Spontaneous transgenesis of human B lymphocytes.

DNA can cross the cell membrane by natural means, but the functional relevance of this phenomenon has not been fully elucidated. Here, we analyzed spontaneous transgenesis of human B cells using plasmid DNA coding for a functional immunoglobulin (Ig) heavy chain gene under the control of a B-cell-specific promoter. Using polymerase chain reaction (PCR), reverse transcriptase-PCR, and flow cytometry in combination, spontaneous transgenesis was documented in Burkitt's lymphoma cell lines, Epstein-Barr virus-transformed cell lines, and peripheral blood B lymphocytes of the mature naïve phenotype (IgM(+)/IgD(+)/CD27(-)). By immunoelectron microscopy, the internalized DNA was seen in the lysosomes/late endosomes and in the cytosol proximal to the nucleus. Importantly, spontaneously transgenic B cells processed and presented to major histocompatibility complex (MHC)-restricted T lymphocytes a peptide expressed in the transgenic product. This is the first demonstration that primary B lymphocytes possess a program for the spontaneous internalization of DNA, which in turn imparts the cell with new immunological functions. As spontaneous transgenesis is obtained using a nonviral vector, does not require prior cell activation, and is not associated with chromosomal integration, the findings reported here open new possibilities for genetic manipulations of mature naïve B lymphocytes for therapy and vaccination.

Cross-priming is under control of the relB gene.

Cross-priming is an important mechanism of intercell transfer of antigenic material leading to the specific activation of cytotoxic T lymphocytes. Dendritic cells (DCs) are considered the central antigen-presenting cell in cross-priming. Here we decided to probe the role of the relB gene, a regulator of DC differentiation, in the in vivo cross-priming of a model tumour antigen, TAP(-/-) murine embryo cells (MEC), expressing human adenovirus type 5 early region 1. To this end, we used relB(-/-) mutant mice to generate bone marrow (BM) chimeras as these possess few residual DC but are capable of initiating CD4+ and CD8+ T-cell responses in vivo. Our results show that relB(-/-) BM chimeras are unable to cross-prime CD8+ T cells, suggesting that the relB gene regulates cross-priming.

Circumvention of MHC class II restriction by genetic immunization.

The fate of T cell responses to peptide-based vaccination is subject to constraints by the major histocompatibility complex (MHC), MHC restriction. Using as a model system of T and B cell epitopes from the circumsporozoite protein of Plasmodium falciparum malaria parasite, we show that vaccination by somatic transgene immunization readily primes Balb/c mice (H-2(d)) a strain previously reported to be non-responder to immunization with a synthetic peptide vaccine encompassing these epitopes. Following genetic vaccination Balb/c mice developed a primary T cell response comparable to that of the responder strain C57Bl/6 (H-2(b)). Following booster immunization on day 45 Balb/c mice responded with a typical T cell memory response. Priming induced the formation of specific antibodies, which rose sharply after booster immunization. These findings suggests that genetic immunization can circumvent MHC class II restriction.

Studies on CD4 T cell immunity using somatic transgene immunization.

This review presents and discusses our recent data on the use of somatic transgene immunization in the induction of CD4 T cell responses in vivo. Somatic transgene immunization is a process that targets B lymphocytes resident in the spleen with DNA coding for antigenized immunoglobulin H chain genes. After transgenesis B lymphocytes function as antigen-producing and antigen-presenting cells. The studies reviewed herein describe the characteristics of the primary and memory CD4 response against a dominant Th cell determinant. In addition, they show how ad hoc modifications of the transgene result in the induction of a CD4 T cell response against Th cell determinants against which a response is normally not obtained. The new concept Th-Th cooperation is discussed.

Functional cooperation between T helper cell determinants.

