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

Current Research into Applications of Tomography for Fusion Diagnostics

Retrieving spatial distribution of plasma emissivity from line integrated measurements on tokamaks presents a challenging task due to ill-posedness of the tomography problem and limited number of the lines of sight. Modern methods of plasma tomography therefore implement a-priori information as well as constraints, in particular some form of penalisation of complexity. In this contribution, the current tomography methods under development (Tikhonov regularisation, Bayesian methods and neural networks) are briefly explained taking into account their potential for integration into the fusion reactor diagnostics. In particular, current development of the Minimum Fisher Regularisation method is exemplified with respect to real-time reconstruction capability, combination with spectral unfolding and other prospective tasks

Functional evaluation of ICM in vitro and in vivo.

<p>A) Complement C1q binding ELISA; ICM were used at the 1∶20 antibody-antigen ratio and the neat sample contained 5 µg/m total protein (ICM) or the equivalent amount for individual components; each bar represents mean value from triplicate assays and the patterns indicate serial dilutions. B) Analysis of binding of ICM to spleen-derived APCs by flow cytometry; shown are the proportions of cells (out of 10,000 counted) that bound either mAb alone or ICM. C) Serum anti-Ag85B IgG responses from mice immunised with an equimolar (1∶1) or a low (1∶20) antibody-antigen ratio, twice at the base of the tail, at 3-week intervals. Mice were culled 3 weeks after the final immunisation. Shown are the mean values and corresponding serial dilutions from a pilot experiment (n = 3 mice).</p

Expression, purification and chemical crosslinking of recombinant proteins.

<p>A) Coomassie Blue staining of purified, His-tagged proteins separated by 12% SDS-PAGE; 1. Acr-Ag85B (50 kDa), 2. Acr (20 kDa) and 3. Ag85B (32 kDa). B) Western blot analysis using antigen-specific antibodies; 1. Acr, 2. Acr-Ag85B and 3, Ag85B (1 and 2 probed with anti-Acr mAb TBG65; 3 probed with rabbit anti-Ag85B serum). C) Chemical crosslinking of Acr; shown is a Coomassie-stained (1, and 2) or Western blot (3 and 4) analysed sample with crosslinker (1 and 3) or without crosslinker (2 and 4). Letters indicate various molecular forms based on expected size (M-monomer, D-dimer, Tr-trimer, Te-tetramer, H-hexamer, O-oligomers). D) Chemical crosslinking of Acr-Ag85B fusion protein; shown is a sample with (1 and 3) or without (2 and 4) crosslinker. Letters indicate various molecular forms as for Acr (C). E) Chemical crosslinking of Ag85B (internal control); Coomassie and Western blot analysis of a sample with (1 and 3) or without (2 and 4) crosslinker.</p

Immune responses and MTB bacterial counts in mice immunised with ICM.

<p>Mice were immunised with 50 µg ICM (1∶20 antibody antigen ratio) or with Ag85B alone (30 µg), Ag85B+CT, BCG and PBS; two weeks after the final immunisation mice were either culled and their tissues (blood and spleens) used for immunological evaluation, or challenged i.n. with 70,000 MTB H37Rv. * Indicates statistically significant difference (p<0.05). A,B) Ag85B (A) and Acr (B) specific IgG, IgG1 and IgG2a serum responses determined by ELISA; shown are the mean values from 3 mice analysed in triplicates and in serial dilutions (indicated by differing patterns). C) Splenocyte proliferation after <i>in vitro</i> stimulation with Ag85B, measured by ³[H]-thymidine incorporation and expressed as stimulation indices (specific/nonspecific proliferation); n = 3 animals. D) IFN-γ release in splenocyte cultures (as in C) measured by an IFN-γ ELISA based kit. E) Lung bacterial counts in immunised mice; shown are the counts for individual mice (n = 6, except in some groups due to death of animals before the end of the experiment) and the means +/− SEM for each group.</p

Schematic representation of immune complex mimics (ICM) based on Acr-Ag85B fusion protein and an anti-Acr mAb (A) and the classical immune complexes (IC) based on Ag85B antigen of MTB and polyclonal Abs (B).

<p>For ICM, the fusion protein is depicted as a trimer, which is one of the predominant molecular forms for Acr in solution. Each mAb molecule must bind to a different monomer unit of Acr (A); in contrast, polyclonal Abs can bind to the same Ag85B molecule (B).</p

Image_1_Spore-FP1 tuberculosis mucosal vaccine candidate is highly protective in guinea pigs but fails to improve on BCG-conferred protection in non-human primates.pdf

Tuberculosis remains a major health threat globally and a more effective vaccine than the current Bacillus Calmette Guerin (BCG) is required, either to replace or boost it. The Spore-FP1 mucosal vaccine candidate is based on the fusion protein of Ag85B-Acr-HBHA/heparin-binding domain, adsorbed on the surface of inactivated Bacillus subtilis spores. The candidate conferred significant protection against Mycobacterium. tuberculosis challenge in naïve guinea pigs and markedly improved protection in the lungs and spleens of animals primed with BCG. We then immunized rhesus macaques with BCG intradermally, and subsequently boosted with one intradermal and one aerosol dose of Spore-FP1, prior to challenge with low dose aerosolized M. tuberculosis Erdman strain. Following vaccination, animals did not show any adverse reactions and displayed higher antigen specific cellular and antibody immune responses compared to BCG alone but this did not translate into significant improvement in disease pathology or bacterial burden in the organs.</p

Image_2_Spore-FP1 tuberculosis mucosal vaccine candidate is highly protective in guinea pigs but fails to improve on BCG-conferred protection in non-human primates.tif

Tuberculosis remains a major health threat globally and a more effective vaccine than the current Bacillus Calmette Guerin (BCG) is required, either to replace or boost it. The Spore-FP1 mucosal vaccine candidate is based on the fusion protein of Ag85B-Acr-HBHA/heparin-binding domain, adsorbed on the surface of inactivated Bacillus subtilis spores. The candidate conferred significant protection against Mycobacterium. tuberculosis challenge in naïve guinea pigs and markedly improved protection in the lungs and spleens of animals primed with BCG. We then immunized rhesus macaques with BCG intradermally, and subsequently boosted with one intradermal and one aerosol dose of Spore-FP1, prior to challenge with low dose aerosolized M. tuberculosis Erdman strain. Following vaccination, animals did not show any adverse reactions and displayed higher antigen specific cellular and antibody immune responses compared to BCG alone but this did not translate into significant improvement in disease pathology or bacterial burden in the organs.</p

Image_5_Spore-FP1 tuberculosis mucosal vaccine candidate is highly protective in guinea pigs but fails to improve on BCG-conferred protection in non-human primates.tif

Tuberculosis remains a major health threat globally and a more effective vaccine than the current Bacillus Calmette Guerin (BCG) is required, either to replace or boost it. The Spore-FP1 mucosal vaccine candidate is based on the fusion protein of Ag85B-Acr-HBHA/heparin-binding domain, adsorbed on the surface of inactivated Bacillus subtilis spores. The candidate conferred significant protection against Mycobacterium. tuberculosis challenge in naïve guinea pigs and markedly improved protection in the lungs and spleens of animals primed with BCG. We then immunized rhesus macaques with BCG intradermally, and subsequently boosted with one intradermal and one aerosol dose of Spore-FP1, prior to challenge with low dose aerosolized M. tuberculosis Erdman strain. Following vaccination, animals did not show any adverse reactions and displayed higher antigen specific cellular and antibody immune responses compared to BCG alone but this did not translate into significant improvement in disease pathology or bacterial burden in the organs.</p