High antibody titres induced by protein subunit vaccines against Buruli ulcer using Mycobacterium ulcerans antigens Hsp18 and MUL_3720

Background Mycobacterium ulcerans is the causative agent of a debilitating skin and soft tissue infection known as Buruli ulcer (BU). There is no vaccine against BU. The purpose of this study was to investigate the vaccine potential of two previously described immunogenic M. ulcerans proteins, MUL_3720 and Hsp18, using a mouse tail infection model of BU. Methods Recombinant versions of the two proteins were each electrostatically coupled with a previously described lipopeptide adjuvant. Seven C57BL/6 and seven BALB/c mice were vaccinated and boosted with each of the formulations. Vaccinated mice were then challenged with M. ulcerans via subcutaneous tail inoculation. Vaccine performance was assessed by time-to-ulceration compared to unvaccinated mice. Results The MUL_3720 and Hsp18 vaccines induced high titres of antigen-specific antibodies that were predominately subtype IgG1. However, all mice developed ulcers by day-40 post-M. ulcerans challenge. No significant difference was observed in the time-to-onset of ulceration between the experimental vaccine groups and unvaccinated animals. Conclusions These data align with previous vaccine experiments using Hsp18 and MUL_3720 that indicated these proteins may not be appropriate vaccine antigens. This work highlights the need to explore alternative vaccine targets and different approaches to understand the role antibodies might play in controlling BU.


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Buruli ulcer (BU) is a disease caused by Mycobacterium ulcerans. M. ulcerans infects 44 subcutaneous tissue and commonly presents as a skin nodule (in Africa) or papule (in Australia), 45 sometimes accompanied by redness; however, oedema is another common initial presentation. 46 As the disease progresses the skin around the infected area breaks down and an ulcer develops 47 [1,2]. Ulcers typically present with deep undermined edges and have a necrotic core comprised 48 of slough of bacteria, dead skin and immune cells [3,4]. Infections are rarely fatal but untreated 49 ulcers can destroy fat tissue, blood vessels, muscles and bone [5,6]. Bellarine and Mornington Peninsulas near the major metropolitan centre of Melbourne [10][11][12]. 59 The disease can affect all age groups and ethnicities [13]. In Australia, ulcers are predominately 60 reported on upper (27%) and lower limbs (70%) [14].

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M. ulcerans is a slow-growing bacterium, with a doubling time of greater than 48 hours. As such, 63 symptoms of BU can take months to appear after primary infection. If diagnosed early, BU can 64 be treated effectively by combination antibiotic therapy [15]. Unfortunately, in many cases the 65 disease can initially be misdiagnosed as other more common skin infections [16,17]. Delayed 66 4 diagnosis and treatment can lead to extensive lesions that leave victims with life-long 67 disfigurement and disability. Reparative surgery is often required for severe cases [18]. A 68 retrospective study in Australia showed that most diagnoses (87%) occurred once ulceration has 69 been reached [19] and in Ghana 66% cases were diagnosed with active lesions [20]. There is 70 currently no protective treatment for BU and no distinct mechanism of transmission. 71 Furthermore, treatment can be difficult to access for those in rural areas. Thus, there is a need to 72 develop an effective vaccine to protect those particularly in highly endemic areas. populations [21][22][23][24][25]. Therefore, the BCG vaccine is the benchmark for assessing potential M. 77 ulcerans vaccines. Some studies have assessed the efficacy of putative BU vaccines although 78 none have reached clinical trials [21,22,[26][27][28][29][30][31][32][33][34][35][36][37]. All these vaccines were tested in murine 79 challenge models and were not capable of preventing the eventual onset of disease.

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One approach to vaccination is to use antigens specific for a specific pathogen (e.g. certain 82 proteins(s) that are recognized by the immune system and induce neutralizing antibodies [38,83 39]. For rapid immune recognition these proteins would ideally be cell surface associated. Two 84 M. ulcerans proteins MUL_3720 and Hsp18 have been identified as potential candidates for 85 vaccine antigens. Hsp18 is a protein associated with biofilm formation and M. ulcerans-infected 86 individuals produce antibodies against Hsp18 [40,41]. MUL_3720 is a highly expressed cell-87 wall associated protein with a putative role in cell-wall biosynthesis [42,43]. As protein antigens may be poorly immunogenic on their own, adjuvants are used to enhance 90 antigenic potency. A lipopeptide adjuvant known as R 4 Pam 2 Cys has been found to increase 91 antigen uptake, increase dendritic cell trafficking to lymph nodes and enhance antibody 92 production against antigens derived from pathogens including influenza and hepatitis C in 93 murine models [44][45][46][47].

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The aim of this study was to try to develop a preventative vaccine against Buruli ulcer, 96 comprising two highly expressed cell-wall associated proteins, MUL_3720 or Hsp18, bound to 97 an R 4 Pam 2 Cys-based lipopeptide adjuvant. Samples were denatured in an equal volume of 2 x sample loading buffer (40% (v/v) 0.5M Tris-130 HCL pH 6.8, 10% glycerol, 1.7% (w/v) SDS, 10% 2-β-mercaptoethanol, 0.13% (w/v) 131 bromophenol blue in distilled water) at 100°C for 5 minutes. Ten microlitres of each sample and 132 SeeBlue® Plus2 pre-stained protein standard (Invitrogen) were loaded into a 0.5mm 12%       Serum was prepared from blood obtained from mice at day 0, day 18, day 33 and day 63.

