Bacterial Vector Research Articles

Attenuated Listeria monocytogenes: a powerful and versatile vector for the future of tumor immunotherapy.

For over a century, inactivated or attenuated bacteria have been employed in the clinic as immunotherapies to treat cancer, starting with the Coley's vaccines in the 19th century and leading to the currently approved bacillus Calmette-Guérin vaccine for bladder cancer. While effective, the inflammation induced by these therapies is transient and not designed to induce long-lasting tumor-specific cytolytic T lymphocyte (CTL) responses that have proven so adept at eradicating tumors. Therefore, in order to maintain the benefits of bacteria-induced acute inflammation but gain long-lasting anti-tumor immunity, many groups have constructed recombinant bacteria expressing tumor-associated antigens (TAAs) for the purpose of activating tumor-specific CTLs. One bacterium has proven particularly adept at inducing powerful anti-tumor immunity, Listeria monocytogenes (Lm). Lm is a gram-positive bacterium that selectively infects antigen-presenting cells wherein it is able to efficiently deliver tumor antigens to both the MHC Class I and II antigen presentation pathways for activation of tumor-targeting CTL-mediated immunity. Lm is a versatile bacterial vector as evidenced by its ability to induce therapeutic immunity against a wide-array of TAAs and specifically infect and kill tumor cells directly. It is for these reasons, among others, that Lm-based immunotherapies have delivered impressive therapeutic efficacy in preclinical models of cancer for two decades and are now showing promise clinically. In this review, we will provide an overview of the history leading up to the development of current Lm-based immunotherapies, the advantages and mechanisms of Lm as a therapeutic vaccine vector, the preclinical experience with Lm-based immunotherapies targeting a number of malignancies, and the recent findings from clinical trials along with concluding remarks on the future of Lm-based tumor immunotherapies.

Recombinant fusion protein of cholera toxin B subunit with YVAD secreted by Lactobacillus caseiinhibits lipopolysaccharide-induced caspase-1 activation and subsequent IL-1 beta secretion in Caco-2 cells

BackgroundLactobacillus species are used as bacterial vectors to deliver functional peptides to the intestine because they are delivered live to the intestine, colonize the mucosal surface, and continue to produce the desired protein. Previously, we generated a recombinant Lactobacillus casei secreting the cholera toxin B subunit (CTB), which can translocate into intestinal epithelial cells (IECs) through GM1 ganglioside. Recombinant fusion proteins of CTB with functional peptides have been used as carriers for the delivery of these peptides to IECs because of the high cell permeation capacity of recombinant CTB (rCTB). However, there have been no reports of rCTB fused with peptides expressed or secreted by Lactobacillus species. In this study, we constructed L. casei secreting a recombinant fusion protein of CTB with YVAD (rCTB–YVAD). YVAD is a tetrapeptide (tyrosine–valine–alanine–aspartic acid) that specifically inhibits caspase-1, which catalyzes the production of interleukin (IL)-1β, an inflammatory cytokine, from its inactive precursor. Here, we examined whether rCTB–YVAD secreted by L. casei binds to GM1 ganglioside and inhibits caspase-1 activation in Caco-2 cells used as a model of IECs.ResultsWe constructed the rCTB–YVAD secretion vector pSCTB–YVAD by modifying the rCTB secretion vector pSCTB. L. casei secreting rCTB–YVAD was generated by transformation with pSCTB–YVAD. Both the culture supernatant of pSCTB–YVAD-transformed L. casei and purified rCTB–YVAD bound to GM1 ganglioside, as did the culture supernatant of pSCTB-transformed L. casei and purified rCTB. Interestingly, although both purified rCTB–YVAD and rCTB translocated into Caco-2 cells, regardless of lipopolysaccharide (LPS), only purified rCTB–YVAD but not rCTB inhibited LPS-induced caspase-1 activation and subsequent IL-1β secretion in Caco-2 cells, without affecting cell viability.ConclusionsThe rCTB protein fused to a functional peptide secreted by L. casei can bind to GM1 ganglioside, like rCTB, and recombinant YVAD secreted by L. casei may exert anti-inflammatory effects in the intestine. Therefore, rCTB secreted by L. casei has potential utility as a vector for the delivery of YVAD to IECs.

