Anti-peptide antibody production elicited by in vitro immunization of human peripheral blood mononuclear cells

Human monoclonal antibodies have great potential for use in the treatment of various diseases. We have established an in vitro immunization protocol for inducing antigen-specific antibody production from human peripheral blood mononuclear cells (PBMCs). In the in vitro immunization protocol, PBMCs are pretreated with L-leucyl-L-leucine methyl ester (LLME) to remove suppressive cells, and are sensitized and cultured with a soluble antigen in the presence of IL-2, IL-4 and muramyl dipeptide for 8 d, and then an antigen-specific antibody is produced. In this study, we examined the novel possibility of an in vitro immunization protocol, specifically, whether LLME-treated PBMCs can be sensitized with a peptide antigen to produce an anti-peptide antibody. The results indicate that antigen-specific immune responses were elicited by a peptide antigen derived from rice allergen, a cholera toxin B subunit, and TNF-alpha as a sensitizing antigen in in vitro immunization. These results suggest that the in vitro immunization protocol is applicable in the generation of an anti-peptide antibody against various antigens, including food allergens, foreign antigens, and self-antigens.     

Identification of genes involved in the suppression of antibody production from human peripheral blood lymphocytes

Pretreatment with L-leucyl-L-leucine methyl ester (LLME) is a prerequisite for peripheral blood mononuclear cells (PBMCs) to produce antigen-specific antibodies when sensitized with an antigen. Little information, however, is available regarding the mechanisms involved in LLME-induced augmentation of antibody production from PBMCs that are antigen sensitized. In the present study, we attempted to identify the genes involved in the suppression of antibody production from PBMCs that was not treated with LLME, but sensitized with an antigen. Using subtractive screening, we obtained 63 independent genes, including 17 EST genes, that are specific for LLME-nontreated PBMC. Among these genes, the expression of heavy chain ferritin (H-ferritin), CC chemokine ligand 18 (CCL18), and matrix metalloproteinase 12 (MMP12) were augmented in LLME-nontreated PBMCs, suggesting that inflammatory factors might be involved in the suppression of antibody production in LLME-nontreated PBMCs.     

In vitro immunization can elicit the expansion of diverse repertoire of B cells from peripheral blood mononuclear cells

We previously developed an in vitro immunization (IVI) protocol of human peripheral blood mononuclear cells (PBMC) for generating antigen-specific human antibodies. In order to clarify whether IVI protocolinduces antigen-specific B cell responses in PBMC, we analyzed family gene usage and sequence of the variable region gene of immunoglobulin heavy chain (VH gene) of the antibody produced from the in vitro immunized PBMC. Sequence homology analyses of VH gene demonstrated that a larger repertoire of B cells can be sensitized with mite-extract than with cholera toxin B subunit and rice allergen. Further, antigen-specific B cells were efficiently expanded by using CpG oligodeoxynucleotide as adjuvant. These results suggest that appropriate combination of sensitizing antigen and adjuvant is primarily important for expansion of antigen-specific B cells in IVI protocol.     

In vitro immunization of Epstein–Barr virus-immortalized B cells augments antigen-specific antibody production

The current method for in vitro immunization (IVI) uses several antigens including toxins, food allergens, pathogenic bacteria, and self-antigen-derived peptides that induce an antigen-specific immune response in peripheral blood mononuclear cells (PBMCs). This protocol, however, requires donor blood collection and preparation of PBMCs before every IVI. In the present study, we aimed to design a more efficient system utilizing B cells immortalized with Epstein–Barr virus (EBV-B) as host cells for IVI to make antigen-specific antibodies. Results showed that previously antigen-sensitized, EBV-B cells exposed to the antigen along with IL-6, CpG oligonucleotides, and CD40 ligand signal produced antigen-specific antibodies. These results provide evidence for a novel and easy method to expand memory-type B cells and produce antigen-specific antibodies.     

