Fusion of the Mycobacterium tuberculosis Antigen 85A to an Oligomerization Domain Enhances Its Immunogenicity in Both Mice and Non-Human Primates

et al. (2012) Fusion of the Mycobacterium tuberculosis Antigen 85A to an Oligomerization Domain Enhances Its Immunogenicity in Both Mice and Non-Human Primates. PLoS ONE 7(3): e33555. doi:10.1371/journal.pone.0033555 Fusion of the Mycobacterium tuberculosis Antigen 85A to an Oligomerization Domain Enhances Its Immunogenicity in Both Mice and Non-Human Primates Alexandra J. Spencer 0 1 Fergal Hill 0 1 Jared D. Honeycutt 0 1 Matthew G. Cottingham 0 1 Migena Bregu 0 1 Christine S. Rollier 0 1 Julie Furze 0 1 Simon J. Draper 0 1 Karen C. Sgaard 0 1 Sarah C. Gilbert 0 1 David H. Wyllie 0 1 Adrian V. S. Hill 0 1 Thomas Jens Scriba, University of Cape Town, South Africa 0 a Current address: Oxford Vaccine Group, CCVTM, Churchill Hospital , Oxford , United Kingdom b Current address: Wellcome Trust Centre for Human Genetics, University of Oxford , Oxford , United Kingdom 1 1 The Jenner Institute, University of Oxford , Oxford , United Kingdom , 2 Imaxio SA, Lyons , France To prevent important infectious diseases such as tuberculosis, malaria and HIV, vaccines inducing greater T cell responses are required. In this study, we investigated whether fusion of the M. tuberculosis antigen 85A to recently described adjuvant IMX313, a hybrid avian C4bp oligomerization domain, could increase T cell responses in pre-clinical vaccine model species. In mice, the fused antigen 85A showed consistent increases in CD4+ and CD8+ T cell responses after DNA and MVA vaccination. In rhesus macaques, higher IFN-c responses were observed in animals vaccinated with MVA-Ag85A IMX313 after both primary and secondary immunizations. In both animal models, fusion to IMX313 induced a quantitative enhancement in the response without altering its quality: multifunctional cytokines were uniformly increased and differentiation into effector and memory T cell subsets was augmented rather than skewed. An extensive in vivo characterization suggests that IMX313 improves the initiation of immune responses as an increase in antigen 85A specific cells was observed as early as day 3 after vaccination. This report demonstrates that antigen multimerization using IMX313 is a simple and effective cross-species method to improve vaccine immunogenicity with potentially broad applicability. - Funding: This work was funded by grants from the Foundation for the National Institute of Health through the Grand Challenges in Global Health Initiative with additional funding from the Wellcome Trust. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have read the journals policy and have the following conflicts: AJS, FH, MGC, SJD and AVSH are named inventors on patent applications for the use of C4bp to increase immunogenicity. FH is an employee of and shareholder in Imaxio SA. This does not alter the authors adherence to all the PLoS ONE policies on sharing data and materials. A major challenge in vaccinology is the development of effective vaccines against intracellular pathogens where cell mediated immunity plays an important protective role. Viral vectored vaccines have a remarkable capacity to induce and boost antigenspecific T cells [1], but higher frequency responses will likely be required to achieve useful protective efficacy [2]. There is, therefore, a need for adjuvants in the next generation of vectored vaccines to increase T cell immunogenicity. Oligomerization is employed by many natural proteins to increase protein valency, binding affinity and structural stability [3], and while a pentameric coiled coil was initially used to improve B-cell responses in mice [4] its sequence is too similar to its human ortholog to be considered safe for use in humans. Recently, a series of homologous oligomerization protein domains were shown to act as adjuvants in mice, resulting in an augmentation of both B and T cell responses [5,6]. These proteins were derived from the domain encoded by the last exon of the complement 4 binding protein (C4bp) a-chain. This exon encodes the only domain not involved in the complement related functions of C4bp and is both essential and sufficient for oligomerization of the seven C4bp alpha chains [7]# and a number of other proteins [8,9,10,11,12]. Fusion of recombinant Plasmodium yoelii MSP-119 protein to C4bp oligomerization domains from a variety of mammalian and avian species was shown to improve antibody responses to this weak immunogen [6]; of the domains tested, a chicken C4bp hybrid with less than 20% homology to human C4bp, called IMX313, was shown to induce the highest titers of MSP-119 specific antibodies, buts its effect on T cell responses was not investigated. Tuberculosis (TB) remains one of the most serious worldwide infections despite the use of the M. bovis strain Bacillus CalmetteGuerin (BCG) as a vaccine since the 1920s. The most advanced sub-unit vaccine in clinical development is a modified vaccinia virus Ankara (MVA) expressing the M. tuberculosis protein 85A. Clinical trials in both the UK and Africa have shown the substantial capacity of MVA-Ag85A to boost T cell responses to BCG in healthy individuals [13,14,15], whether this capacity is maintained in HIV-infected individuals is unclear. Although there is currently no clear correlate of protection, T cells have been shown to play an important role [16,17,18] and therefore strategies to increase the level of vaccine induced T cells could have a substantial impact. In this study we have assessed whether fusion to the IMX313 domain could enhance the T cell mediated response to the M. tuberculosis antigen 85A in two pre-clinical animal species, mice and rhesus macaques. In both animal models, IFN-c ELISpot and multi-parameter flow cytometry were used to investigate the effects of fusion to IMX313 on the overall quality of the immune response in terms of cytokine secretion and generation of effector or memory T cell subsets. Fusion of antigens to the IMX313 domain is a straightforward method and its ability to enhance T cell immune responses could have broad applicability across a variety of animal species and disease settings. In the first instance, we intend to undertake a direct comparison in humans of MVAAg85A with MVA-Ag85A IMX313 to confirm the results described here in two very distinct animal species. Fusion to IMX313 multimerizes antigen 85A To confirm that fusion of IMX313 resulted in formation of disulphide-linked multimers without affecting protein expression, cell lysates from MVA infected BHK cells were analyzed by Western blots of reducing and non-reducing SDS-PAGE gels (Figure 1). Under reducing conditions, both Ag85A and Ag85A IMX313 proteins migrated at the expected apparent molecular weight of the mature monomeric peptide (35.5 kDa for Ag85A or 40.4 kDa for Ag85A IMX313) (Figure 1d). For both proteins, an additional higher molecular weight band (,45 kDa for Ag85A and ,50 kDa for Ag85A IMX313) w (...truncated)