Approaches for sampling human antibodies for repertoire analysis and reagent development (VAC6P.953)

Abstract Purpose: Majority of vaccines rely on Human humoral response for protection. Sampling antibody response is useful for understanding protection correlates of vaccine and isolating reagent antibodies for lifecycle management. We used 3 approaches delineated below to isolate Rubella neutralizing antibodies from plasmablast B-cells separated from human subjects recently vaccinated with MMRII vaccine. Direct B cell cloning: Plasmablast B cells isolated from donors at day 7 after vaccination were used directly as templates in the RT-PCR amplification of antibody genes. Antigen-specific Fabs were isolated following molecular cloning and ELISA screening. Deep Sequencing: RNA extracted from plasmablast B cells was used as template in RT-PCR amplification. The PCR products of antibody genes were subjected to deep sequencing, retrieved antibody VH, VL and VK sequences were rank ordered based on the frequency and, top antibody sequences in each category were expressed as Fabs and tested. Immune Library Construction/Phage Display: Antibody heavy and light chain variable regions from plasmablast B cells were cloned into bacterial phage display vectors to make immune libraries, specific Fabs were identified following bio-panning. Result: All together 25 specific antibodies against Rubella were isolated through the above approaches, the best Fab ELISA binder had an EC50 of 3.5ng/mL (0.07nM). Two of the Fabs demonstrated neutralizing activity with EC50’s of 1µg/mL (20nM) and 4µg/mL (80nM).

Vaccine self-assembling immune matrix (VacSIM) is a non-viral delivery platform that augments responses to recombinant protein vaccines (VAC6P.940)

Abstract Vaccination remains the most effective public health tool to prevent infectious disease. However, many vaccines remain marginally effective, especially for immune-compromised populations. To enhance vaccine immunogenicity, we exploited the biphasic property of certain synthetic oligopeptides to create a new vaccine delivery method, VacSIM (vaccine self-assembling immune matrix). Vaccine components are easily mixed with the VacSIM solution for injection, after which the peptides self-assemble into hydrated nanofiber gel-matrices, forming a vaccine depot. Thus, we have a non-viral, inert, biodegradable delivery system, not requiring formulation, which we can use to deliver a multitude of vaccines. Analogous to a treat-dispensing dog toy, VacSIM is a carrier for vaccine components, allowing for gradual release, rather than immediate administration. We believe this depot attracts antigen presenting cells, driving enhanced vaccine-specific responses that improve both the quantity and quality of the response. We have tested this delivery platform with live bacterial vectors, inactivated virus and multiple recombinant protein vaccines. Shown here, VacSIM augments vaccine responses to the recombinant Hepatitis B surface antigen and recombinant HIV Envelope in 2 strains of mice. Enhancement of vaccine responses by VacSIM administration could represent a fundamental paradigm shift in how vaccines are delivered.

Cell-penetrating peptides mediated protein cross-membrane delivery and its use in bacterial vector vaccine

