Induction Of Cell-mediated Immunity Research Articles

Effects of ultra-filtration purification of infectious bursal disease virus on immune responses and cytokine activation in specific pathogen free chickens.

Purification is an important step in the production of viral vaccines that strongly affects product recovery and subsequent immune responses. The present study was carried out with the aim of improving the purification of infectious bursal disease virus (IBDV) by the tangential flow filtration (TFF) method. Then, the effect of the purified virus on the induction of immune responses against IBDV in specific pathogen free (SPF) chickens was investigated. The IBD07IR strain was propagated in embryonated SPF eggs. The virus was purified using a 100 kDa cassette. The quality of the recovered viruses was evaluated by titration. A total number of 60 SPF chickens were randomly divided into three groups (n = 20) and received the concentrated viral antigen, commercial live IBDV vaccine and phosphate-buffered saline at the age of 3 weeks by eye drop method. The bursa of Fabricius was examined histopathologically for possible changes. Sera were collected at 1-week intervals from day 0 until the end of 6 weeks after vaccination. The IBDV-specific antibody levels, induction of cell-mediated immunity and mRNA expression levels of cytokines were evaluated. The results showed that despite a relative raise in virus titer from 7.66 to 8.17 embryo infectious dose (EID)50 mL-1 following purification, both the purified IBDV and commercial vaccine are able to induce strong immune responses against the virus. Within a context of egg-based IBDV vaccine production, a single-step TFF can be applied for the relatively purification. This platform requires a further study in the selection of multiple membranes to optimize the operating conditions and final product.

Adjuvant effect of allogeneic blood in vaccines against edwardsiellosis in ginbuna crucian carp Carassius auratus langsdorfii

Edwardsiella tarda (E. tarda), an intracellular pathogen, has caused severe economic losses in aquaculture. Effective vaccine development for E. tarda prevention is urgently needed. A previous study indicates that cell-mediated immunity (CMI) might play an important role in E. tarda infection. We believe that the involvement of allograft rejection and CMI has now been well documented in mammals and some fishes. However, there is still little research on the application of blood allograft rejection in vaccine development. In the current study, we investigate the immune response and vaccine effect in fish vaccinated with allogeneic blood + formalin-killed cells vaccine (FKC), allogeneic blood + phosphate-buffered saline (PBS), PBS + FKC and PBS + PBS. In the challenge test, the relative percentage survival (RPS) of the allogeneic + FKC, the allogeneic blood + PBS and the PBS + FKC group was 61.46, 35.41, and 30.63 % respectively. The up-regulated expression of Th1-related genes IFN-γ 1, IFN-γ 1rel2, IL-12p35 and T-bet suggests the protection is via CMI induction. Only in the allogeneic + FKC group, gene expression of IFN-γ 1, IL-12p35 and T-bet is significantly higher, indicating synergy between the two substances. Furthermore, among the fish injected with the allogeneic blood cells, syngeneic blood cells and PBS group, only in the fish of the allogenic blood cells injection group, did expression of IFN-γ 1, IFN-γ 2 and IFN-γ rel2 gene expression significantly increased. The results indicate that the rejection was induced by allogeneic components. Thus, our findings might provide essential information and insights into vaccine development in aquaculture.

Older adults lack SARS CoV-2 cross-reactive T lymphocytes directed to human coronaviruses OC43 and NL63

Currently, infections with SARS-Coronavirus-2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, are responsible for substantial morbidity and mortality worldwide. Older adults subjects > 60 years of age account for > 95% of the over one million fatal cases reported to date. It is unclear why in this age group SARS-CoV-2 infection causes more severe disease than in young adults. We hypothesized that differences in SARS-CoV-2 cross-reactive cellular immunity induced after infection with human coronaviruses (HCoVs), like OC43 and NL63, were at the basis of the differential mortality (and morbidity) observed after SARS-CoV-2 infection, because a small proportion of HCoV-specific T cells cross-react with SARS-CoV-2. Our data demonstrate that pre-existing T cell immunity induced by circulating human alpha- and beta-HCoVs is present in young adult individuals, but virtually absent in older adult subjects. Consequently, the frequency of cross-reactive T cells directed to the novel pandemic SARS-CoV-2 was minimal in most older adults. To the best of our knowledge, this is the first time that the presence of cross-reactive T cells to SARS-CoV-2 is compared in young and older adults. Our findings provide at least a partial explanation for the more severe clinical outcome of SARS-CoV-2 infection observed in the elderly. Moreover, this information could help to design efficacious vaccines for this age group, aiming at the induction of cell-mediated immunity.