Induction of Systemic and Mucosal Immune Responses to Human Immunodeficiency Virus Type 1 by a DNA Vaccine Formulated with QS-21 Saponin Adjuvant via Intramuscular and Intranasal Routes

Induction of mucosal and cell-mediated immunity is critical for development of an effective vaccine against human immunodeficiency virus (HIV). We compared intramuscular and intranasal immunizations with a DNA vaccine encoding env of HIV-1 and evaluated the QS-21 saponin adjuvant for augmentation of the systemic and mucosal immune responses to HIV-1 in a murine model. Vaccination via the two routes elicited comparable systemic immune responses, and QS-21 consistently enhanced antigen-specific serum immunoglobulin G2a (IgG2a) production, delayed-type hypersensitivity reaction, and cytolytic activity of splenocytes. Intestinal secretory IgA production and cytolytic activity of the mesenteric lymph node cells are preferentially elicited by intranasal immunization, and QS-21 augmented these activities as well. This adjuvant augmented production of interleukin-2 (IL-2) and gamma interferon (IFN-γ) associated with decrease in IL-4 synthesis by antigen-restimulated splenocytes. The serum immunoglobulin subtype profile showed a dominant IgG2a response and less strong IgG1 and IgE production in a QS-21 dose-dependent manner. As expected, enhancements of humoral and cell-mediated immune responses by QS-21 were abrogated by treatment with anti-IL-2 and anti-IFN-γ monoclonal antibodies. These results suggest that the intranasal route of DNA immunization is more efficient than the intramuscular route in inducing mucosal immunity mediated by sIgA and mesenteric lymphocytes. Furthermore, QS-21 is able to act as a mucosal adjuvant in DNA vaccination and demonstrates its immunomodulatory property via stimulation of the Th1 subset. This study emphasizes the importance of the route of immunization and the use of an adjuvant for effective DNA vaccination against HIV-1.     

Involvement of IL-10 in the suppression of antibody production by in vitro immunized peripheral blood mononuclear cells

Previously, we have established an in vitro immunization method to induce antigen-specific antibody-producing B cells. In the present study, we have attempted to clarify the mechanisms that regulate antibody production by in vitro immunized peripheral blood mononuclear cells (PBMC). Freshly isolated PBMC did not induce antibody production following in vitro immunization, but expressed the interleukin (IL)-10 gene. On the other hand, PBMC pretreated with l-leucyl-l-leucine methyl ester (LLME) induced antibody production, but did not express the IL-10 gene. IL-10 induced functional impairment of CD4⁺ Th cells and CD11c⁺ DC, resulting in the suppression of antibody production by in vitro immunized PBMC.     

Generation of a Human Anti-Tumor Necrosis Factor-α Monoclonal Antibody by in Vitro Immunization with a Multiple Antigen Peptide

We developed the in vitro immunization method to induce antigen-specific immune responses in human peripheral blood mononuclear cells (PBMCs). However, when we used a peptide as sensitizing antigen, the antigen-specific immune response was found to be weak, and hence, we could not effectively obtain the antigen-specific antibody gene. In the present study, we attempted to improve the in vitro immunization method by augmenting the immune response to the peptide antigen. We used a multiple antigen peptide for sensitization. In vitro immunization of the multivalent antigen elicited a strong antigen-specific immune response in the PBMCs, and we succeeded in obtaining antigen-specific antibody genes by the phage-display method. Further, by combining the variable-region genes and constant-region genes of human IgG, we obtained four independent human monoclonal antibodies specific for tumor necrosis factor-α. This might be a good strategy for generating antigen-specific human monoclonal antibodies using a peptide antigen.     

Adjuvant effect of killed Propionibacterium acnes on mouse peritoneal B-1 lymphocytes and their early phagocyte differentiation

