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.

Figure 1. IMAC purified polyketide synthase domains and Ag85A (MUL4987) separated by SDS-PAGE.

Recombinant proteins (2,5 µg each) were separated by 15% (left) or 12% (right) SDS-PAGE and stained with PageBlue™. Lane 1, molecular weight markers (kD); 2, ACP1; 3, ACP2; 4, ACP3; 5, KR A; 6, molecular weight markers (kD); 7, ATac1; 8, Atac2; 9,ATp; 10, ER; 11, KS; 12, Ag85A (MUL4987).

Figure 2. Antigen-specific IgG antibodies in naïve and vaccinated mice.

IgG antibodies in C57BL/6 mice vaccinated twice with pDNA encoding the nine Pks synthase domains and boosted with the homologous recombinant protein. Sera were collected three weeks after the protein boost and tested by ELISA, using serial twofold dilutions, starting at 1∶50. Open circles: naive mice, closed circles vaccinated mice. Results presented as mean O.D. values ± SD of 4–6 mice tested individually.

Figure 3. Antigen-specific IL-2 production in naïve and vaccinated mice, as tested by ELISA.

Interleukin-2 levels in 24 hr spleen cell culture supernatants of C57BL/6 mice vaccinated against the 9 PkS domains using the pDNA prime/protein boost protocol and stimulated in vitro with the corresponding recombinant protein antigen (5 µg/ml). Results represent mean ± SD IL-2 values (pg/ml) of 4–6 mice tested individually. Data are representative of one of three experiments.

Figure 4. Antigen-specific IFN-γ production in naïve and vaccinated mice, as tested by ELISA.

IFN-γ levels in 72 hr spleen cell culture supernatants of C57BL/6 mice vaccinated against the 9 PkS domains using the pDNA prime/protein boost protocol and stimulated in vitro with the corresponding recombinant protein antigen (5 µg/ml). Results represent mean ± SD IFN-γvalues (pg/ml) of 4–6 mice tested individually. Data are representative of one of three experiments.

Figure 5. Antigen-specific IFN-γ production in naïve and vaccinated mice, as tested by ELISPOT.

Number of IFN-γ producing cells in C57BL/6 mice vaccinated against the 9 PkS domains using the pDNA prime/protein boost protocol and stimulated in vitro with the corresponding recombinant protein antigen (5 µg/ml). Results represent mean ± SD of spot forming cells/10⁶ cells of 4–6 mice tested individually as detected in a 48 hr ELISPOT. Data are representative of one of two experiments. White columns: cells without Ag stimulation; black columns: cells stimulated for 48 hr with corresponding antigen.

Figure 6. Number of AFB in footpad of naïve or vaccinated mice, six weeks after challenge.

Enumeration of acid fast bacteria (AFB) in footpad of naïve or vaccinated mice, challenged 6 weeks after the protein boost with 10⁵ AFB of M. ulcerans 1615 and sacrificed another 6 weeks later. Bars represent mean AFB ± SD in footpad of 5 mice tested individually. Numbers were converted to log₁₀ AFB/footpad for statistical analysis.

Figure 7. Survival of C57BL/6 mice vaccinated against Pks domains and challenged with M. ulcerans 1615.

Ten mice/group were challenged 6 weeks after the protein boost with 10⁵ AFB of M. ulcerans 1615 and subsequently monitored for 112 days for footpad swelling after M. ulcerans 1615 infection. Animals were euthanized when footpad swelling exceeded 4 mm. Figure shows the percentage of surviving mice during the 112 day follow-up. A: Survival curves of unvaccinated (ctrl) mice or mice vaccinated with ACP1, ACP2, ACP3, ATac1, ATac2 or M. bovis BCG. B: Survival curves of unvaccinated (ctrl) mice or mice vaccinated with ATp, ER, KR A, KS, Ag85A or M. bovis BCG.