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MUL_3720 preferentially bound to R 4 Pam 2 Cys compared to E 8 Pam 2 Cys ( Fig. 2A). This is 243 shown as a gradual increase in optical density following the addition of increasing amounts of 244 R 4 Pam 2 Cys to a constant amount of MUL_3720. At a 5-fold molar excess of protein to 245 lipopeptide the OD 450 plateaued, suggesting MUL_3720 bound most strongly to R 4 Pam 2 Cys at a 246 1:5 protein to lipopeptide ratio. Conversely, when E 8 Pam 2 Cys was added to MUL_3720 the 247 optical density remained static and did not increase with increasing lipopeptide concentrations, 248 indicating a lack of binding. Hsp18 also appeared to bind preferentially to R 4 Pam 2 Cys and also at 249 a 1:5 ratio of Hsp18 to R 4 Pam 2 Cys ( Figure 2B). Therefore, two protein-adjuvant formulations 250 were prepared using MUL_3720 with R 4 Pam 2 Cys and Hsp18 with R 4 Pam 2 Cys, both at a 1:5 251 protein to lipopeptide molar ratio.  Vaccination with MUL_3720 recombinant protein alone or MUL_3720 + R 4 Pam 2 Cys were 261 capable of inducing MUL_3720-specific antibody titres in both BALB/c and C57BL/6 strains of 262 13 mice (Fig. 3A, B). Primary vaccination with MUL_3720 protein alone induced MUL_3720-263 specific antibody responses that significantly increased (p < 0.0001) following a vaccine boost (p 264 = 0.0234). Additionally, MUL_3720 + R 4 Pam 2 Cys generated MUL_3720 specific antibody 265 responses after primary vaccination (p < 0.0001 in BALB/c and C57BL/6), which were increased 266 after the secondary boost (p < 0.0001 in BALB/c and not statistically significant in C57BL/6).

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The titres after the boost in particular were greater than MUL_3720 alone vaccination (p =  Quantifying levels of IgG antibody shows that the predominant isotypes produced by 288 MUL_3720 were IgG 1 and IgG2 b (Fig. 3E) with no significant difference between these isotype 289 titres. Vaccination with MUL_3720 + R 4 Pam 2 Cys produces significantly more IgG 1 and IgG2 b 290 antibodies (p = 0.0076). The antibody titres for both isotypes were highest prior to infection with 291 M. ulcerans (day 33) and decreased after infection by day 63. This vaccine was capable of 292 inducing IgG2a antibodies, which was detected also on day 33, however in smaller amounts than 293 IgG 1 and IgG2 b (p = 0.0399 for MUL_3720 + R 4 Pam 2 Cys) (Fig. 3E).

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Similar to vaccination with MUL_3720, Hsp18 was also capable of inducing strong IgG 296 antibody titres. The predominant isotype was IgG 1 which Hsp18 + R 4 Pam 2 Cys elicited more than 297 any other isotype (Fig. 3F) including IgG2 a and IgG2 b , Again, these titres was highest at day 33  clinical symptoms for Buruli ulcer in this model (Fig. 4). Once ulceration has been reached the 307 15 disease would likely continue until the tail became necrotic. Therefore, the experimental 308 endpoint was deemed to be the point of ulceration.  (Table 1 and Table 2) and all mice reached ulceration by day 63, 30 days post-M. ulcerans 319 challenge (Fig. 5A, B).  (Fig. 3A, C) the antibody titres at day 63 were lower than after the secondary 329 response prior to challenge (p < 0.0001 for both Hsp18 + R 4 Pam 2 Cys and MUL_3720 + 330 R 4 Pam 2 Cys) but remained significantly higher than at day 0 (p < 0.0001 for both Hsp18 + 331 R 4 Pam 2 Cys and MUL_3720 + R 4 Pam 2 Cys). In C57BL/6 mice ( Fig. 3B and 3D), antibody titres 332 against MUL_3720 or Hsp18 from mice vaccinated with either protein alone or protein plus 333 lipopeptide adjuvant were also significantly decreased at day 63 compared to the secondary 334 response at day 35 (p < 0.0001 and p = 0.0406 for MUL_3720 + R 4 Pam 2 Cys and Hsp18 + 335 R 4 Pam 2 Cys, respectively). Similar to BALB/c mice, the day 63 respective protein-specific 336 antibodies for MUL_3720 + R 4 Pam 2 Cys and Hsp18 + R 4 Pam 2 Cys were significantly higher than 337 at day 0 (p < 0.0001 and p = 0.0004 for MUL_3720 + R 4 Pam 2 Cys and Hsp18 + R 4 Pam 2 Cys, 338 respectively). responses are much lower than the protein-specific antibody responses generated from 347 MUL_3720 or Hsp18 vaccinated mice, particularly in C57/BL6 mice (p < 0.0001) (Fig. 6).

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Animals from both mouse strains that were vaccinated with R 4 Pam 2 Cys alone or BCG showed 349 no increase in protein-specific antibody responses against either recombinant MUL_3720 and This study aimed to develop a vaccine against M. ulcerans utilizing two previously described 355 cell-wall associated proteins, Hsp18 and MUL_3720 [40][41][42][43]. Both the MUL_3720 and Hsp18-356 based vaccines were capable of inducing high antibody titres, but these responses were not 357 associated with protection (Fig. 5). This may indicate that these proteins, while strongly  The greatest antibody responses were of the IgG1 subclass. Typical antibody responses against 395 proteins occur via B cell isotype switching from IgM (non-specific antibody isotype) to IgG.

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There are 4 subclasses of IgG (IgG1, IgG2, IgG3 and IgG4) and isotype switching to 397 predominantly IgG1 suggests refinement of immune responses to respond specifically to either 398 MUL_3720 or Hsp18, as IgG1 is capable of binding to protein antigens [67]. IgG1 can also bind 399 all forms of FcγR which is required to elicit and mediate effector immune functions as described