B-1 lymphocytes are the predominant cells in mouse peritoneal cavity. They express macrophage and lymphocyte markers and are divided into B-1a, B-1b and B-1c subtypes. The role of B-1 cells is not completely clear, but they are responsible for natural IgM production and seem to play a regulatory role. An enriched B-1b cell population can be obtained from non-adherent peritoneal cell cultures, and we have previously demonstrated that these cells undergo differentiation to acquire a mononuclear phagocyte phenotype upon attachment to the substrate in vitro. Nevertheless, the B-1 cell response to antigens or adjuvants has been poorly investigated. Because killed Propionibacterium acnes exhibits immunomodulatory effects on both macrophages and B-2 lymphocytes, we analyzed whether a killed bacterial suspension or its soluble polysaccharide (PS) could modulate the absolute number of peritoneal B-1 cells in BALB/c mice, the activation status of these cells and their ability to differentiate into phagocytes in vitro. In vivo, P. acnes treatment elevated the absolute number of all B-1 subsets, whereas PS only increased B-1c. Moreover, the bacterium increased the number of B-1b cells that were positive for MHC II, TLR2, TLR4, TLR9, IL-4, IL-5 and IL-12, in addition to up-regulating TLR9, CD80 and CD86 expression. PS increased B-1b cell expression of TLR4, TLR9, CD40 and CD86, as well as IL-10 and IL-12 synthesis. Both of the treatments decreased the absolute number of B-1b cells in vitro, suggesting their early differentiation into B-1 cell-derived phagocytes (B-1CDP). We also observed a higher phagocytic activity from the phagocytes that were derived from B-1b cells after P. acnes and PS treatment. The adjuvant effect that P. acnes has on B-1 cells, mainly the B-1b subtype, reinforces the importance of B-1 cells in the innate and adaptive immune responses.     

IL-10 augments antibody production in in vitro immunized lymphocytes by inducing a Th2-type response and B cell maturation

An in vitro immunization (IVI) protocol enables antigen specific antibody production from L-Leucyl-L-Leucine methyl ester (LLME)-treated human peripheral blood lymphocytes (PBL) upon antigen stimulation in the presence of IL-2, IL-4, and muramyl dipeptide. In the course of our studies, we have found that IL-10 added at the antigen sensitization significantly augmented antibody production level from the LLME-treated PBL. In the present study, we tried to demonstrate the role of IL-10 in the augmentation of antibody production in an IVI protocol by clarifying the cytokine expression profiles in CD4(+) and CD8(+) T cells. The results showed that IL-10 skewed the Th1/Th2 balance to Th2-type responses by suppressing Th1-type cytokine production and augmenting Th2-type cytokine production in CD4(+) and CD8(+) T cells, as well as in CD19(+) B cells. Furthermore, IL-10 augmented the expression of CD38, an antigen marker of plasma cells, on B cells, which clearly indicates that IL-10 promoted differentiation and maturation of B cells in an IVI protocol. These results indicate that IL-10 plays an important role in setting the cellular milieu to produce antibodies in an IVI protocol.     

High yields of specific hybridomas obtained by electrofusion of murine lymphocytes immunized in vivo or in vitro

We have studied the use of electrofusion to obtain hybridomas producing antigen-specific antibodies after immunization of murine lymphocytes in vitro. Under optimal conditions fusion frequencies of the order of magnitude of 10(-3) were obtained, which is approximately 80-fold higher than the mean value obtained with fusion induced by polyethylene glycol. The number of antigen-specific hybridomas was also increased in a comparable way. The high yields of specific hybridomas observed with electrofusion were independent of the immunization procedure, the antigen or the hapten of interest, or the sources of the lymphocytes. The data presented in this paper indicate that electrofusion may be an extremely attractive alternative method for immortalization of human lymphocytes following immunization in vitro.     

The Effect of IFN-gamma, Alum and Complete Freund Adjuvant on TNP-KLH Induced Ig.G(1), IgE and IgG(2a) Responses in Mice

Adjuvants are considered to play an important role in directing the isotype and amount of antibodies produced upon immunization by conducting the development of either Th-1 or Th-2 cells upon T-cell stimulation. This is based on the different cytokine production patterns that were observed after in vitro resttmulation of T cells isolated from mice immunized with antigen either adsorbed on alum or emulsified in complete Freund adjuvant (CFA). However, other studies suggest that primarily the type of antigen determines which isotypes are produced and to what extent. In these studies, however, IgE was not determined. Therefore, this study examined whether alum and CFA influenced the amount and/or ratio of IgG(1), IgE and IgG(2a) produced after TNP-KLH immunization. Similar levels of IgG(1), IgE and IgG(2a) antibodies were found upon immunization with TNP-KLH either adsorbed on alum or emulsified in CFA. Moreover, administration of IFN-gamma in combination with TNP-KLH adsorbed on alum did not increase the amount of IgG(2a) produced. IFN-gamma treatment resulted in an increased IL-6 and decreased IFN-gamma production by spleen cells upon Con A stimulation, whereas it did not change the IL-4 production in similar conditions. The presented results suggest that upon immunization with TNP-KLH high IL-4 levels are produced, resulting in an antibody response that is dominated by IgG(1), independent of the adjuvant employed. The IL-4 inducing property of TNP-KLH is substantiated by the finding that repeated immunization of mice with TNP-KI, without adjuvant, increases the serum total IgE level. The presented data suggest that the carrier part of TNP-KLH preferentially results in Th-2 cell activity after which the adjuvant merely enhances the antibody responses generated.     