Elucidation of the Interleukin 12 Production Mechanism during Intracellular Bacterial Infection in Amberjack, Seriola dumerili.

Intracellular bacterial infections affect all vertebrates. Cultured fish are particularly vulnerable because no effective protection measures have been established since such infections emerged approximately 50 years ago. As in other vertebrates, the induction of cell-mediated immunity (CMI) plays an important role in protecting fish against infection. However, details of the mechanism of CMI induction in fish have not been clarified. In the present study, we focused on the production of interleukin 12 (IL-12), an important factor in CMI induction in fish. Using several different approaches, we investigated IL-12 regulation in amberjack (Seriola dumerili), the species most vulnerable to intracellular bacterial disease. The results of promoter assays and transcription factor gene expression analyses showed that the expression of interferon regulatory factor-1 (IRF-1) and activator protein-1 (AP-1) is necessary for IL-12 production. Phagocytosis of living cells (LCs) of Nocardia seriolae bacteria induced IL-12 production in neutrophils, accompanied by IRF-1 and AP-1 gene expression. Bacteria in which the exported repetitive protein (Erp)-like gene was deleted (Δerp-L) could not establish intracellular parasitism or induce IRF-1 and AP-1 expression or IL-12 production, despite being phagocytosed by neutrophils. These data suggest that IL-12 production is regulated by (i) two transcription factors, IRF-1 and AP-1, (ii) phagocytosis of LCs by neutrophils, and (iii) one or more cell components of LCs. Our results enhance the understanding of the immune response to intracellular bacterial infections in vertebrates and could facilitate the discovery of new agents to prevent intracellular bacterial disease.

Adjuvant effect of recombinant interleukin-12 in the Nocardiosis formalin-killed vaccine of the amberjack Seriola dumerili

Nocardiosis causes serious economic damage in the fish farming of Japanese yellowtail fish. Nocardia seriolae identified as pathogenic bacterium is an intracellular-pathogen. In general, induction of cell-mediated immunity (CMI) is effective in infection defense against intracellular-pathogen. However, the conventional formalin-killed N. seriolae (FKC) vaccine induces humoral immunity. Interleukin-12 (IL-12) is Th1 type heterodimeric cytokine and induces cell differentiation in mammals. Our previous study showed that recombinant amberjack IL-12 has a role in CMI induction in vitro and could be a possible CMI inducing adjuvant. However, its adjuvant effect of fish IL-12 was not studied. In the present study, six types of amberjack recombinant IL-12 (rIL-12) were mixed and injected into amberjack with FKC. Firstly, we analyzed Th1- and Th2- related gene expression and monitored Th1/Th2 status followed by investigation of antibody titer. As a result, Th1-type immunity was induced in FKC + rIL-12 vaccinated fish. Secondly, we checked Th1/Th2 status of vaccinated fish after 10 days of N. seriolae infection using the expression of related genes. High T-bet/GATA-3 ratio was observed in FKC + rIL-12 vaccinated fish, suggesting that Th1 cells possesing antigen memory were induced against N. seriolae infection. Finally, the survival rate in challenge test showed that 88% of FKC + rIL-12 vaccinated fish was survived at 34 days after N. seriolae injection whereas PBS (control) and FKC only were exterminated. These result suggest that i) rIL-12 is viable CMI inducible adjuvant and ii) production of Th1 cells having antigen memory resulting from activation of IL-12 signaling pathway is important for defense against N. seriolae infection.

Identification and functional characterization of multiple interleukin 12 in amberjack (Seriola dumerili).

