Passive immunoprotection targeting a secreted CAMP factor of Propionibacterium acnes as a novel immunotherapeutic for acne vulgaris

Abstract

Propionibacterium acnes (P. acnes) bacteria play a key role in the pathogenesis of acne vulgaris. Although our previous studies have demonstrated that vaccines targeting a surface sialidase or bacterial particles exhibit a preventive effect against P. acnes, the lack of therapeutic activities and incapability of neutralizing secretory virulence factors motivate us to generate novel immunotherapeutics. In this study, we develop an immunotherapeutic antibody to secretory Christie-Atkins-Munch-Peterson (CAMP) factor of P. acnes. Via agroinfiltration, P. acnes CAMP factor was encapsulated into the leaves of radishes. ICR mice intranasally immunized with whole leaves expressing CAMP factor successfully produced neutralizing antibodies that efficiently attenuated P. acnes-induced ear swelling and production of macrophage-inflammatory protein-2. Passive neutralization of CAMP factor enhanced immunity to eradicate P. acnes at the infection site without influencing bacterial growth elsewhere. We propose that CAMP factor is a novel therapeutic target for the treatment of various P. acnes-associated diseases and highlight the concept of neutralizing P. acnes virulence without disturbing the bacterial commensalism in human microbiome.

Fig. 1

P. acnes CAMP factor exerted virulence activity. Ears of ICR mice were injected intradermally with recombinant GFP (left ear) and CAMP factors (right ear). (A) Inflammation-induced ear redness (arrow) was visualized 24 hours after injection. (B) Ear swelling was observed in an H&E-stained frozen tissue section of GFP- (a) or CAMP factor- (b) injected ear. The magnification 4× images indicated the dilated veins filled with erythrocytes (arrowheads). Bars (A)= 1 cm. Bars [B(a)]= 2 mm. Bars [B(b)]= 0.5 mm.

Fig. 2

CAMP factors and GUS transiently expressed in radish leaves. (A) Leaves of radish (Raphanw sativus L.) were infiltrated with A. tumefaciens (LBA4404 strains) transforming a 35S::GUS construct (right). Leaves infiltrated with non-transformed LBA4404 cells (left) served as negative controls. Dotted circles indicate locations of syringe infiltration with A. tumefaciens. Blue stained areas indicate the GUS expression. The dynamic pattern of GUS expression in radish leaves from 1 to 5 days after infiltration was analyzed by (B) histochemical and (C) GUS activity assays. (*p<0.05 and **p<0.005, by Student’s t-test). (D) Detection of CAMP factor expression by Western blot analysis. Ground radish leaves (20 µg) infiltrated with A. tumefaciens carrying a 35S::CAMP factor-His (CAMP factor-His), a 35S::SCAP-MBP-His (SCAP-MBP-His) or 15 µg recombinant GUS (rGUS) were run on a 10% (w/v) SDS-PAGE and blotted onto a nitrocellulose membrane. The membranes were then probed with anti-CAMP factor serum produced by mice immunized with UV-irradiated E. coli, BL21 (DE3) over-expressing CAMP factor. An arrow indicates CAMP factor appearing at a molecular weight of 29 kDa. Bar = 6 mm.

Fig. 3

Mice Immunized with CAMP factor-encapsulated whole leaves producing CAMP factor specific antibodies. Purified CAMP factor from leaf extracts (65 µg) run on a 10% (w/v) SDS-PAGE was blotted onto a nitrocellulose membrane and immuno-reacted to sera obtained from mice immunized with 25 µl of whole leaves containing GUS (left) or CAMP factors (right). A single band with 29 kDa indicates the purified CAMP factor reactive to serum from CAMP factor-immunized mice, verifying the immunogenicity of CAMP factor.

Fig. 4

Passive immunization of mice with neutralizing antibody to CAMP factor diminished P. acnes-induced inflammation. (A) 5 % (v/v) anti-GUS (open bar) or anti-CAMP factor (solid bar) serum-treated P. acnes (1 × 10⁷ CFU) was inoculated into the right ears of ICR mice to induce an increase in ear thickness as described in the “Materials and Methods”. As a control, an equal volume of PBS was injected into the left ears of the same mice. Ear thickness was measured with a micro-caliper at the indicated times after bacteria injection. The ear thickness of P. acnes-injected ear was calculated as % of a PBS-injected control. Error bars represented mean ± SE of four mice (**p<0.005, by Student’s t-test). (B) Ear redness (arrows) was visualized 3 days after injection with anti-GUS serum (a) or anti-CAMP factor (b) serum treated-P. acnes (1 × 10⁷ CFU). Bar = 1 cm. (C) Ear inflammation was observed in an H&E-stained frozen tissue section of ear injected with PBS alone (a) or P. acnes treated with anti-GUS (b) or anti-CAMP factor (c) serum. The granulamatous reactions (arrowheads) were visualized under magnification 4× (a, b, c; bars= 2 mm).

Fig. 5

Passive neutralization of P. acnes CAMP factor reduced the production of pro-inflammatory MIP-2 cytokine and bacterial colonization without altering P. acnes survival at other body site. (A) Measurement of pro-inflammatory MIP-2 cytokine was carried out by a sandwich ELISA using a Quantikine M mouse MIP-2 set. Compared to the neutralization with anti-GUS serum (open bar), passive neutralization with anti-CAMP factor serum (solid bar) markedly suppressed the P. acnes-induced increase in MIP-2. (B) The left ears of mice were injected with P. acnes (1 × 10⁷ CFU) in the presence of anti-GUS serum (open bar) or anti-CAMP factor serum (solid bar). (C) The right ears of both mice from (B) were injected with live P. acnes (1 × 10⁷ CFU) alone. Bacterial colonization (CFU) was quantified in agar plates as described in “Materials and Methods. Error bars represent mean ± SE of four mice (*p<0.05, by Student’s t-test).