Antigen-specific humoral and cellular immune responses can be modulated in rhesus macaques through the use of IFN-gamma, IL-12, or IL-18 gene adjuvants

DNA or nucleic acid immunization has been shown to induce both antigen-specific cellular and humoral immune responses in vivo. Moreover, immune responses induced by DNA immunization can be enhanced and modulated by the use of molecular adjuvants. To engineer the immune response in vivo towards more T-helper (Th)1-type cellular responses, we investigated the co-delivery of inteferon (IFN)-gamma, interleukin (IL)-12, and IL-18 genes along with DNA vaccine constructs. We observed that both antigen-specific humoral and cellular immune responses can be modulated through the use of cytokine adjuvants in mice. Most of this work has been performed in rodent models. There has been little confirmation of this technology in primates. We also evaluated the immunomodulatory effects of this approach in rhesus macaques, since non-human primates represent the most relevant animal models for human immunodeficiency virus (HIV) vaccine studies. As in the murine studies, we also observed that each Th1 cytokine adjuvant distinctively regulated the level of immune responses generated. Co-immunization of IFN-gamma and IL-18 in macaques enhanced the level of antigen-specific antibody responses. Similarly, co-delivery of IL-12 and IL-18 also enhanced the level of antigen-specific Th proliferative responses. These results extend this adjuvant strategy in a more relevant primate model and support the potential utility of these molecular adjuvants in DNA vaccine regimens.     

Immunological properties of Propionibacterium acnes. I. Potentiation and Suppression on antibody response to sheep and hamster erythrocytes in mice.

Adjuvant activity of phenol-treated cells of Propionibacterium acnes C-7 in antibody response was investigated in ICR mice. Simultaneous administration (day 0) of P. acnes (i.p.) and sheep red blood cells (SRBC) (i.v.) enhanced the formation of direct plaque-forming cells (PFC) on days 2, and the formation of indirect PFC response on day 7 and thereafter. Conversely, pretreatment from 11 to 14 days before antigen injection suppressed markedly the antibody response. The potentiation and the suppression of immune response depended on doses of antigen and of P. acnes, the timing of adjuvant injection and the time of assay. The two opposite phenomena caused by P. acnes were also confirmed in antibody response against hamster red blood cells (HRBC). Pretreatment with P. acnes 1 to 14 days before antigen injection suppressed markedly anti-HRBC antibody response, whereas P. acnes injected simultaneously with HRBC or one day after injection of the antigen induced prolongation of antibody response and the production of 2-mercaptoethanol-resistant antibody.     

Antibody response to crude cell lysate of propionibacterium acnes and induction of pro-inflammatory cytokines in patients with acne and normal healthy subjects

Propionibacterium acnes (P. acnes) plays an important role in the disease pathogenesis of acne vulgaris, a disorder of pilosebaceous follicles, seen primarily in the adolescent age group. In the present study, the presence of antibodies against P. acnes (MTCC1951) were detected in acne patient (n=50) and disease free controls (n=25) using dot-ELISA and Western blot assay. The ability of P. acnes to induce pro-inflammatory cytokines by human peripheral blood mononuclear cells (PBMCs), obtained from acne patients and healthy subjects, were also analysed. The patients (n=26) who were culture positive for skin swab culture, were found to have a more advanced disease and higher antibody titres (1:4000 to > 1:16000) compared to the P. acnes negative patients (n=24) and normal controls (n=25). An analysis of patients' sera by western blot assay recognized a number of antigenic components of P. acnes, ranging from 29 to 205 kDa. The major reactive component was an approximately 96 kDa polypeptide, which was recognised in 92% (24 of 26) of the patients sera. Further, the P. acnes culture supernatant, crude cell lysate and heat killed P. acnes whole cells, obtained from 72-h incubation culture, were observed to be able to induce significant amounts of IL-8 and tumor necrosis factor alpha (TNF-alpha) by the PBMCs in both the healthy subjects and patients, as analysed by cytokine-ELISA. The levels of cytokines were significantly higher in the patients than the healthy subjects. A major 96 kDa polypeptide reactant was eluted from the gel and was found to cause dose dependent stimulation of the productions of IL-8 and TNF-alpha. Thus, the above results suggest that both humoral and pro-inflammatory responses play major roles in the pathogenesis of acne.     