Interleukin (IL) -12 is a heterodimeric cytokine mainly produced by monocytes, macrophages, and dendritic cells in mammals. IL-12p70 composed of IL-12p35 and IL-12p40, is known to play a crucial role in promoting cell-mediated immunity (CMI) through Th1 differentiation and IFN-γ production. Although two types of IL-12p35 (p35a, p35b) and three types of IL-12p40 (p40a, p40b and p40c) have been identified in several fish species, the knowledge on functional characteristics of teleost IL-12 is still limited. In the present study, we cloned two types of IL-12p35 and three types of IL-12p40 genes in amberjack and yellowtail, and analyzed their expressions in response to stimulation with Nocardia seriolae in amberjack. As a result, four types of IL-12 (IL-12p35a, p35b, p40a and p40b) and IFN-γ mRNA were increased by live-N.seriolae stimulation but not by formalin-killed N.seriolae, suggesting that four types of IL-12 (p35, p35b, p40a and p40c) participate in promoting CMI. Subsequently, we produced six types of recombinant IL-12p70 (rIL12p70) protein in insect cells. Head kidney leukocytes were cultured with formalin-killed N.seriolae and six types of rIL-12p70 to elucidate the role of amberjack IL-12p70 in induction of CMI. After stimulation, IFN-γ expression was elevated whereas IL-10 expression was suppressed in Head kidney leukocytes stimulated with four types of rIL-12 (p40a/p35a, p40c/p35a, p40a/p35b, p40a/p35b). On the other hand, two types of rIL-12 (p40b/p35a, p40b/p35b) only elicited down regulation of IL-10 expression. These results indicate that all amberjack IL-12p70 isoforms are involved in Th1 -differentiation and promotion of CMI with different manners. Fish IL-12 has a potential for the promising vaccine adjuvant.

Characterization and functional analysis of interleukin-12 in amberjack Seriola dumerili

Interleukin-12 is proinflammatory cytokine mainly produced by monocytes, macrophages and dendritic cells. In mammal, IL-12p70 is composed of IL-12p35 and IL-12p40, is known to play crucial role in promoting cell-mediated immunity (CMI) through Th1 differentiation and IFNγ production. Although fish possess two types of IL-12p35 (p35a, p35b) and three types of IL-12p40 (p40a, p40b and p40c) genes, the knowledge on functional characteristics of teleost IL-12 is still limited. In the present study, we have cloned two types of IL-12p35 and three types of IL-12p40 genes from amberjack. To elucidate the role of amberjack IL-12p70 in CMI, six types of recombinant amberjack IL-12p70 protein generated in insect cells HighFive, were purified by Ni-NTA column. Head kidney leukocytes were cultured with formalin-killed Nocardia seriolae and rIL-12. As a result of gene expression analysis after stimulation, IFN-γ was induced whereas IL-10 expression was suppressed in head kidney leucocytes stimulated with four types of rIL-12 (p35a/p40a, p35a/p40c, p35b/p40a, p35b/p40c). On the other hand, two types of rIL-12 (p35a/p40b, p35b/p40b) only elicited down-regulation of IL-10 expression. In addition, leukocytes stimulated with formalin-killed N.seriolae (without rIL-12) showed suppression of IFN-γ and up-regulation of IL-10 gene. These data suggest that all amberjack IL-12p70 isoforms are involved in promotion of CMI with different manner. Furthermore, there is no modulation of IFN-γ and IL-10 genes in the leukocytes stimulated with amberjack rIL-12 alone, indicating that induction of CMI by IL-12 requires antigen stimulation. This is the first report to show that in teleosts multiple IL-12 protein are involved in induction of CMI. KEYWARD IL-12, IFNγ, Amberjack, cell-mediated immunity, IL-10 *Corresponding author, Tel.: +81 992864162; Fax: +81992864162. E-mail address: araki@fish.kagoshima-u.ac.jp

Synthetic long peptide-based vaccine formulations for induction of cell mediated immunity: A comparative study of cationic liposomes and PLGA nanoparticles

Nanoparticulate formulations for synthetic long peptide (SLP)-cancer vaccines as alternative to clinically used Montanide ISA 51- and squalene-based emulsions are investigated in this study. SLPs were loaded into TLR ligand-adjuvanted cationic liposomes and PLGA nanoparticles (NPs) to potentially induce cell-mediated immune responses. The liposomal and PLGA NP formulations were successfully loaded with up to four different compounds and were able to enhance antigen uptake by dendritic cells (DCs) and subsequent activation of T cells in vitro. Subcutaneous vaccination of mice with the different formulations showed that the SLP-loaded cationic liposomes were the most efficient for the induction of functional antigen-T cells in vivo, followed by PLGA NPs which were as potent as or even more than the Montanide and squalene emulsions. Moreover, after transfer of antigen-specific target cells in immunized mice, liposomes induced the highest in vivo killing capacity. These findings, considering also the inadequate safety profile of the currently clinically used adjuvant Montanide ISA-51, make these two particulate, biodegradable delivery systems promising candidates as delivery platforms for SLP-based immunotherapy of cancer.

