A mycobacterial growth inhibition assay (MGIA) for bovine TB vaccine development

Human tuberculosis remains a significant cause of mortality and morbidity throughout the world. The global economic impact of bovine TB is considerable. An effective vaccine would be the most cost-effective way to control both epidemics, particularly in emerging economies. TB vaccine research would benefit from the identification of an immune correlate of protection with which vaccines could be gated at both preclinical and clinical levels. In-vitro mycobacterial growth inhibition assays (MGIA) are functional assays that include most aspects of the complex host immune response to mycobacteria, and they may serve as functional immune correlates for vaccine development. We applied to cattle an MGIA that was developed for use with human and murine samples. Several technical difficulties were encountered while transferring it to the cattle model. However, our data demonstrate that the assay was not discriminatory in cattle and further work is needed before using it for bovine TB vaccine development

First characterization of the CD4 and CD8 T-cell responses to QuantiFERON-TB Plus

Summary Introduction QuantiFERON ® -TB Gold Plus (QFT-Plus) is the new generation of QuantiFERON-TB Gold In-Tube test to identify latent tuberculosis infection (LTBI). QFT-Plus includes TB1 and TB2 tubes which contain selected Mycobacterium tuberculosis (Mtb) peptides designed to stimulate both CD4 and CD8 T-cells. Aim of this study is the flow cytometric characterization of the specific CD4 and CD8 T-cell responses to Mtb antigens contained within QFT-Plus. Methods We enrolled 27 active tuberculosis (TB) patients and 30 LTBI individuals. Following stimulation with TB1 and TB2, antigen-specific T-cells were characterized by flow cytometry. Data were also correlated with the grade of TB severity. Results TB1 mainly elicited a CD4 T-cell response while TB2 induced both CD4 and CD8 responses. Moreover, the TB2-specific CD4 response was detected for both active TB and LTBI patients, whereas the TB2-specific CD8 response was primarily associated with active TB (p = 0.01). Conclusions To our knowledge, we report the first characterization of the CD4 and CD8 T-cell response to QFT-Plus. CD8 T-cell response is mainly due to TB2 stimulation which is largely associated to active TB. These results provide a better knowledge on the use of this assay

Ex vivo mycobacterial growth inhibition assay (MGIA) for tuberculosis vaccine testing - a protocol for mouse splenocytes

The testing of vaccines for tuberculosis is costly and time-consuming, and dependent on preclinical animal challenge models and clinical trials. We have recently developed a mycobacterial growth inhibition assay (MGIA) to test vaccine efficacy ex vivo. This assay measures the summative effect of the host immune response and may serve as a novel tool to facilitate vaccine testing. It has generated much interest recently, and to facilitate technology transfer and reproducibility between laboratories, we here describe a detailed protocol for an ex vivo MGIA in mouse splenocytes.</jats:p

Immune-Complex Mimics as a Molecular Platform for Adjuvant-Free Vaccine Delivery

Protein-based vaccine development faces the difficult challenge of finding robust yet non-toxic adjuvants suitable for humans. Here, using a molecular engineering approach, we have developed a molecular platform for generating self-adjuvanting immunogens that do not depend on exogenous adjuvants for induction of immune responses. These are based on the concept of Immune Complex Mimics (ICM), structures that are formed between an oligomeric antigen and a monoclonal antibody (mAb) to that antigen. In this way, the roles of antigens and antibodies within the structure of immune complexes are reversed, so that a single monoclonal antibody, rather than polyclonal sera or expensive mAb cocktails can be used. We tested this approach in the context of Mycobacterium tuberculosis (MTB) infection by linking the highly immunogenic and potentially protective Ag85B with the oligomeric Acr (alpha crystallin, HspX) antigen. When combined with an anti-Acr monoclonal antibody, the fusion protein formed ICM which bound to C1q component of the complement system and were readily taken up by antigen-presenting cells in vitro. ICM induced a strong Th1/Th2 mixed type antibody response, which was comparable to cholera toxin adjuvanted antigen, but only moderate levels of T cell proliferation and IFN-γ secretion. Unfortunately, the systemic administration of ICM did not confer statistically significant protection against intranasal MTB challenge, although a small BCG-boosting effect was observed. We conclude that ICM are capable of inducing strong humoral responses to incorporated antigens and may be a suitable vaccination approach for pathogens other than MTB, where antibody-based immunity may play a more protective role