IL-12-assisted immunization generates CD4+ T cell-mediated immunity to Listeria monocytogenes

Mice infected with virulent Listeria monocytogenes develop long-lived acquired immunity. We previously reported that acquired immunity to Listeria could also be elicited by immunizing mice with non-viable Listeria or listerial proteins/peptides in combination with IL-12. Here we show that this IL-12-assisted immunization strategy was effective in class I but not in class II MHC-deficient mice, suggesting that antigen-specific CD4(+) T cells are selectively generated using this adjuvant system. We have also evaluated the importance of endogenous production of IFN-gamma and IL-12 for the efficacy of IL-12-assisted immunization. IFN-gamma-deficient mice immunized with HKLM and IL-12 failed to produce effective Listeria-specific responses. In contrast, IL-12-deficient mice were able to generate protective antigen-specific T cell responses in response to immunization with HKLM and IL-12, indicating that exogenous IL-12 is sufficient to initiate a cytokine cascade that results in a potent T(H)1 response. IL-12-assisted immunization provides a model in which both the generation and effector mechanisms of anti-bacterial antigen-specific CD4(+) effector cells can be analyzed.     

Requirement for TLR9 in the immunomodulatory activity of Propionibacterium acnes

Propionibacterium acnes (formerly Corynebacterium parvum) is part of the human flora and, as such, is associated with several human pathologies. It possesses strong immunomodulatory activities, which makes this bacterium interesting for prophylactic and therapeutic vaccination. The bacterial component(s) and the host receptor(s) involved in the induction of these activities are poorly understood. We show in this study that TLR9 is crucial in generating the characteristic effects of killed P. acnes priming in the spleen, such as extramedullary hemopoiesis and organ enlargement, and granuloma formation in the liver. Furthermore, the ability to overproduce TNF-alpha and IFN-gamma in response to LPS, lipid A, synthetic lipopeptide Pam(3)CysK(4), or whole killed bacteria was present in P. acnes-primed wild-type, but not TLR9(-/-), mice. Finally, P. acnes priming failed to induce enhanced resistance to murine typhoid fever in TLR9(-/-) mice. Thus, TLR9 plays an essential role in the induction of immunomodulatory effects by P. acnes. Because IFN-gamma is a key mediator of these effects, and enhanced IFN-gamma mRNA expression was absent in spleen and liver of P. acnes-primed TLR9(-/-) mice, we conclude that TLR9 is required for the induction of IFN-gamma by P. acnes.     

Native flagellin does not protect mice against an experimental Proteus mirabilis ascending urinary tract infection and neutralizes the protective effect of MrpA fimbrial protein

Proteus mirabilis expresses several virulence factors including MR/P fimbriae and flagella. Bacterial flagellin has frequently shown interesting adjuvant and protective properties in vaccine formulations. However, native P. mirabilis flagellin has not been analyzed so far. Native P. mirabilis flagellin was evaluated as a protective antigen and as an adjuvant in co-immunizations with MrpA (structural subunit of MR/P fimbriae) using an ascending UTI model in the mouse. Four groups of mice were intranasally treated with either MrpA, native flagellin, both proteins and PBS. Urine and blood samples were collected before and after immunization for specific antibodies determination. Cytokine production was assessed in immunized mice splenocytes cultures. Mice were challenged with P. mirabilis, and bacteria quantified in kidneys and bladders. MrpA immunization induced serum and urine specific anti-MrpA antibodies while MrpA coadministered with native flagellin did not. None of the animals developed significant anti-flagellin antibodies. Only MrpA-immunized mice showed a significant decrease of P. mirabilis in bladders and kidneys. Instead, infection levels in MrpA-flagellin or flagellin-treated mice showed no significant differences with the control group. IL-10 was significantly induced in splenocytes of mice that received native flagellin or MrpA-flagellin. Native P. mirabilis flagellin did not protect mice against an ascending UTI. Moreover, it showed an immunomodulatory effect, neutralizing the protective role of MrpA. P. mirabilis flagellin exhibits particular immunological properties compared to other bacterial flagellins.     