Vaccine potential of plasma bead-based dual antigen delivery system against experimental murine candidiasis

The development of prophylactic anti-candidal vaccine comprising the Candida albicans cytosolic proteins (Cp) as antigen and plasma beads (PB) prepared from plasma as sustained delivery system, is described. The immune-prophylactic potential of various PBs-based dual antigen delivery systems, co-entrapping Cp pre-entrapped in PLGA microspheres were tested in the murine model. Induction of cell mediated immunity was measured by assaying DTH and NO production as well as in vitro proliferation of lymphocytes derived from the immunized animals. Expression of surface markers on APCs (CD80, CD86) and T-cells (CD4+, CD8+) was also evaluated. Humoral immune response was studied by measuring circulating anti-Cp antibodies and their subclasses. When the prophylactic efficacy of the vaccines was tested in mice challenged with virulent C. albicans, the PB-based formulation (PB-PLGA-Cp vaccine) was found to be most effective in the generation of desirable immune response, in terms of suppression of fungal load and facilitating the survival of the immunized animals.

A novel DNA vaccine for reduction of PRRSV-induced negative immunomodulatory effects: A proof of concept

BackgroundViral-induced interleukin (IL)-10 and regulatory T lymphocytes (Tregs) are believed to play a major role in shaping the immunological and clinical outcomes following Porcine Reproductive and Respiratory Syndrome virus (PRRSV) infection. Recently, it has been shown that PRRSV nucleocapsid (N) protein can induce IL-10 production which is essential for induction of PRRSV-specific Tregs. We hypothesized that immunity to N protein should reduce PRRSV-induced negative immunomodulatory effects which will be essential for establishing proper anti-PRRSV immunity in infected pigs. ObjectivesTo investigate the immunomodulatory effects of DNA vaccine encoding a linearized, truncated form of PRRSV-N protein (pORF7t) which was designed to preferentially induce cell-mediated immunity against PRRSV N protein. MethodImmunomodulatory effects of the novel DNA vaccine were investigated in an experimental vaccinated-challenged model. In addition, long-term immunomodulatory effects of the DNA vaccine were investigated in vaccinated pigs kept at the PRRSV-positive environment until the end of the fattening period. Pigs were vaccinated either prior to or following natural PRRSV infection. ResultThe results indicated that pORF7t could modulate the anti-PRRSV immune responses and promote the control of viral replication in the vaccinated-challenged pigs. Immunized pigs exhibited increased numbers of PRRSV-specific activated CD4+CD25+ lymphocytes, reduced numbers of PRRSV-specific Tregs, and rapid viral clearance following infection. In a long-term study, regardless of the time of vaccination, DNA vaccine could modulate the host immune responses, resulted in enhanced PRRSV-specific IFN-γ producing cells, and reduced numbers of PRRSV-specific Tregs, without evidence of enhanced antibody responses. No vaccine adverse reaction was observed throughout the study. ConclusionThis study revealed the novel concept that PRRSV-specific immunity can be modulated by induction of cell-mediated immunity against the nucleocapsid protein. This concept could potentially benefit the development of PRRSV management and control strategies.

Priming by a novel universal influenza vaccine (Multimeric-001)—A gateway for improving immune response in the elderly population

