Analysis of the Vaccine Potential of Plasmid DNA Encoding Nine Mycolactone Polyketide Synthase Domains in Mycobacterium ulcerans Infected Mice

Abstract

There is no effective vaccine against Buruli ulcer. In experimental footpad infection of C57BL/6 mice with M. ulcerans, a prime-boost vaccination protocol using plasmid DNA encoding mycolyltransferase Ag85A of M. ulcerans and a homologous protein boost has shown significant, albeit transient protection, comparable to the one induced by M. bovis BCG. The mycolactone toxin is an obvious candidate for a vaccine, but by virtue of its chemical structure, this toxin is not immunogenic in itself. However, antibodies against some of the polyketide synthase domains involved in mycolactone synthesis, were found in Buruli ulcer patients and healthy controls from the same endemic region, suggesting that these domains are indeed immunogenic. Here we have analyzed the vaccine potential of nine polyketide synthase domains using a DNA prime/protein boost strategy. C57BL/6 mice were vaccinated against the following domains: acyl carrier protein 1, 2, and 3, acyltransferase (acetate) 1 and 2, acyltransferase (propionate), enoylreductase, ketoreductase A, and ketosynthase load module. As positive controls, mice were vaccinated with DNA encoding Ag85A or with M. bovis BCG. Strongest antigen specific antibodies could be detected in response to acyltransferase (propionate) and enoylreductase. Antigen-specific Th1 type cytokine responses (IL-2 or IFN-γ) were induced by vaccination against all antigens, and were strongest against acyltransferase (propionate). Finally, vaccination against acyltransferase (propionate) and enoylreductase conferred some protection against challenge with virulent M. ulcerans 1615. However, protection was weaker than the one conferred by vaccination with Ag85A or M. bovis BCG. Combinations of these polyketide synthase domains with the vaccine targeting Ag85A, of which the latter is involved in the integrity of the cell wall of the pathogen, and/or with live attenuated M. bovis BCG or mycolactone negative M. ulcerans may eventually lead to the development of an efficacious BU vaccine.

Vaccine-specific immune responses againstMycobacterium ulceransinfection in a low-dose murine challenge model

“…Mycolactone is the key virulence factor produced by M. ulcerans and an attractive vaccine target, but the molecule is poorly immunogenic (55). However, the PKS enzymes used by the bacterium to synthesize mycolactones – are highly conserved and immunogenic (49, 56-58). Therefore, we hypothesized that targeting the conserved enzymatic domains of the mycolactone PKS could be an effective vaccine strategy.…”

“…One domain in particular, the enoyl reductase (ER) protein domain, elicits serum antibodies in BU patients and healthy controls in BU endemic regions (57). The ER protein expressed as an antigen in a DNA-protein prime-boost vaccine has also been shown to reduce bacterial burden in a mouse footpad M. ulcerans challenge model (49). Here, we utilised the ER protein to create a novel BU vaccine candidate by electrostatically associating it with the TLR-2 agonist-based lipopeptide R 4 Pam 2 Cys.…”

“…Within each PKS are enzymatic domains that form the mycolactone molecule. Some of these domains have been found to be immunogenic and in particular immune responses against the enoyl reductase (ER) domain have previously been shown to significantly reduce M. ulcerans bacterial load in the footpad of a murine prime-boost vaccination study (49). Based on the immunogenic and protective qualities of ER, we have utilised it as a target antigen for a BU subunit vaccine.…”

The Immunology of Buruli Ulcer

“…Whether in the form of a plasmid DNA vaccine or as a recombinant protein, vaccination strategies with Ag85A conferred some degree of protection by eliciting a cellular response with high levels of IFN-γ, IL-2 and Ag85-specific IgGs, but, similarly to BCG, eventually failed to deter disease development [163][164][165][166]. Likewise, a vaccine employing DNA plasmids of polyketide synthase domains of Mycobacterium leprae Hsp65, previously tested in a murine model of tuberculosis, conferred protection against BU only for a few months, despite the high homology of the amino acid sequence with the M. ulcerans orthologue [167,168]. Therefore, better targets for immunization are still to be found and, to this end, a comparative genomics screening study identified several essential components of M. ulcerans that could provide important hints to direct future vaccination strategies [169].…”

Overexpression of a Mycobacterium ulcerans Ag85B-EsxH Fusion Protein in Recombinant BCG Improves Experimental Buruli Ulcer Vaccine Efficacy

“…Priming with a DNA-based vaccine encoding multiple MU polyketide synthase modules and boosting with recombinant protein by Roupie et al . yielded differential levels of antigen-specific IgG responses, as well as IFNγ and IL-2 secretion upon recombinant MU antigen stimulation [12]. However, no improvement in protection over the level conferred by BCG vaccination was observed.…”