Immunogenic Properties of Colloidal Gold

We studied the capacity of colloidal gold for enhancing specific and nonspecific immune response in laboratory animals (rabbits, rats, and mice) immunized with antigens of various nature. The antibody titers obtained with colloidal gold as a carrier were higher as compared to the standard immunization techniques (free antigen or its combination with Freund's adjuvant). Application of colloidal gold also enhanced nonspecific immune responses, such as lysozyme concentration in the blood, activity of the complement system proteins, as well as phagocytic and bactericidal activities. The antibodies were tested by immunodot assay using gold markers. Immunization of the animals with colloidal gold conjugates with haptens or complete antigens (without other adjuvants) was shown to induce the production of highly active antibodies. In addition, the amount of antigen used for animal immunization with colloidal gold was an order of magnitude lower, compared to immunization with complete Freund's adjuvant. This fact can be evidence for adjuvant properties of colloidal gold proper.     

Adjuvant properties of thermal component of hyperthermia enhanced transdermal immunization: effect on dendritic cells

Hyperthermia enhanced transdermal (HET) immunization is a novel needle free immunization strategy employing application of antigen along with mild local hyperthermia (42°C) to intact skin resulting in detectable antigen specific Ig in serum. In the present study, we investigated the adjuvant effect of thermal component of HET immunization in terms of maturation of dendritic cells and its implication on the quality of the immune outcome in terms of antibody production upon HET immunization with tetanus toxoid (TT). We have shown that in vitro hyperthermia exposure at 42°C for 30 minutes up regulates the surface expression of maturation markers on bone marrow derived DCs. This observation correlated in vivo with an increased and accelerated expression of maturation markers on DCs in the draining lymph node upon HET immunization in mice. This effect was found to be independent of the antigen delivered and depends only on the thermal component of HET immunization. In vitro hyperthermia also led to enhanced capacity to stimulate CD4+ T cells in allo MLR and promotes the secretion of IL-10 by BMDCs, suggesting a potential for Th2 skewing of T cell response. HET immunization also induced a systemic T cell response to TT, as suggested by proliferation of splenocytes from immunized animal upon in vitro stimulation by TT. Exposure to heat during primary immunization led to generation of mainly IgG class of antibodies upon boosting, similar to the use of conventional alum adjuvant, thus highlighting the adjuvant potential of heat during HET immunization. Lastly, we have shown that mice immunized by tetanus toxoid using HET route exhibited protection against challenge with a lethal dose of tetanus toxin. Thus, in addition to being a painless, needle free delivery system it also has an immune modulatory potential.     

Sublingual vaccination with fusion protein consisting of the functional domain of hemagglutinin A of Porphyromonas gingivalis and Escherichia coli maltose-binding protein elicits protective immunity in the oral cavity

This study demonstrated that sublingual immunization with a fusion protein, 25k-hagA-MBP, which consists of a 25-kDa antigenic region of hemagglutinin A purified from Porphyromonas gingivalis fused to maltose-binding protein (MBP) originating from Escherichia coli as an adjuvant, elicited protective immune responses. Immunization with 25k-hagA-MBP induced high levels of antigen-specific serum IgG and IgA, as well as salivary IgA. High level titers of serum IgG and IgA were also induced for almost 1 year. In an IgG subclass analysis, sublingual immunization with 25k-hagA-MBP induced both IgG1 and IgG2b antibody responses. Additionally, numerous antigen-specific IgA antibody-forming cells were detected from the salivary gland 7 days after the final immunization. Mononuclear cells isolated from submandibular lymph nodes (SMLs) showed significant levels of proliferation upon restimulation with 25k-hagA-MBP. An analysis of cytokine responses showed that antigen-specific mononuclear cells isolated from SMLs produced significantly high levels of IL-4, IFN-γ, and TGF-β. These results indicate that sublingual immunization with 25k-hagA-MBP induces efficient protective immunity against P. gingivalis infection in the oral cavity via Th1-type and Th2-type cytokine production.