The immune response to T helper (Th) cell determinants of a variety of antigens is often poor and limits severely the potential efficacy of current therapeutic measures through vaccination. Here, we report that an immunologically silent tumor determinant can be rendered immunogenic if linked with a dominant determinant of a parasite antigen, suggesting the existence of functional Th-Th cooperation in vivo. This phenomenon could be mimicked in part by signaling either through CD40 to the antigen-presenting cells or through OX40 to the tumor-determinant reactive T cells, with maximal effects obtained by combined anti-CD40 and anti-OX40 treatment in vivo. The data suggest that CD4 T cells reactive with a dominant determinant provide help to other CD4 T cells through up-regulating the costimulatory ability of antigen-presenting cells, in much the same way as help for CD8 cells. CD4 help for CD4 T cells represents a new immunological principle and offers new practical solutions for vaccine therapy against cancer and other diseases in which antigenic help is limiting.

Activation of CD4 T cells by somatic transgenesis induces generalized immunity of uncommitted T cells and immunologic memory.

Cellular immune responses were analyzed in vivo after a single intraspleen inoculation of DNA coding for a 12-residue Th cell determinant associated with a 12-residue B cell epitope, a process termed somatic transgene immunization. We show that CD4 T cells are readily activated and produce IL-2, IFN-gamma and IL-4, characteristics of an uncommitted phenotype. Linked recognition of the two epitopes coded in the same transgene promoted IgM-IgG1 switch and enhanced the total Ab response but had no effect on IgG2a Abs. Although originating in the spleen, T cell responsiveness was found to spread immediately and with similar characteristics to all lymph nodes in the body. A single inoculation was also effective in establishing long term immunologic memory as determined by limiting dilution analysis, with memory T cells displaying a cytokine profile different from that of primary effector T cells. These studies provide evidence that by initiating immunity directly in secondary lymphoid organs, an immune response is generated with characteristics that differ from those using vaccines of conventional DNA or protein in adjuvant administered in peripheral sites. Somatic transgene immunization can therefore be used to probe T cell responsiveness in vivo and represents a tool to further understanding of the nature of the adaptive immune response.

A transphyletic anti-infectious control strategy based on the killer phenomenon.

A strategy for the prevention and control of candidiasis, pneumocystosis, and tuberculosis, based on the idiotypic network of the yeast killer effect has been envisaged. Anti-idiotypic antibodies representing the internal image of a candidacidal, pneumocysticidal, and mycobactericidal killer toxin from Pichia anomala and idiotypes of killer toxin-neutralizing monoclonal antibodies mimicking the specific cell wall receptor of sensitive microorganisms might provide a unique approach for engineering innovative antibiotics and vaccines active against taxonomically unrelated pathogenic microorganisms. The rationale of the strategy relies on a phenomenon of microbial competition which has been mutated by the immune system in the response to natural infections.

Immunological memory after somatic transgene immunization is positively affected by priming with GM-CSF and does not require bone marrow-derived dendritic cells.

Inoculation of plasmid DNA is a promising vaccination approach but optimal regimes and ways to enhance immunogenicity remain to be established. Among natural immunological adjuvants, granulocyte-macrophage colony-stimulating factor (GM-CSF) was shown to increase the potency of immunization against tumor cells and protein antigens. Here we studied the effect of GM-CSF on memory responses against a 12-mer B cell epitope in mice primed with a single DNA inoculation. The results show that GM-CSF given at priming as a DNA/GM-CSF chimeric vaccine enhances the magnitude of the anamnestic response irrespective of the form of antigen used subsequently in the booster immunization. Using mice lacking bone marrow-derived dendritic cells we also determined that the enhancing effect is not strictly dependent on these cells. These results expand our understanding of the activity of GM-CSF in vivo as a modulator of the immune response including immunological memory.

Durable immunity and immunologic memory to a parasite antigen induced by somatic transgene immunization.