It is an attractive strategy to develop a recombinant bacterial vector vaccine by expressing exogenous protective antigen to induce the immune response, and the main concern is how to enhance the cellular internalization of antigen produced by bacterial vector. Cell-penetrating peptides (CPPs) are short cationic/amphipathic peptides which facilitate cellular uptake of various molecular cargoes and therefore have great potentials in vector vaccine design. In this work, eleven different CPPs were fused to the C-terminus of EGFP respectively, and the resultant EGFP-CPP fusion proteins were expressed and purified to assay their cross-membrane transport in macrophage J774 A.1 cells. Among the tested CPPs, TAT showed an excellent capability to deliver the cargo protein EGFP into cytoplasm. In order to establish an efficient antigen delivery system in Escherichia coli, the EGFP-TAT synthesis circuit was combined with an in vivo inducible lysis circuit PviuA-E in E. coli to form an integrated antigen delivery system, the resultant E. coli was proved to be able to lyse upon the induction of a mimic in vivo signal and thus release intracellular EGFP-TAT intensively, which were assumed to undergo a more efficient intracellular delivery by CPP to evoke protective immune responses. Based on the established antigen delivery system, the protective antigen gene flgD from an invasive intracellular fish pathogen Edwardsiella tarda EIB202, was applied to establish an E. coli recombinant vector vaccine. This E. coli vector vaccine presented superior immune protection (RPS = 63%) under the challenge with E. tarda EIB202, suggesting that the novel antigen delivery system had great potential in bacterial vector vaccine applications.

Precise and in situ genetic humanization of 6 Mb of mouse immunoglobulin genes

Genetic humanization, which involves replacing mouse genes with their human counterparts, can create powerful animal models for the study of human genes and diseases. One important example of genetic humanization involves mice humanized for their Ig genes, allowing for human antibody responses within a mouse background (HumAb mice) and also providing a valuable platform for the generation of fully human antibodies as therapeutics. However, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which they were genetically humanized. Heretofore, most genetic humanizations have involved disruption of the endogenous mouse gene with simultaneous introduction of a human transgene at a new and random location (so-called KO-plus-transgenic humanization). More recent efforts have attempted to replace mouse genes with their human counterparts at the same genetic location (in situ humanization), but such efforts involved laborious procedures and were limited in size and precision. We describe a general and efficient method for very large, in situ, and precise genetic humanization using large compound bacterial artificial chromosome-based targeting vectors introduced into mouse ES cells. We applied this method to genetically humanize 3-Mb segments of both the mouse heavy and κ light chain Ig loci, by far the largest genetic humanizations ever described. This paper provides a detailed description of our genetic humanization approach, and the companion paper reports that the humoral immune systems of mice bearing these genetically humanized loci function as efficiently as those of WT mice.

Optimization of a One-Step Heat-Inducible In Vivo Mini DNA Vector Production System

While safer than their viral counterparts, conventional circular covalently closed (CCC) plasmid DNA vectors offer a limited safety profile. They often result in the transfer of unwanted prokaryotic sequences, antibiotic resistance genes, and bacterial origins of replication that may lead to unwanted immunostimulatory responses. Furthermore, such vectors may impart the potential for chromosomal integration, thus potentiating oncogenesis. Linear covalently closed (LCC), bacterial sequence free DNA vectors have shown promising clinical improvements in vitro and in vivo. However, the generation of such minivectors has been limited by in vitro enzymatic reactions hindering their downstream application in clinical trials. We previously characterized an in vivo temperature-inducible expression system, governed by the phage λ pL promoter and regulated by the thermolabile λ CI[Ts]857 repressor to produce recombinant protelomerase enzymes in E. coli. In this expression system, induction of recombinant protelomerase was achieved by increasing culture temperature above the 37°C threshold temperature. Overexpression of protelomerase led to enzymatic reactions, acting on genetically engineered multi-target sites called “Super Sequences” that serve to convert conventional CCC plasmid DNA into LCC DNA minivectors. Temperature up-shift, however, can result in intracellular stress responses and may alter plasmid replication rates; both of which may be detrimental to LCC minivector production. We sought to optimize our one-step in vivo DNA minivector production system under various induction schedules in combination with genetic modifications influencing plasmid replication, processing rates, and cellular heat stress responses. We assessed different culture growth techniques, growth media compositions, heat induction scheduling and temperature, induction duration, post-induction temperature, and E. coli genetic background to improve the productivity and scalability of our system, achieving an overall LCC DNA minivector production efficiency of ∼90%.We optimized a robust technology conferring rapid, scalable, one-step in vivo production of LCC DNA minivectors with potential application to gene transfer-mediated therapeutics.