Recombinant immune complexes as a novel molecular platform for the delivery of mycobacterial antigens

This work describes the generation of recombinant immune complexes (RICs) and the evaluation of their vaccine potential in the context of tuberculosis (TB). Novel vaccine candidates based on recombinant immune complexes were synthesised and tested in mice for the immunogenic potential and protective capacity against My- cobacterium tuberculosis infection. Two different approaches were employed to make these molecules: i) utilising a plant expression system which is amenable for expression of complex molecules and ii) utilizing the unique polymeric properties of the Acr (X- crystalline-like) antigen. E. coli recombinant immune complexes (ERICs) were initially generated in the study, and their functionality was demonstrated in vitro by a complement (Clq component) binding ELISA assay. In addition, they bound efficiently to the surface of spleen-derived antigen- presenting cells, suggesting that these molecules could be used as a novel molecu- lar platform for vaccine delivery in vivo. The vaccine potential of ERICs was evaluated by testing their immunogenicity and protective capacity in the mouse model of TB infection. The study demonstrated that ERICs induced strong humoral responses to both Ag85B and Acr, but only a moderate cellular immune response and IFN-y production. Homol- ogous prime-boost subcutaneous immunisation of mice with ERICs conferred little or no protection against M. tuberculosis intranasal challenge; however, some protection (greater than that with BCG alone) was observed when they were used as a booster vaccine in BCG primed mice. This suggests that ERICs could be used as a novel BCG-boosting vaccine candidate against TB infection and also, could be considered for other infections, especially those where protection requires a strong humoral response. 1 • For preparation of plant recombinant immune complexes (RlCs), the ability of Nico- tiana tabacum plants to express a functional TBG65 IgG 1 antibody was initially demon- strated. To express RICs in planta, the DNA construct comprising TBG65y chain se- quence, in frame with Acr or Acr-Ag85B coding sequences, was inserted into two different plant expression vectors, one suitable for transient expression and the other to gener- ate transgenic tobacco plants. Expression of both constructs was obtained and, while proceeding with the lengthy process of generation of stably transformed plants, RICs purification protocols were tested on transiently expressed material. The presence of im- mune complexes in the RICs preparation was verified by Clq-binding ELISA assay, and a preliminary immunisation experiment demonstrated a boosting effect of RICs in BCG- immunised mice. These data complement the current data available on plant RlCs adding important information about their vaccine potential, but further work is now required to optimise production and purification of plant-RICs on a scale suitable for in-depth vaccine studies.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Recombinant immune complexes as a novel molecular platform for the delivery of mycobacterial antigens