Somatic transgene immunization (STI) is an alternative approach to immunization mediated by inoculation of plasmid DNA. In the experiments presented here we show that inoculation of plasmid DNA carrying an immunoglobulin heavy chain gene under the control of tissue-specific regulatory elements, leads to immunity and persistent immunologic memory against a peptide epitope encoded in the third complementarity-determining region. The epitope consists in three repeats of the tetrapeptide Asn-Ala-Asn-Pro (NANP) and is the immunodominant B cell epitope expressed at the surface of Plasmodium falciparum malaria parasite. When inoculated directly in the spleen the plasmid DNA initiated a specific anti-NANP response which lasted for 2 years. During the initial phase of priming the anti-NANP response was higher than that induced by immunization with recombinant protein in immunologic adjuvants. The establishment of immunologic memory was probed by single booster injection at various times after priming. We found that STI induces persistent immunologic memory up to 2 years. The immunologic characteristics of this new model are examined with respect to the requirement for the induction of B cell memory.

DNA immunization in relB-deficient mice discloses a role for dendritic cells in IgM-->IgG1 switch in vivo.

A single intraspleen inoculation of plasmid DNA coding for an immunoglobulin heavy chain gene initiates immunity and establishes immunologic memory against the antigenic determinants of transgenic immunoglobulins, somatic transgene immunization. During priming mice produce IgM but not IgG1 antibodies. Since IgM --> IgG1 class switch occurs spontaneously during the primary immune response to protein antigens we investigated possible mechanisms for failure of spontaneous isotype switch in vivo in this model of immunity. We found that inoculation of plasmid DNA in the form of a chimeric gene coding for granulocyte-macrophage colony-stimulating factor (GM-CSF) was able to drive IgG1 class switch readily after priming. Since GM-CSF activates cells of the dendritic lineage we tested the possibility that dendritic cells (DC) may be involved in regulating IgM --> IgG1 switch. To this end we used bone marrow chimeras constructed from mice carrying the null mutation for the relB member of the NF-kappaB/Rel family as these mice lack bone marrow-derived mature DC. RelB (-/-) mice and (-/-) bone marrow chimeras inoculated with DNA/GM-CSF did not produce IgG1 antibodies during the primary immune response. Since relB (-/-) bone marrow chimeras lack DC of donor origin but possess resident follicular dendritic cells we conclude that Ig class switch in vivo is regulated by the function of interdigitating dendritic cells (IDC). Thus, IDC may contribute to the qualitative aspects of the emerging immune response.

Mycobactericidal activity of human natural, monoclonal, and recombinant yeast killer toxin-like antibodies.

Human natural (KTAb), murine monoclonal (KTMAb), and single-chain recombinant (KTScFv) candidacidal antibodies representing the internal image of a killer toxin from the yeast Pichia anomala (KT), characterized by a wide spectrum of antibiotic activity, exerted a lethal effect against a KT-sensitive multidrug-resistant isolate of Mycobacterium tuberculosis. KTMAb and KTScFv were produced by the hybridoma and DNA technologies, respectively, from the spleen lymphocytes of animals immunized with the idiotype of a KT-neutralizing MAb (MAb KT4), while KTAb were purified against MAb KT4 from the vaginal fluid of women infected with Candida albicans cells bearing an idiotype-like KT cell wall receptor. Mycobactericidal activity was related to the binding of KTAb, KTMAb, and KTScFv to the cell surface of KT-sensitive bacterial cells and was prevented by specific absorption of KT-like antibodies onto MAb KT4. These data identify a novel potentially useful immunotherapeutic approach to tuberculosis.

Immunity to Plasmodium falciparum malaria sporozoites by somatic transgene immunization.

Immunity against the human malaria parasite Plasmodium falciparum was induced using somatic transgene immunization, a method to effectively target B lymphocytes in vivo. A single inoculation of plasmid DNA containing an immunoglobulin heavy-chain gene coding in the complementarity-determining region 3 for three repeats of the sequence Asn-Ala-Asn-Pro (NANP), a B-cell epitope of P.falciparum sporozoites, induced antibodies against NANP in all mice. A booster with an antibody antigenized with the NANP peptide, or challenge with P. falciparum sporozoites, demonstrated the establishment of immunologic memory. Immunity to a parasite antigen can be induced by exploiting mechanisms in which B lymphocytes are both the source of the immunogen as well as the effector mechanism of immunity. The results indicate that somatic transgene immunization is a potential approach for vaccination against foreign pathogens.