BackgroundA new vaccine, “Multimeric-001” (M-001) has been recently developed, containing conserved, common linear influenza epitopes that activate both cellular and humoral arms of the immune system against a wide variety of influenza A and B strains. Apart from its direct action, M-001 is an attractive candidate for priming immune responses to seasonal influenza vaccine for the elderly population. The current clinical study was designed to assess M-001's standalone and priming action in participants over 65 years old. Evaluation of standalone action is based on induction of cell mediated immunity (CMI), since M-001 alone does not induce hemagglutinin inhibition (HAI) antibodies. MethodsThis was a two-center, randomized, placebo-controlled study. 120 participants were randomized 1:1:1:1 into four groups to receive either two sequential non-adjuvanted or a single non-adjuvanted or a single adjuvanted intramuscular injection of 500mcg M-001 (treatment), or one placebo (saline) injection, before receiving the trivalent inactivated influenza vaccine (TIV). Due to visual differences between placebo and treatment the study was partially blinded. HAI was evaluated at baseline and 3 weeks after standard TIV vaccination as a measure of M-001's efficacy. CMI responses were evaluated in a subset (10/group) of the participants. Participants were monitored for safety throughout the study. ResultsOverall the treatment was well-tolerated and safe, though sample sizes allowed only limited statistical analysis. M-001 priming resulted in enhanced seroconversion towards all three TIV strains, compared to priming with placebo. Significant elevation of influenza-specific CMI was observed following immunization with M-001 alone. ConclusionsThe standalone and priming actions of M-001 were demonstrated in elderly participants despite the limitations of small population size and pre-existing HAI antibody titers in some participants. As a standalone vaccine, M-001 induced significant CMI to multiple strains and as a primer, M-001 enhanced HAI responses. Larger scale studies are warranted. ClinicalTrials.gov registry numberNCT01419925.

Polyanhydride nanovaccine platform enhances antigen-specific cytotoxic T cell responses

Polyanhydride nanoparticle-based vaccines (or nanovaccines) stabilize protein antigens, provide sustained antigen release leading to prolonged antigen presence, enhance activation of antigen presenting cells, and elicit protective immunity against respiratory infections upon challenge. However, induction of cell-mediated immunity when mice are immunized with polyanhydride nanovaccines has not been evaluated. Using a transgenic ovalbumin-specific T cell adoptive transfer model, we report the induction of antigen-specific cytotoxic CD8+ T cells expressing an effector memory phenotype by seven days after immunization with nanovaccine formulations. Furthermore, mice immunized with polyanhydride nanovaccines demonstrated enhanced recall responses after antigen re-exposure 35 days post-immunization indicating the activation and recruitment of antigen-specific memory CD8+ T cells to the site of antigen deposition.

Immunostimulatory responses to crude extracts of Warburgia ugandensis (Sprague) subsp ugandensis (Canellaceae) by BALB/c mice infected with Leishmania major.

IntroductionTo determine the immunostimulatory potential of crude extracts of Warburgia ugandensis subsp. ugandensis with a soluble leishmanial antigen in vaccinating BALB/c mice.MethodsSeventy two female BALB/c mice were randomly assigned into six groups. The mice were vaccinated with soluble leishmania antigens (SLA) alone, hexane, ethyl acetate, and dichloromethane extract co-administered with SLA. Unvaccinated mice formed the control group. The induction of cell-mediated immunity following vaccination was determined by measuring in vitro lymphocyte proliferation and the production of interleukin (IL)-4 and gamma interferon (IFN-γ) determined by flow cytometry. Protection against L. major was determined by quantifying parasite burdens in L. major infected footpads using a limiting dilution assay and by measuring lesion sizes of the infected footpad compared to the contralateral uninfected footpad.ResultsOn vaccination with extracts of W. ugandensis subsp. ugandensis alone or as adjuvants when used in combination with Leishmania antigens, the hexane extract and the dichloromethane extract plus SLA stimulated moderate production of IFN-γ and low levels of IL-4.These mice were partially protected from cutaneous leishmaniasis as shown by the slow development of lesions and comparatively less parasite burdens.ConclusionThese data suggest that extracts of W. ugandensis subsp. ugandensis are suitable adjuvants for Leishmania vaccines. However, since W. ugandensis subsp. ugandensis has been shown to be effective against Leishmania parasites in vitro and in vivo, further studies ought to be conducted to determine its immunochemotherapeutic potential when co-administered with a soluble leishmanial antigen in vaccinating BALB/c mice.

Role of CD4+ and CD8α+ T cells in protective immunity against Edwardsiella tarda infection of ginbuna crucian carp, Carassius auratus langsdorfii

Edwardsiella tarda is an intracellular pathogen that causes edwardsiellosis in fish. Our previous study suggests that cell-mediated immunity (CMI) plays an essential role in protection against E. tarda infection. In the present study, we adoptively transferred T-cell subsets sensitized with E. tarda to isogenic naïve ginbuna crucian carp to determination the T-cell subsets involved in protecting fish from E. tarda infection. Recipients of CD4+ and CD8α+ cells acquired significant resistance to infection with E. tarda 8 days after sensitization, indicating that helper T cells and cytotoxic T lymphocytes plays crucial roles in protective immunity to E. tarda. Moreover, transfer of sensitized CD8α+ cells up-regulated the expression of genes encoding interferon-γ (IFN-γ) and perforin, suggesting that protective immunity to E. tarda involves cell-mediated cytotoxicity and interferon-γ-mediated induction of CMI. The results establish that CMI plays a crucial role in immunity against E. tarda. These findings provide novel insights into understanding the role of CMI to intracellular pathogens of fish.