Engineering Hydrogen Gas Production from Formate in a Hyperthermophile by Heterologous Production of an 18-Subunit Membrane-bound Complex

Biohydrogen gas has enormous potential as a source of reductant for the microbial production of biofuels, but its low solubility and poor gas mass transfer rates are limiting factors. These limitations could be circumvented by engineering biofuel production in microorganisms that are also capable of generating H2 from highly soluble chemicals such as formate, which can function as an electron donor. Herein, the model hyperthermophile, Pyrococcus furiosus, which grows optimally near 100 °C by fermenting sugars to produce H2, has been engineered to also efficiently convert formate to H2. Using a bacterial artificial chromosome vector, the 16.9-kb 18-gene cluster encoding the membrane-bound, respiratory formate hydrogen lyase complex of Thermococcus onnurineus was inserted into the P. furiosus chromosome and expressed as a functional unit. This enabled P. furiosus to utilize formate as well as sugars as an H2 source and to do so at both 80° and 95 °C, near the optimum growth temperature of the donor (T. onnurineus) and engineered host (P. furiosus), respectively. This accomplishment also demonstrates the versatility of P. furiosus for metabolic engineering applications.

Expression of Porcine Respiratory and Reproductive Syndrome Virus Membrane-Associated Proteins in Listeria ivanovii via a Genome Site-Specific Integration and Expression System

Listeria ivanovii (LI) possesses a facultative intracellular life cycle in macrophages and non-professional phagocytes thus making it a potential novel vaccine vector. We have developed a genome site-specific integration system as a universal tool for constructing the live LI-based vaccines. Using this system, the porcine reproductive and respiratory syndrome virus (PRRSV) orf5 and orf6 genes, coding for PRRSV membrane-associated protein GP5 and M, respectively, were integrated into the LI genome. Genome PCR and sequencing results showed that the orf5 or orf6 gene had been integrated into the LI genome downstream of orfXYZ or mpl gene. Western blot analysis of the non-secreted and secreted bacterial protein samples showed the expression and secretion of GP5 or M protein from the recombinant LI strains. The secretion of the heterologous proteins is a key step for bacterial vaccine vectors to deliver the antigens to the immune system to stimulate antigen-specific immune responses. Therefore, this integration system may be a potential tool for constructing novel vaccines using live LI as vector.

Processionary Caterpillar Setae and Equine Fetal Loss

Six pregnant Standardbred mares aged between 6 and 14 years were gavaged with 50 g or 100 g of suspended emulsified whole Processionary caterpillars (Ochrogaster lunifer) for 5 days during 2 experiments undertaken to study the etiology of equine amnionitis and fetal loss (EAFL). The 6 treated mares and 1 untreated mare were between 128 and 252 days gestation. Mare 1 (untreated) was euthanized on day 5 of the treatment period, while the treated mares were euthanized on days 2, 4, 8, 10, 12, and 24 days from their first treatment. Caterpillar setae were not found in the untreated mare. Setal fragments were present in all regions of the gastrointestinal tracts in all treated mares, the uteri and mesenteric lymph nodes of 5 mares, and the liver of 4 mares. Acute gastroenteritis of varying severity was present in all treated mares, and 5 of 6 mares had acute colitis and endometritis. Focal hyperplastic serositis was found in the duodenum, cecum, dorsal colon, and uteri of various mares occasionally with embedded setal fragments. Setal invasion of the mucosa evoked a range of lesions including superficial erosion to deep ulceration. Inflammation in deeper tissues ranged from unapparent to neutrophilic (microabscesses), eosinophilic, or mononuclear (microgranulomas). The finding of setal fragments within the uterus of experimental mares suggests that direct migration of setal fragments acting as a bacterial vector is a likely mechanism for the bacterial abortions that characterize equine amnionitis and fetal loss.

