble protein extract of dried transgenic ginseng hairy roots. Specific humoral and cell-mediated immune responses against BVDV were detected following immunization.ConclusionThese results demonstrated that the Erns glycoprotein could be expressed in ginseng hairy roots and that plant-derived glycoprotein Erns retained its antigenicity and immunogenicity.

Immunization with Ty21a live oral typhoid vaccine elicits crossreactive multifunctional CD8+ T-cell responses against Salmonella enterica serovar Typhi, S. Paratyphi A, and S. Paratyphi B in humans.

Previously we have extensively characterized Salmonella enterica serovar Typhi (S. Typhi)-specific cell-mediated immune responses (CMI) in volunteers orally immunized with the licensed Ty21a typhoid vaccine. In this study we measured Salmonella-specific multifunctional (MF) CD8+ T cell responses to further investigate whether Ty21a elicits cross reactive CMI against S. Paratyphi A and S. Paratyphi B, which also cause enteric fever. Ty21a elicited cross-reactive CMI against all three Salmonella serotypes were predominantly observed in CD8+ T effector/memory (TEM) and, to a lesser extent, in CD8+CD45RA+ TEM (TEMRA) subsets. These CD8+ T cell responses were largely mediated by MF cells co producing IFN-γ, MIP-1β and expressing CD107a with or without TNF-α. Significant proportions of Salmonella-specific MF cells expressed the gut homing molecule integrin α4β7. In most subjects similar MF responses were observed to S. Typhi and S. Paratyphi B, but not to S. Paratyphi A. These results suggest that Ty21a elicits MF CMI against Salmonella which could be critical in clearing the infection. Moreover, because S. Paratyphi A is a major public concern and Ty21a was shown in field studies not to afford cross-protection to S. Paratyphi A, these results will be important in developing a S. Typhi/S. Paratyphi A bivalent vaccine against enteric fevers.

Dialyzable Leukocyte Extracts Activate TLR-2 on Monocytes

Dialyzable leukocyte extracts (DLE) transfer specific cell-mediated immune responses from sensitized donors to non-immune recipients. In addition, DLE have several immunomodulatory effects and are used for the treatment of several infectious and non-infectious diseases. Previous studies showed that human DLE obtained from virus-infected leukocytes and bovine DLE decrease the production of the pro-inflammatory cytokine TNF-alpha in response to bacterial lipopolysaccharide, in vitro and in vivo. In the present work, we inquire as to whether DLE from uninfected human leukocytes have the ability to regulate cytokine production in peripheral blood mononuclear cells (PBMC) in vitro. We observed that PBMC from healthy individuals were able to produce TNF-alpha, IL-12 and IL-10 after stimulation with DLE. Moreover, we identified monocytes as the main cell population that produced TNF-alpha after DLE stimulation. Interestingly, we found that DLE contain unidentified ligands that activate Toll-like receptor (TLR)-2. Finally, we observed that DLE directly activated monocytes through TLR-2. These results reveal a new biological activity of DLE, and suggest that part of the immunomodulatory properties of DLE could be attributed to TLR-2 activation on monocytes and to the induction of a pro-inflammatory environment that is crucial for control of infectious diseases.

Respiratory syncytial virus (RSV)-specific cell-mediated immune responses in human peripheral blood (VIR2P.1025)

Abstract Respiratory syncytial virus (RSV) is the leading cause of respiratory tract infections and hospitalization in infants, but no vaccine exists, primarily because the immune response is poorly understood and immune correlates of protection have not been adequately defined. Antiviral lymphocytes, along with secreted immunoglobulins (Ig) are likely the most important components of effective anti-RSV immunity RSV-specific assays to quantify the effective cell-mediated immune response to naturally-occurring RSV infections are imperative. We therefore quantified IFN-γ secreting human peripheral blood mononuclear cells (PBMCs) with and without stimulation by whole RSV from different clades, and with highly purified RSV-peptide pools. PBMCs from donors demonstrated between-donor variable levels of immune response to the RSV peptide pools and the whole virus preparations. Robust IFN-γ T-cell responses were observed at 72 hours in all PBMCs, thus allowing a single time point assay evaluation. Higher responses were seen in the CD4+ population of PBMCs compared to the CD8+ population. Responses of CD8+ PBMCs to whole virus preparations and to mock infection control preparations were similar and responses to RSV peptide pools were not statistically significant from the negative peptide control. IFN-γ based ELISPOTS and FACS assays are promising tools to quantify RSV-specific T-cell-mediated immune responses in human blood.

