Final Report Summary - ADJUTUB (Rational design of a lipopolysaccharide adjuvant for tuberculosis vaccines)
Tuberculosis (TB), a disease caused by the intracellular pathogen Mycobacterium tuberculosis, is the second leading cause of death of adults from an infectious disease worldwide. Although an attenuated strain of M. bovis called BCG is widely used as vaccine against TB, its effectiveness is generally considered as insufficient. Dendritic cells and CD4+ T lymphocytes with a Th1 phenotype are key players in the immune response against TB. As a new approach for BCG improvement we have proposed to add a novel type of adjuvant, based on genetically engineered versions of the lipopolysaccharide (LPS) from Neisseria meningitidis (Nm). LPS modifications aim at specific targeting dendritic cells where an immune response is initiated and at reducing the toxicity of LPS by making the lipid A part less active for the LPS receptor TLR4/MD2. The inactivation of the lgtB gene results in Nm LPS with a truncated oligosaccharide lacking the terminal galactose, which specifically binds to the receptor DC-SIGN on human dendritic cells. This binding skews the resulting T-cell response in a Th1-direction. Heterologous expression in N. meningitidis of the pagL gene from Bordetella bronchiseptica, which encodes a lipid A 3-O-deacylase, results in pentaacylated Nm LPS of strongly reduced TLR4-activating ability. We have designed and obtained an lgtB- pagL+ Nm mutant combining both modifications. We have characterized in detail the molecular composition and heterogeneity of the intact LPS purified from lgtB- pagL+ Nm bacteria by using high-resolution electrospray ionization mass spectrometry (ESI-MS). We found that most LPS species contain pentaacylated meningococcal lipid A structures that are substituted with three phosphates and one or two phosphoethanolamine groups and lack the 3-hydroxydodecanoic acid (C12OH) that is ester-linked to the C3-position of the lipid A glucosamine disaccharide. Although hexaacylated LPS species were also present, these were only of minor abundance. Consequently, a highly efficient conversion of the Nm LPS from the hexaacyl to the 3-O-desacyl pentaacyl form was achieved. MS analyses provided evidence of the expression of only one major oligosaccharide species lacking the terminal galactose residue of the lacto-N-neotetraose outer core. This is in agreement with an effective inactivation of the lgtB gene and the absence of oligosaccharide structural variation due to phase-variable expression of LPS biosynthetic genes. In conclusion, the heterogeneity of the LPS obtained was relatively minor and much lower than initially anticipated, which is advantageous for the future application of this LPS as vaccine adjuvant since this will facilitate LPS quality control and more reproducible manufacturing of LPS preparations. The toxic pro-inflammatory and immune stimulatory potential of the purified lgtB- pagL+ Nm LPS was determined in-vitro in the human monocytic cell line Mono-Mac6 (MM6). The capacity of pentaacylated lgtB- pagL+ LPS to induce the production of the pro-inflammatory cytokine interleukin-6 (IL-6) was markedly reduced as compared to that of an LPS of lgtB- Nm carrying a wild-type hexaacylated lipid A, thus indicating the much lower potential toxicity of pentaacylated lgtB- pagL+ LPS. By contrast, the concomitant reduction in secretion of the chemokine interferon-gamma-induced protein-10 (IP-10) and anti-inflammatory cytokine interleukin 10 (IL-10) was far less pronounced. This retained capacity of pentaacylated lgtB- pagL+ LPS to induce the secretion of the chemokine IP-10 suggests a bias towards the TRIF over the MyD88 signaling pathway after TLR4 activation, which is considered to be favorable for the development of adaptive immunity. Furthermore, we have evaluated the effect of different proportions of pentaacylated lgtB- pagL+ LPS and hexaacylated lgtB- LPS on the activation of human monocytic MM6 cells, as measured by the secretion of the cytokine IL-6. It was found that addition of the pentaacylated lgtB- pagL+ LPS at an excess weight ratio of 6:1 to the hexaacylated lgtB- LPS drastically decreased the IL-6 inducing activity of hexaacylated LPS in MM6 cells. Hence, the pentaacylated lgtB- pagL+ LPS