Induction of Protein Expression Within Escherichia coli Vector for Entry into Mammalian Cells

Direct protein delivery into the cytosol of mammalian cells by invasive Escherichia coli (E. coli) bacterial vector will bypass the need to achieve nuclear entry and transcription of DNA, a major hurdle that is known to seriously limit gene transfer. The bacterial vector is induced to express the protein during its growth phase, before presentation for entry into mammalian cells and release of its content into the cellular environment. For this class of vector, crossing the plasma membrane becomes the primary step that determines the success of protein delivery. Yet, how the mechanics of protein expression within the vector affect its entry into the host is poorly understood. We found the vector's effectiveness to enter HeLa cells diminished together with its viability when phage N15 protelomerase (TelN) expression was induced continuously in the invasive E. coli despite producing an abundant amount of functional protein. By comparison, shorter induction, even as little as 3 hr, produced sufficient amounts of functional TelN and showed more effective invasion of HeLa cells, comparable to that of uninduced invasive E. coli. These results demonstrate that brief induction of protein expression during vector growth is essential for optimal entry into mammalian cells, an important step for achieving bacteria-mediated protein delivery.

Expression and characterization of transforming protein E7 from cervical cancer-associated human papillomavirus type 31

E7 protein is a major oncogenic factor of human papillomaviruses (HPVs) that plays a key role in virus-associated human cervical carcinogenesis. To determine the biochemical properties of the E7 protein of high-risk HPV type 31, the gene encoding the protein was cloned into a bacterial vector, pET-32a (+), to allow expression of HPV-31E7 as a thioredoxin (Trx) fusion protein in Escherichia coli BL21 (DE3). The resulting expression level of the fusion protein reached 15 ∼ 20% of the total cell protein and more than 60% of the target proteins were in soluble form upon cultivation for 6 h at 30°C in the presence of 0.5 mM IPTG. The fusion protein Trx-HPV-31E7 was effectively purified by Ni2+-chelating chromatography and analyzed by SDS-PAGE and Western blotting. After release from the fusion protein by enterokinase cleavage and purification to homogeneity, the recombinant HPV-31E7 (rHPV-31E7) was investigated for in vitro interaction with the pocket protein p107, which is known to interact with the amino-terminal portion of the protein. The immunoprecipitation studies revealed strong interactions of rHPV-31E7 protein with p107, suggesting it had binding activities and retained its conformational properties.

Polymyxin B treatment improves bactofection efficacy and reduces cytotoxicity.

Improvements to bacterial vectors have resulted in nonviral gene therapy vehicles that are easily prepared and can achieve high levels of transfection efficacy. However, these vectors are plagued by potential cytotoxicity and immunogenicity, prompting means of attenuation to reduce unwanted biological outcomes while maintaining transfection efficiency. In this study, listeriolysin O (LLO) producing Escherichia coli BL21(DE3) strains were pretreated with polymyxin B (PLB), a pore-forming antibiotic, and tested as a delivery vector for gene transfer to a murine RAW264.7 macrophage cell line using a 96-well high-throughput assay. PLB treatment resulted in statistically significant higher levels of gene delivery and lower cytotoxicity. The results suggest a fine balance between bacterial cellular damage, heightened gene and protein release, and increased mammalian cell gene delivery. Overall, the approach presented provides a simple and effective way to enhance bacterial gene delivery while simultaneously reducing unwanted outcomes as a function of using a biological vector.

Androgen Regulation of the TMPRSS2 Gene and the Effect of a SNP in an Androgen Response Element

More than 50% of prostate cancers have undergone a genomic reorganization that juxtaposes the androgen-regulated promoter of TMPRSS2 and the protein coding parts of several ETS oncogenes. These gene fusions lead to prostate-specific and androgen-induced ETS expression and are associated with aggressive lesions, poor prognosis, and early-onset prostate cancer. In this study, we showed that an enhancer at 13 kb upstream of the TMPRSS2 transcription start site is crucial for the androgen regulation of the TMPRSS2 gene when tested in bacterial artificial chromosomal vectors. Within this enhancer, we identified the exact androgen receptor binding sequence. This newly identified androgen response element is situated next to two binding sites for the pioneer factor GATA2, which were identified by DNase I footprinting. Both the androgen response element and the GATA-2 binding sites are involved in the enhancer activity. Importantly, a single nucleotide polymorphism (rs8134378) within this androgen response element reduces binding and transactivation by the androgen receptor. The presence of this SNP might have implications on the expression and/or formation levels of TMPRSS2 fusions, because both have been shown to be influenced by androgens.