Modulation of HIV peptide antigen specific cellular immune response by synthetic α- and β-defensin peptides

Defensin peptides have their direct role in host defense against microbial infection as innate molecules and also thought to contribute to adaptive immunity by recruiting naïve T-cells and immature dendritic cells at the site of infection through CCR6 receptor. The main aim of the present study is to investigate the efficacy of defensins for the induction of cell mediated immune response against the peptide antigen of HIV-1 encapsulated in PLG microparticles through intranasal (IN) route in mice model. To characterized, we have analyzed T-cell proliferation, Th1/Th2 cytokines, β-chemokines production and IFN-γ/perforin secretion from CD4+/CD8+ T-cells in response to HIV immunogen alone and with defensins at different mucosal site i.e. lamina propria (LP), spleen (SP) and peyer's patches (PP). The cellular immunogenicity of HIV peptide with defensin formulations showed a significantly higher (p<0.001) proliferation response as compared to individual HIV peptide. The enhanced cytokines measurement profile showed mixed Th1 and Th2 type of peptide specific immune response by the incorporation of defensins. In the continuation, enhancement in MIP-1α and RANTES level was also observed in HIV peptide–defensin formulations. The FACS data had revealed that CD4+/CD8+ T-cells showed significantly (p<0.001) higher IFN-γ and perforin secretion in HIV with defensin peptide formulations than HIV antigen alone group. Thus, the study emphasized here that defensin peptides have a potential role as mucosal adjuvant, might be responsible for the induction of cell mediated immunity when administered in mice through IN route with HIV peptide antigen.

Plasma beads loaded with Candida albicans cytosolic proteins impart protection against the fungal infection in BALB/c mice

The development of a prophylactic vaccine against systemic candidiasis, employing Candida albicans cytosolic proteins (Cp) as antigen and fibrin cross-linked plasma beads as an antigen bearing dual delivery system is described. Groups of mice were administered either with free Cp, or Cp entrapped in plasma beads, Cp entrapped in liposomes or liposome encapsulated Cp further entrapped in plasma beads. Humoral immunity was studied by measuring the anti-Cp antibody titers in the sera of the immunized animals. Induction of cell-mediated immunity was assessed by delayed type hypersensitivity (DTH), NO production, up-regulation of co-stimulatory molecules viz. CD80, CD86 on APCs on one hand and T-cells proliferation as well as induction of IFN-γ and IL-4 on the other. The efficacy of various vaccine formulations in protecting mice against a lethal challenge with C. albicans, was assessed by determining animal survival rate and fungal burden in the systemic circulation and vital organs. Among various Cp-based vaccines investigated, the preparation containing liposomized Cp entrapped in plasma beads imparted superior protection in the immunized mice as compared to other antigens delivery systems.

The recombinant globular head domain of the measles virus hemagglutinin protein as a subunit vaccine against measles

Despite the availability of live attenuated measles virus (MV) vaccines, a large number of measles-associated deaths occur among infants in developing countries. The development of a measles subunit vaccine may circumvent the limitations associated with the current live attenuated vaccines and eventually contribute to global measles eradication. Therefore, the goal of this study was to test the feasibility of producing the recombinant globular head domain of the MV hemagglutinin (H) protein by stably transfected human cells and to examine the ability of this recombinant protein to elicit MV-specific immune responses. The recombinant protein was purified from the culture supernatant of stably transfected HEK293T cells secreting a tagged version of the protein. Two subcutaneous immunizations with the purified recombinant protein alone resulted in the production of MV-specific serum IgG and neutralizing antibodies in mice. Formulation of the protein with adjuvants (polyphosphazene or alum) further enhanced the humoral immune response and in addition resulted in the induction of cell-mediated immunity as measured by the production of MV H-specific interferon gamma (IFN-γ) and interleukin 5 (IL-5) by in vitro re-stimulated splenocytes. Furthermore, the inclusion of polyphosphazene into the vaccine formulation induced a mixed Th1/Th2-type immune response. In addition, the purified recombinant protein retained its immunogenicity even after storage at 37°C for 2 weeks.