Engineering vaccines with heterologous B and T cell epitopes using immunoglobulin genes.

Antibodies engineered in their variable domain to express epitopes of heterologous antigens-antigenized antibodies-function as immunogens. Only the third complementarity-determining region (CDR3) of the H chain has been used as the site of epitope expression, as this loop has the highest natural variability in length and amino acid composition. We demonstrate that the CDR2 can be engineered to express a 12-amino acid peptide, which is a T-cell determinant that enhances the response to a B-cell epitope peptide of Plasmodium falciparum expressed in the CDR3 of the same variable domain. Mice with this gene inoculated into the spleen mounted an antibody response against the B-cell epitope higher than mice receiving the gene coding for the B-cell epitope only. In vitro studies established that the two epitopes were independently immunogenic in vivo.

Yeast killer systems.

The killer phenomenon in yeasts has been revealed to be a multicentric model for molecular biologists, virologists, phytopathologists, epidemiologists, industrial and medical microbiologists, mycologists, and pharmacologists. The surprisingly widespread occurrence of the killer phenomenon among taxonomically unrelated microorganisms, including prokaryotic and eukaryotic pathogens, has engendered a new interest in its biological significance as well as its theoretical and practical applications. The search for therapeutic opportunities by using yeast killer systems has conceptually opened new avenues for the prevention and control of life-threatening fungal diseases through the idiotypic network that is apparently exploited by the immune system in the course of natural infections. In this review, the biology, ecology, epidemiology, therapeutics, serology, and idiotypy of yeast killer systems are discussed.

In vivo role of B lymphocytes in somatic transgene immunization.

Immunity generated by in vivo inoculation of plasmid DNA is a straightforward and potentially valuable new approach to immunization. Little is known about the type of cells involved, the various immunological aspects, and the destiny of the transgene. In this report, we describe a system in which immunity is the result of in vivo targeting of B lymphocytes. This was accomplished using plasmid DNA encoding an immunoglobulin heavy-chain gene under the control of immunoglobulin promoter and enhancer elements. We show persistence of the transgene in splenic B lymphocytes for at least 3 months, i.e., the average life span of long-lived B lymphocytes in the mouse. The transgene could not be detected in any other lymphoid or nonlymphoid organs over a period of 6 months. We also established that the transgene is integrated in the host DNA. These studies bring new understanding to the events underlying the in vivo use of plasmid DNA. Moreover, the characteristics of this new approach make somatic transgene immunization a model system to study the immunogenicity of endogenous antigens in adult animals.

Somatic transgene immunization with DNA encoding an immunoglobulin heavy chain.

A plasmid DNA containing a chimeric immunoglobulin heavy-chain gene with tissue-specific promoter and enhancer elements was used as a model system to study the events triggered by a single intraspleen DNA inoculation in adult C57Bl/6 mice. A single intraspleen inoculation was followed in a week by secretion of transgene immunoglobulins and production of immunoglobulin M (IgM) anti-immunoglobulins. Their kinetics of serum appearance were almost superimposable. While anti-immunoglobulin antibodies remained detectable for over 6 months, transgene immunoglobulins disappeared after 3-4 weeks. However, transgene mRNA was detected in the spleen for 4 months. A multiplex polymerase chain reaction (PCR) analysis on splenic genomic DNA 17 days after inoculation demonstrated that the transgene was integrated in the host chromosomal DNA. The nucleotide sequence of the rearranged VDJ region from splenic genomic DNA was identical to that of the parental plasmid DNA, hence ruling out that hypermutation had occurred. A booster injection of immunoglobulin encoded by the transgene on day 200 elicited a typical secondary immune response with IgG1 and IgG2b antibodies. These results demonstrate that a single inoculation of an immunoglobulin heavy-chain DNA targeted to spleen lymphocytes leads to spontaneous integration of the transgene into the host DNA, and that this is sufficient to initiate immunity and establish immunologic memory. Our data also show that minute amounts (<100 ng/ml) of an endogenously produced protein secreted in the microenvironment of a lymphoid tissue generate immunity and establish immunologic memory rather than tolerance.