Protective effects of oral immunization with live Lactococcus lactis expressing Eimeria tenella 3-1E protein

The codon-optimized Eimeria tenella 3-1E gene was introduced into the lactic acid bacterial vector pTX8048 to construct plasmid pTX8048-3-1E. The plasmid pTX8048-3-1E was transformed into Lactococcus lactis NZ9000 by electroporation to create the strain of L. lactis pTX8048-3-1E. The expression of objective protein was verified by Western blot. The live bacteria L. lactis pTX8048-3-1E were administered orally, and an animal challenge experiment was carried out to evaluate the protective efficacy. The results indicated the strain of L. lactis pTX8048-3-1E was constructed successfully. Oral immunization to specific pathogen-free (SPF) chickens with L. lactis pTX8048-3-1E provided partial protection against homologous challenge including significant increased oocyst decrease ratio, reduced average lesion score in cecum, and improved body weight gain compared to control bacteria L. lactis pTX8048. These results demonstrate the use of Lactococcus as live vector for delivery of Eimeria antigen is feasible and promising method to control coccidiosis in poultry.

In vivo reprogramming in inflammatory bowel disease

The direct reprogramming of somatic cells has immense implications in various areas of medicine. Although remarkable progress has been made in developing novel reprogramming methods, the efficiency and fidelity of reprogramming still need to be improved. Inflammatory bowel disease (IBD) involves chronic inflammatory diseases of the gastrointestinal tract with a complex etiology caused by various genetic, immunological and environmental factors. Recently, the role of stem cells has been proposed in pathogenesis and therapy of IBD. However, the efficiency and the safety of the stem cell treatments depend on the origin of the stem cell and the administration method. We hypothesize that the reprogramming of the intestinal cells into a pluripotent state is of huge importance for IBD therapy and prevention. The vectors carrying reprogramming genes encoding pluripotency factors can be transferred to the damaged tissue, in this case the intestine, by means of invasive bacterial vectors able to colonize colon mucosa. Reconstruction of tissues in vivo might avoid problems encountered in tissue rebuilding in vitro because of lack of appropriate scaffolds and microenvironments. Herein we present a review of recent literature and a perspective of in vivo reprogramming in IBD using bacterial vectors and analyze the rationale for such approach.

Ants in a hospital environment and their potential as mechanical bacterial vectors

We studied the richness and abundance of ant species, their bacteria and the bacteria isolated from patient clinical samples. Ants were collected with baited traps at 64 sites in a public hospital in São Luis, State of Maranhão, Brazil. In total, 1,659 ants from 14 species were captured. The most frequent species were Crematogaster victima, Solenopsis saevissima, Tapinoma melanocephalum, Camponotus vittatus and Paratrechina fulva. Forty-one species of bacteria were isolated from the ants and 18 from patients. Ants are potential vehicles for pathogenic and opportunistic bacteria, and they can represent a risk factor in nosocomial infections.