structure not only proved to have weak pro-inflammatory IL-6 inducing activity, but also was able to antagonize hexaacylated LPS. On the other hand, addition of increasing proportions of the hexaacylated lgtB- LPS to the lgtB- pagL+ LPS produced increasing expression of IL-6 in MM6 cells. Therefore, by controlling the proportion of hexa-to-penta LPS it was possible to finely control the level of activation of human monocytic cells. We also addressed the question of how much the different molecular species present in the pentaacylated LPS from the lgtB- pagL+ Nm mutant contributed to the toxicity and immune stimulating properties of this preparation. The relatively high homogeneity of this LPS preparation precluded the application of SDS-PAGE and liquid chromatography separation methods for separating different intact LPS species. In particular, the question remained whether the cytokine inducing activity of the lgtB- pagL+ LPS was due to the presence of minor hexaacylated LPS species in this preparation. To answer this question, we next performed a very mild alkaline hydrolysis of the LPS in 5% triethylamine to specifically remove residual 3-O-acylated C12OH fatty acids in LPS. As a result, mass spectra of LPS after mild alkaline hydrolysis showed major signals corresponding to intact pentaacylated LPS species while minor signals of hexaacylated LPS species were absent. The bioactivity of this highly pure pentaacylated LPS preparation from lgtB- pagL+ Nm was tested in MM6 cells. It was found that the IL-6 inducing activity of this LPS was reduced even further after mild alkaline hydrolysis, but it was not completely nullified. The stimulating activity of this LPS in terms of IP-10 and IL-10 secretion also decreased following mild alkaline hydrolysis, but to a much lower extent than in terms of IL-6 production. These results indicated that minor hexaacylated LPS species present in the mostly pentaacylated lgtB- pagL+ LPS could effectively modulate the stimulatory activity of this LPS preparation and secondly, that pure pentaacylated lgtB- pagL+ LPS possess itself a beneficial immune stimulating capacity, it induces much less toxic pro-inflammatory effects in-vitro and is still capable of activating human innate immune cells. We then evaluated the effect of the lgtB- pagL+ LPS on the immune response of human monocytic cells to BCG in-vitro. We studied the activation of human MM6 monocytic cells by mixtures of BCG and LPS, as measured by the production of IL-6. The addition of either the pentaacylated lgtB- pagL+ LPS mutant or the hexaacylated lgtB- LPS mutant of Nm to a BCG lysate generally produced additive effects on the induction of the production of the pro-inflammatory cytokine IL-6. Synergistic effects were also observed when high LPS concentrations were combined with high BCG concentrations. Finally, we have investigated the ability of the lgtB- pagL+ Nm LPS to enhance the immune response against BCG in an immunization experiment in mice. For this proof of concept study, T-cell responses against BCG antigens were determined after immunization with BCG combined with LPS. Overall, the results of this project represent a major advance in the development of novel Nm LPS adjuvant candidates for BCG vaccine improvement. The information obtained on the structural and immune stimulating properties of the Nm LPS and the analytical methods implemented will provide criteria to establish in-process and quality control assays, which guarantee batch-to-batch reproducibility of the properties of the produced GMP-grade Nm LPS adjuvant. Furthermore, it gives hints on how to improve further the final preparation and its manufacturing process. The project has allowed the fellow to extend and deepen his experience with LPS molecular analysis and to acquire new practical skills and theoretical knowledge in microbiology and especially immunology. This has strengthened and diversified the position of the fellow as a researcher and helped to consolidate his incorporation to the European Research Community. Ultimately, this project has contributed into making Europe an attractive working environment for researchers who want to contribute to eradicate major infectious diseases through the application of innovative biotechnological research.