Monoclonal yeast killer toxin-like candidacidal anti-idiotypic antibodies.

Rat monoclonal yeast killer toxin (KT)-like immunoglobulin M (IgM) anti-idiotypic antibodies (KT-IdAbs) were produced by idiotypic vaccination with a mouse monoclonal antibody (MAb; MAb KT4) that neutralized a Pichia anomala KT characterized by a wide spectrum of antimicrobial activity. The characteristics of the KT-IdAbs were demonstrated by their capacity to compete with the KT to the idiotype of MAb KT4 and to interact with putative KT cell wall receptors (KTRs) of sensitive Candida albicans cells. The internal-image properties of KT-IdAbs were proven by their killer activity against KT-sensitive yeasts. This lethal effect was abolished by prior adsorption of KT-IdAbs with MAb KT4. These findings stressed the potential importance of antibody-mediated immunoprotection against candidiasis and suggested a feasible experimental approach for producing antimicrobial receptor antibodies without purifying the receptor. KT-IdAbs might represent the basis for producing engineered derivatives with a high potential for effective therapeutic antifungal activity.

Therapeutic potential of antiidiotypic single chain antibodies with yeast killer toxin activity.

Single chain fragment (ScFv) antiidiotypic antibodies (antilds) of a killer toxin (KT) from the yeast Pichia anomala have been produced by recombinant DNA methodology from the splenic lymphocytes of mice immunized by idiotypic vaccination with a KT-neutralizing monoclonal antibody (Mab KT4). ScFv KT-like antilds (KTIdAb) react with specific Candida albicans KT cell wall receptors (KTR) exerting a candidacidal activity in vitro could be neutralized by adsorption with Mab KT4. ScFv KTIdAb displayed an effective therapeutic activity in an experimental model of rat candidal vaginitis.

Candida albicans stress mannoprotein, SMP200, enhances tumour necrosis factor secretion in the murine macrophage cell line ANA-1.

Tumour necrosis factor (TNF) secretory activity has been studied in the murine macrophage cell line ANA-1 following in vitro exposure to Candida albicans 200 kDa stress mannoprotein (SMP200). Treatment of ANA-1 murine macrophages with 200 kDa stress mannoprotein results in increased TNF secretion. The phenomenon is (i) dose- and time-dependent, (ii) abrogated by 200 kDa stress mannoprotein preincubation with a specific monoclonal antibody, and (iii) dependent on intact murine macrophage Ca2+/calmodulin-dependent protein kinase function.

Human natural yeast killer toxin-like candidacidal antibodies.

A murine mAb (mAbKT4, IgG1) that neutralized in vitro the anti-Candida activity of a killer toxic (KT) from the yeast Pichia anomala acted as an idiotypic (Id) vaccine in eliciting anti-Id Abs with toxin-like activity (KT-IdAb) in a rat vaginitis model. In this study, we demonstrate that intravaginal or intragastric inoculations of Candida albicans bearing a receptor for the toxin was able to recall KT-IdAb production in the vagina of the animals primarily immunized with mAbKT4 and also to elicit by themselves an Ab that functionally mimicked the KT (KTAb). Anti-Id-like, KT-like Abs were also consistently found in the vaginal fluid of human vaginitis patients who were infected by Candida but who had never been exposed to the Id vaccine. These Abs were as candidacidal in vitro as those raised in rat vagina by the Id vaccination, and, likewise, their cytocidal effect was totally neutralized by previous reaction with mAbKT4. Importantly, they were also able to confer a significant anticandidal protection in the rat vaginitis model, comparable to that achievable by KT-IdAb passively transferred to naive rats from Id-vaccinated animals. Thus, candidacidal Abs representing the internal image of a yeast KT are part of the Ab repertoire that follows infection or immunization with Candida. It is speculated that the host's immune system response may exploit the KT receptor of microbial pathogens to produce microbicidal Abs, possibly mirroring competition events among microorganisms in natural habitats.