Characterization of Outer Membrane Vesicles fromBrucella melitensisand Protection Induced in Mice

The outer membrane vesicles (OMVs) from smooth B. melitensis 16 M and a derived rough mutant, VTRM1 strain, were purified and characterized with respect to protein content and induction of immune responses in mice. Proteomic analysis showed 29 proteins present in OMVs from B. melitensis 16 M; some of them are well-known Brucella immunogens such as SOD, GroES, Omp31, Omp25, Omp19, bp26, and Omp16. OMVs from a rough VTRM1 induced significantly higher expression of IL-12, TNFα, and IFNγ genes in bone marrow dendritic cells than OMVs from smooth strain 16 M. Relative to saline control group, mice immunized intramuscularly with rough and smooth OMVs were protected from challenge with virulent strain B. melitensis 16 M just as well as the group immunized with live strain B. melitensis Rev1 (P < 0.005). Additionally, the levels of serum IgG2a increased in mice vaccinated with OMVs from rough strain VTRM1 consistent with the induction of cell-mediated immunity.

Exposure of CD34+ precursors to cytostatic anthraquinone-derivatives induces rapid dendritic cell differentiation: implications for cancer immunotherapy.

Appropriate activation of dendritic cells (DC) is essential for successful active vaccination and induction of cell-mediated immunity. The scarcity of precursor cells, as well as long culture methods, have hampered wide-scale application of DC vaccines derived from CD34(+) precursors, despite their suggested superior efficacy over the more commonly applied monocyte-derived DC (MoDC). Here, employing the CD34(+)/CD14(+) AML-derived human DC progenitor cell line MUTZ3, we show that cytostatic anthraquinone-derivatives (i.e., the anthracenedione mitoxantrone and the related anthracyclin doxorubicin) induce rapid differentiation of CD34(+) DC precursors into functional antigen-presenting cells (APC) in a three-day protocol. The drugs were found to act specifically on CD34(+), and not on CD14(+) DC precursors. Importantly, these observations were confirmed for primary CD34(+) and CD14(+) DC precursors from peripheral blood. Mitoxantrone-generated DC were fully differentiated within three days and after an additional 24 h of maturation, were as capable as standard 9-day differentiated and matured DC to migrate toward the lymph node-homing chemokines CCL19 and CCL21, to induce primary allogeneic T cell proliferation, and to prime functional MART1-specific CD8(+) T lymphocytes. Our finding that anthraquinone-derivatives like mitoxantrone support rapid high-efficiency differentiation of DC precursors may have consequences for in vitro production of DC vaccines as well as for novel immunochemotherapy strategies.

Porcine circovirus type 2 (PCV2) Cap and Rep proteins are involved in the development of cell-mediated immunity upon PCV2 infection

The aim of the present study was to investigate the role of the capside (Cap) and replicase (Rep) proteins of Porcine circovirus type 2 (PCV2) as well as the whole PCV2 (both PCV2a and PCV2b genotypes) in the induction of cell-mediated immunity upon infection. At 6 weeks of age, six pigs were intranasally inoculated with the Stoon 1010 (Stoon) isolate (PCV2a) and seven with the Sp-7-10-54-13 (Sp) isolate (PCV2b). None of the pigs developed clinical disease but the Sp group had significantly higher proportion of pigs with PCV2-associated lesions and PCV2 load in tissues compared to the Stoon group. In both groups, development of IFN-γ secreting cells (SC) in response to the whole PCV2 and Cap protein was detected by means of an ELISPOT from day 7 post-inoculation (PI) to the end of the study (21 days PI). Significant responses against Rep protein were only detected in Sp-inoculated pigs. No differences in ELISPOT results were seen when either PCV2a or PCV2b was used in vitro to recall peripheral blood mononuclear cells (PBMC) in any group. Stimulation of PBMC with the whole virus but not with Cap or Rep protein induced IL-10-SC in all pigs regardless of their PCV2 infection status, indicating an innate origin of this response. The results from this study demonstrate that PCV2-infected pigs developed cell-mediated immunity to Cap and Rep proteins and that, in the course of a sub-clinical infection, development and strength of such responses are possibly related to the levels of PCV2 replication.