Generation of the bovine viral diarrhea virus e0 protein in transgenic astragalus and its immunogenicity in sika deer.

The bovine viral diarrhea virus (BVDV), a single-stranded RNA virus, can cause fatal diarrhea syndrome, respiratory problems, and reproductive disorders in herds. Over the past few years, it has become clear that the BVDV infection rates are increasing and it is likely that an effective vaccine for BVDV will be needed. In this study, transgenic Astragalus was used as an alternative productive platform for the expression of glycoprotein E0. The immunogenicity of glycoprotein E0 expressed in transgenic Astragalus was detected in deer. The presence of pBI121-E0 was confirmed by polymerase chain reaction (PCR), transcription was verified by reverse transcription- (RT-) PCR, and recombinant protein expression was confirmed by ELISA and Western blot analyses. Deer that were immunized subcutaneously with the transgenic plant vaccine developed specific humoral and cell-mediated immune responses against BVDV. This study provides a new method for a protein with weak immunogenicity to be used as part of a transgenic plant vaccine.

Specific cell-mediated immune response in rabbits immunised and infected with Trichophyton mentagrophytes

Abstract Deep crusty dermatophytosis was demonstrated in 10-week-old rabbits naturally and experimentally infected with T. mentagrophytes. Moreover, specific cell-mediated immune response in T. mentagrophytes-infected rabbits occurrd in two phases. During the presence of clinical symptoms of trichophytosis, slightly positive results of leukocyte migration inhibition factor (LMIF) assay were noted, whereas the highest values were observed during spontaneous regression of fungal lesions and complete recovery. The slightly positive LMIF values in rabbits with fungal lesions reflect the mechanism of cell-mediated immune response during T. mentagrophytes infection. On the other hand, the highest positive LMIF values during spontaneous recovery indicate that regression of fungal lesions is attributed to acquired specific cellular immunity. The findings also confirm that the used vaccine against trichophytosis quite strongly stimulates the cell-mediated immune mechanisms, which is confirmed by positive LMIF values in immunised rabbits.

Leukocyte Proliferation and Immune Modulator Production in Patients with Chronic Kidney Disease

IntroductionIn Chronic Kidney Disease (CKD), immune cells are affected by uremic retention toxins. Given this effect, we analyzed lymphocyte proliferative response and immune modulators production following in vitro stimulation.MethodsWhole blood was drawn from healthy controls, patients with eGFR <20 ml/min/1.73 m2 (Pre-dialysis, CKD stages 4 and 5) and hemodialysis patients (stage 5D). Peripheral cells were incubated for six days with pokeweed mitogen, concanavalin A, Staphylococcus enterotoxin A or influenza A vaccine. Peripheral lymphocyte proliferation was then analyzed by the “Flow-cytometric Assay of Specific Cell-mediated Immune response in Activated whole blood” (FASCIA) method, and cytokine profile in the cell supernatants was analyzed by the Milliplex multi-array method.ResultsThe absolute number of lymphoblasts in response to mitogenic stimulation and the number of cells in each CD4+ and CD8+ subpopulation were similar comparing the three groups, except for a single decline in number of lymphoblasts after stimulation with Staphylococcus enterotoxin A, comparing dialysis patients with healthy controls. Levels of interleukin (IL)-2 (p=0.026), -10 (p=0.019) and -15 (p=0.027) in the Staphylococcus enterotoxin A-stimulated supernatant were lower in hemodialysis patients compared to healthy controls. Levels of IL-15 (p=0.017) from pre-dialysis patients and levels of IL-5 (p=0.019) from hemodialysis patients in influenza A vaccine-stimulated supernatants were also lower compared to controls. In pokeweed mitogen–stimulated supernatant, IL-2 levels (p=0.013) were lower in hemodialysis patients compared to pre-dialysis patients. TNF-α, IL-10, IL-12, IL-15, IL-8, MCP-1, IP-10, IFN-α2, IL-1α and eotaxin levels were all significantly higher in plasma obtained from CKD patients.ConclusionOur results suggest that T-cells from CKD patients have similar proliferative response to stimulation compared with healthy individuals. Moreover, however the immune cells show inability to produce selected cytokines, most likely due to the uremic milieu or dialysis procedure.