Immune-complex mimics as a molecular platform for adjuvant-free vaccine delivery

Abstract

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.

Figure 1. 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).

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).

Figure 2. Expression, purification and chemical crosslinking of recombinant proteins.

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.

Figure 3. Functional evaluation of ICM in vitro and in vivo.

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).

Figure 4. Immune responses and MTB bacterial counts in mice immunised with ICM.

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 in vitro 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.

Figure 5. Immune responses and MTB bacterial counts in mice immunised with BCG and boosted with ICM.

Mice were immunised s.c. with BCG and twice boosted with ICM six and eight weeks later; 2 weeks after the final boost, mice were either culled and their tissues (blood and spleens) used for immunological evaluation, or challenged i.n. with 70,000 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 in vitro 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) and the means +/− SEM for each group.