This work describes the generation of recombinant immune complexes (RICs) and the evaluation of their vaccine potential in the context of tuberculosis (TB). Novel vaccine candidates based on recombinant immune complexes were synthesised and tested in mice for the immunogenic potential and protective capacity against My- cobacterium tuberculosis infection. Two different approaches were employed to make these molecules: i) utilising a plant expression system which is amenable for expression of complex molecules and ii) utilizing the unique polymeric properties of the Acr (X- crystalline-like) antigen. E. coli recombinant immune complexes (ERICs) were initially generated in the study, and their functionality was demonstrated in vitro by a complement (Clq component) binding ELISA assay. In addition, they bound efficiently to the surface of spleen-derived antigen- presenting cells, suggesting that these molecules could be used as a novel molecu- lar platform for vaccine delivery in vivo. The vaccine potential of ERICs was evaluated by testing their immunogenicity and protective capacity in the mouse model of TB infection. The study demonstrated that ERICs induced strong humoral responses to both Ag85B and Acr, but only a moderate cellular immune response and IFN-y production. Homol- ogous prime-boost subcutaneous immunisation of mice with ERICs conferred little or no protection against M. tuberculosis intranasal challenge; however, some protection (greater than that with BCG alone) was observed when they were used as a booster vaccine in BCG primed mice. This suggests that ERICs could be used as a novel BCG-boosting vaccine candidate against TB infection and also, could be considered for other infections, especially those where protection requires a strong humoral response. 1 • For preparation of plant recombinant immune complexes (RlCs), the ability of Nico- tiana tabacum plants to express a functional TBG65 IgG 1 antibody was initially demon- strated. To express RICs in planta, the DNA construct comprising TBG65y chain se- quence, in frame with Acr or Acr-Ag85B coding sequences, was inserted into two different plant expression vectors, one suitable for transient expression and the other to gener- ate transgenic tobacco plants. Expression of both constructs was obtained and, while proceeding with the lengthy process of generation of stably transformed plants, RICs purification protocols were tested on transiently expressed material. The presence of im- mune complexes in the RICs preparation was verified by Clq-binding ELISA assay, and a preliminary immunisation experiment demonstrated a boosting effect of RICs in BCG- immunised mice. These data complement the current data available on plant RlCs adding important information about their vaccine potential, but further work is now required to optimise production and purification of plant-RICs on a scale suitable for in-depth vaccine studies.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Hierarchical thermoplastic rippled nanostructures regulate Schwann Cell adhesion, morphology and spatial organization

Periodic ripples are a variety of anisotropic nanostructures that can be realized by ion beam irradiation on a wide class of solid surfaces. Only few authors have investigated these surfaces for tuning the response of biological systems, probably because it is challenging to directly produce them in materials that well sustain long-term cellular cultures. Here, hierarchical rippled nanotopographies with lateral periodicity of ∽300 nm are produced from a gold-irradiated germanium mold in polyethylene terephthalate (PET), a biocompatible polymer approved by the US Food and Drug Administration for clinical applications, by a novel three-steps embossing process. The effects of nano-ripples on Schwann Cells (SCs) are studied in view of their possible use for nerve-repair applications. Data demonstrate that nano-ripples can enhance short-term SC adhesion and proliferation (3-24h from seeding), drive their actin cytoskeleton spatial organization and sustain long-term cell growth. Notably, SCs orient perpendicularly with respect to the nanopattern lines. These results provide information about the possible use of hierarchical nano-rippled elements for nerve-regeneration protocols

Mechanosensing at the nanoscale: the influence of thermoplastic nanostructures on neural cells

It is well established that the behavior of neural cells is influenced by geometrical patterns in the micrometric and sub-micrometric range. Here we present two different types of periodical patterns in the nanometric range (i.e. with a typical features having a lateral size ≤ 100 nm) and their impact on cell contact guidance. In the first case, hierarchical periodic nano-rippled structure (i.e. nano-ripples) made by ion-bombardment technique were replicated on top of polyethylene terephtalate (PET) films. We demonstrated that Schwann cells actively interact with these nanorippled surfaces, showing perpendicular contact guidance and improved adhesion and proliferation with respect to standard flat substrates. The second type of scaffolds here presented consist in cyclic-olefin-copolymer (COC) nanogratings with periodicity (down to 200nm -50% duty cycle), obtained by hot embossing from photoresist molds fabricated by interference lithography. In this case, we coupled the substrates with the PC12 neuronal cell line and measured the neurite alignment and focal adhesion (FA) morphometric parameters. We show optimal contact guidance in the case of periodicity > 400nm, while a progressive degradation of polarized alignment appears by further decreasingthe grating lateral dimensions, correlating with FA shaping. These results set for the first time a lower limit in grating periodicity for effective neurite contact guidance. Altogether thesestudies provide interesting elements for regenerative medicine applications and for developing artificial neural interfaces