Protection of Mice with a Tuberculosis Subunit Vaccine Based on a Fusion Protein of Antigen 85B and ESAT-6

Abstract

In this study, we investigated the potential of a tuberculosis subunit vaccine based on fusion proteins of the immunodominant antigens ESAT-6 and antigen 85B. When the fusion proteins were administered to mice in the adjuvant combination dimethyl dioctadecylammonium bromide-monophosphoryl lipid A, a strong dosedependent immune response was induced to both single components as well as to the fusion proteins. The immune response induced was accompanied by high levels of protective immunity and reached the level of Mycobacterium bovis BCG-induced protection over a broad dose range. The vaccine induced efficient immunological memory, which remained stable 30 weeks postvaccination.Tuberculosis (TB) is the leading infectious disease in the developing world, and the World Health Organization estimates 80 million new cases of tuberculosis in this decade (8). The current vaccine against Mycobacterium tuberculosis, M. bovis Bacillus Calmette-Gúerin (BCG), has been extensively evaluated and demonstrated variable protective efficacies ranging from 0 to 85% in different field trials (13). An improved second-generation vaccine is therefore urgently needed. Alternative strategies in TB vaccine development such as subunit vaccines (2, 16, 23), genetic immunization (17, 27), and attenuated strains of M. tuberculosis (14) are currently being explored in many laboratories. Due to the complexity of the host immune response against tuberculosis and the genetic restriction imposed by major histocompatibility complex molecules, it has become clear that an effective subunit vaccine containing multiple epitopes may be required to ensure a broad coverage of a genetically heterogeneous population. We and others have previously demonstrated that vaccines based on a mixture of culture filtrate antigens can induce levels of protection similar to BCG in mice (2, 16, 23), but so far only a few experimental vaccines based on a single antigen have proved successful in animal models (6,17,27).The strategy being explored in our laboratory is the molecular engineering of recombinant fusion proteins. Compared to mixtures of proteins extracted from cultures or cell lysates, the fusion protein approach offers at least two substantial advantages: (i) it is a more defined product and (ii) it reduces the number of recombinant expression and purification steps. The purpose of our study was to evaluate the potential of a subunit vaccine based on a fusion protein between two immunodominant antigens, Ag85B and the 6-kDa early secretory antigenic target (ESAT-6). In this study, we show that this approach is very promising and promotes an efficient immune response which is highly protective against TB in the mouse model. MATERIALS AND METHODSAnimals. Specific-pathogen-free female C57BL/6J (H-2 b ) and B6CBAF1 (H-2 b,k ) mice were purchased from Bomholtgaard (Ry, Denmark). All mice used were 6-to 12 weeks of age and were housed in cages contained within a BL-3 laminar flow safety enclosure. Animals were allowed free access to water and standard mouse chow. Bact...

Mtb32 is a promising tuberculosis antigen for DNA vaccination in pre- and post-exposure mouse models

“…For instance, one study showed that the early secreted antigen, ESAT-6, affords protection, as evident from the significant reduction in the bacterial number in the lungs, while other groups reported minimal protective activity with no statistical significance in mice. 23,24 In therapeutic settings, HSP65 and Ag85A are the most extensively tested antigens, but their protective efficacies remain a subject of controversy. 21,22,25 Head-to-head comparison of potent TB antigens under a single experimental condition may be the ideal approach for identifying protective antigens, especially when infection models are diverse like TB.…”

Mycobacterium tuberculosis antigen 85B and ESAT-6 expressed as a recombinant fusion protein in Mycobacterium smegmatis elicits cell-mediated immune response in a murine vaccination model

“…The pMCT6 plasmid containing the gene fusion Ag85B and ESAT-6 (Hybrid-1) was constructed as previously described (Weinrich Olsen et al, 2001). The hybrid-1 fusion was amplified from pMCT6 using, Ag85B-HB-forward primer (5'-CGC GGA TCC ATG CAC CAC CAC CAC CAC CAC TTC TCC CGG CCG GGG CTG CC-3') and ESAT-6-SpeI-reverse primer (5'-GGA CTA GTC TAT GCG AAC ATC CCA GTG ACG TTG CCT TC-3').…”

“…Protein concentration of the final pooled purified sample of hybrid 1 was determined using the BCA method (Piercenet). H1 (EC, SSI) was produced and purified as previously described (Weinrich Olsen et al, 2001). …”

“…Immunogenicity of the M. smegmatis derived (H1 (MS, IC)) and E. coli derived (H1 (EC, IC)) Hybrid1 proteins were tested, together with an E. coli Hybrid-1 preparation previously published (H1 (EC, SSI)) (Weinrich Olsen et al, 2001). The experiments on all three antigens were done using the methods outlined recently (Agger et al, 2008).…”

“…Among these candidates is the fusion protein Hybrid-1 or H1, which is based on the immunodominant antigens Antigen 85B (Ag85B) and the Early Secretory Antigenic Target (ESAT-6) from M. tuberculosis. The Hybrid-1 fusion protein is at the forefront of candidate vaccines against TB and has been extensively tested in a number of studies (Agger et al, 2008;Dietrich et al, 2007;Langermans et al, 2005;Olsen et al, 2004;van Dissel et al, 2010;van Dissel et al, 2011;Weinrich Olsen et al, 2001). A recent variant has also been fused to the latency associated protein Rv2660c (H56) that promises efficacy against pre-and post exposure (latent) TB (Aagaard et al, 2011).…”