Hybrid Viral Vectors for Vaccine and Antibody Production in Plants

Plants have a demonstrated potential for large-scale, rapid production of recombinant proteins for diverse product applications, including subunit vaccines and monoclonal antibodies. In this field, the accent has recently shifted from the engineering of "edible" vaccines based on stable expression of target protein in transgenic or transplastomic plants to the development of purified formulated vaccines that are delivered via injection. The injectable vaccines are commonly produced using transient expression of target gene delivered into genetically unmodified plant host via viral or bacterial vectors. Most viral vectors are based on plant RNA viruses, where nonessential sequences are replaced with the gene of interest. Utilization of viral hybrids that consist of genes and regulatory elements of different virus species, or transcomplementation systems (vector/transgene) had a substantial impact on the level of target protein expression. Development and introduction of agroviral hybrid vectors that combine genetic elements of bacterial binary plasmids and plant viral vectors, and agroinfiltration as a tool of the vector delivery have resulted in significant progress in large-scale production of recombinant vaccines and monoclonal antibodies in plants. This article presents an overview of plant hybrid viral vector expression systems developed so far.

A new recombinant immunotoxin hscFv-ETA’ demonstrates specific cytotoxicity against chronic myeloid leukemia cells in vitro

Antibodies against cell surface antigens of tumor have attracted increasing attention in immunotherapy for tumor diagnosis and treatment. Recently, we constructed a new recombinant immunotoxin for possible clinical application in patients with chronic myeloid leukemia (CML). A functional humanized single chain variable fragment (hscFv) against CML patient cells was previously obtained from an anti-CML cell hybridoma derived monoclonal antibody. By insertion into the bacterial vector pWW20, the hscFv was fused with a deletion mutant of Pseudomonas exotoxin A (ETA’). Then the fusion fragment was inserted into the bacterial vector pET32a(+). After isopropyl β-d-thiogalactoside (IPTG) induction, the 6× His tagged hscFv-ETA’ protein was periplasmically expressed and purified by Ni2+-NTA column. The characteristics of the recombinant protein were assessed by cell membrane-ELISA, flow cytometry, and toxicity assays in CML cell lines and CML patient cells. The recombinant immunotoxin showed significant toxicity toward the CML cell lines K562 and KU812 as tested by MTT and apoptosis assay. Approximately 37% of leukemia cells of CML patients were driven into apoptosis by hscFv-ETA’ as measured by flow cytometric analysis. In conclusion, the hscFv-ETA’ is efficacious against CML in vitro, providing the basis for a novel therapeutic strategy for the treatment of CML patients.

The Attenuated Brucella abortus Strain 19 Invades, Persists in, and Activates Human Dendritic Cells, and Induces the Secretion of IL-12p70 but Not IL-23

BackgroundBacterial vectors have been proposed as novel vaccine strategies to induce strong cellular immunity. Attenuated strains of Brucella abortus comprise promising vector candidates since they have the potential to induce strong CD4+ and CD8+ T-cell mediated immune responses in the absence of excessive inflammation as observed with other Gram-negative bacteria. However, some Brucella strains interfere with the maturation of dendritic cells (DCs), which is essential for antigen-specific T-cell priming. In the present study, we investigated the interaction of human monocyte-derived DCs with the smooth attenuated B. abortus strain (S) 19, which has previously been employed successfully to vaccinate cattle.Methodology/Principal findingsWe first looked into the potential of S19 to hamper the cytokine-induced maturation of DCs; however, infected cells expressed CD25, CD40, CD80, and CD86 to a comparable extent as uninfected, cytokine-matured DCs. Furthermore, S19 activated DCs in the absence of exogeneous stimuli, enhanced the expression of HLA-ABC and HLA-DR, and was able to persist intracellularly without causing cytotoxicity. Thus, DCs provide a cellular niche for persisting brucellae in vivo as a permanent source of antigen. S19-infected DCs produced IL-12/23p40, IL-12p70, and IL-10, but not IL-23. While heat-killed bacteria also activated DCs, soluble mediators were not involved in S19-induced activation of human DCs. HEK 293 transfectants revealed cellular activation by S19 primarily through engagement of Toll-like receptor (TLR)2.Conclusions/SignificanceThus, as an immunological prerequisite for vaccine efficacy, B. abortus S19 potently infects and potently activates (most likely via TLR2) human DCs to produce Th1-promoting cytokines.