Vaccine 33 (2015) 3512–3517
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Vaccination against Staphylococcus aureus experimental endocarditis using recombinant Lactococcus lactis expressing ClfA or FnbpA Tiago Rafael Veloso a,1 , Stefano Mancini a , Marlyse Giddey a , Jacques Vouillamoz a , Yok-Ai Que b , Philippe Moreillon a , José Manuel Entenza a,∗ a b
Department of Fundamental Microbiology, University of Lausanne, CH-1015 Lausanne, Switzerland Department of Intensive Care Medicine, University Hospital Medical Centre (CHUV) and University of Lausanne, CH-1011 Lausanne, Switzerland
a r t i c l e
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Article history: Received 3 March 2015 Received in revised form 6 May 2015 Accepted 22 May 2015 Available online 3 June 2015 Keywords: Staphylococcus aureus Vaccine Lactococcus lactis immunization ClfA FnbpA Endocarditis Rat
a b s t r a c t Staphylococcus aureus is a major cause of serious infections in humans and animals and a vaccine is becoming a necessity. Lactococcus lactis is a non-pathogenic bacterium that can be used as a vector for the delivery of antigens. We investigated the ability of non-living L. lactis heterologously expressing S. aureus clumping factor A (ClfA) and fibronectin-binding protein A (FnbpA), alone or together, to elicit an immune response in rats and protect them from S. aureus experimental infective endocarditis (IE). L. lactis ClfA was used for immunization against S. aureus Newman (expressing ClfA but not FnbpA), while L. lactis ClfA, L. lactis FnbpA, as well as L. lactis ClfA/FnbpA, were used against S. aureus P8 (expressing ClfA and FnbpA). Vaccination of rats with L. lactis ClfA elicited antibodies that inhibited binding of S. aureus Newman to fibrinogen, triggered the production of IL-17A and conferred protection to 13/19 (68%) of the animals from IE (P < 0.05). Immunization with L. lactis ClfA, L. lactis FnbpA or L. lactis ClfA/FnbpA also produced antibodies against the target proteins, but these did not prevent binding of S. aureus P8 to fibrinogen or fibronectin and did not protect animals against S. aureus P8 IE. Moreover, immunization with constructs containing FnbpA did not increase IL-17A production. These results indicate that L. lactis is a valuable antigen delivery system able to elicit efficient humoral and cellular responses. However, the most appropriate antigens affording protection against S. aureus IE are yet to be elucidated. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Staphylococcus aureus is a major pathogen responsible for a variety of diseases, ranging from benign skin infections to lifethreatening sepsis, pneumonia and infective endocarditis (IE) [1]. Given the high morbidity and mortality rates associated with S. aureus infections and the limited therapeutic options, the development of a safe and effective vaccine against S. aureus represents a genuine need for the medical community. Several capsular polysaccharides components, cell-wall associated proteins, secreted clotting factors and secreted proteins have been tested as vaccine candidates for S. aureus infections [2–5]. However, despite promising results obtained in pre-clinical and
∗ Corresponding author. Tel.: +41 21 6925612. E-mail address: [email protected] (J.M. Entenza). 1 Present address: Cardiovascular Developmental Biology, Department of Cardiovascular Sciences, Katholieke Universiteit Leuven, 3000 Leuven, Belgium. http://dx.doi.org/10.1016/j.vaccine.2015.05.060 0264-410X/© 2015 Elsevier Ltd. All rights reserved.
initial clinical studies, no licensed S. aureus vaccines are available so far. A novel recombinant alum-adjuvanted vaccine (NDV-3) is currently under human clinical evaluation [6,7]. S. aureus produces numerous infections that imply different pathogenic mechanisms and different types of host defences. Therefore, one approach to immunize against S. aureus would be to design vaccines targeting specific diseases [8]. Some of them have very specific features, as for instance IE, where initial adherence of circulating bacteria to damaged heart valves is pathognomonic of disease establishment. S. aureus ClfA (fibrinogen/fibrin-binding protein clumping factor A) and FnbpA (fibronectin binding protein A) are critical virulence factors for IE [9–11]. Thus, they represent intuitively logical targets for vaccine development against this disease. However, previous immunization studies showed that they were not totally satisfactory in animal models [12,13]. These failures could relate to several reasons including the nature of the presented antigens, the plethora of S. aureus surface proteins, or even the inoculation method used in the in vivo infection model.
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Lactococcus lactis has recently emerged as a promising candidate to be used as a vehicle for the delivery of antigens against bacterial infections [14–16]. A momentous advantage conferred by this system is the capability to safely deliver the antigens to the immune system, possibly in their native conformation [17]. We therefore explored the capacity of non-living recombinant L. lactis, heterologously expressing on its surface ClfA, FnbpA, or ClfA together with FnbpA, to induce both humoral and cellular responses and to protect against S. aureus IE. These immunization studies were conducted in a rat model of endocarditis induced by low-grade bacteremia, a setting that better mimics the infection process of S. aureus in humans [18]. We first tested the concept by immunizing rats with L. lactis ClfA against S. aureus Newman. This organism lacks FnbpA [19], which carries both fibronectin- and fibrinogen-binding domains. We then evaluated the protective effect of immunizing with L. lactis ClfA, L. lactis FnbpA, and of L. lactis ClfA/FnpbA against S. aureus P8, an endocarditis isolate that expresses both ClfA and FnbpA on its surface. The results indicate that L. lactis was an efficient delivery system of S. aureus ClfA and FnbpA antigens and elicited both humoral and cellular responses. However, protection efficacies were different against S. aureus strains carrying only ClfA or ClfA plus FnbpA.
2. Materials and methods 2.1. Bacterial strains and growth conditions L. lactis heterologously expressing S. aureus ClfA and FnbpA on their surface via the pIL253-based expression plasmid, either individually (L. lactis ClfA and L. lactis FnbpA) or together (L. lactis ClfA/FnbpA) [9,20,21], were used for immunization. L. lactis carrying the empty vector pIL253 was used as a control. S. aureus Newman [22] and S. aureus P8 [23] were used to induce experimental endocarditis. All L. lactis strains were grown at 30 ◦ C in M17 broth medium (Difco; Becton Dickinson, Sparks, MD) or on M17 agar plates supplemented with 0.5% glucose and 5 g/ml erythromycin. S. aureus isolates were grown at 37 ◦ C in tryptic soy broth (Difco) or on tryptic soy agar plates (Difco). 2.2. Recombinant proteins The S. aureus recombinant proteins ClfA40 (rClfA), corresponding to the A domain (amino acid 40 to 559) of the S. aureus Newman ClfA [24], and FnbpA-BCD (rFnbpA), corresponding to the BCD domains (amino acid 476 to 838) of the FnbpA of S. aureus 83254 [25] were used to generate polyclonal antibodies in rats. The Escherichia coli TOPP3 (rClfA) and XL1-Blue (rFnbpA) strains containing the pQE30 plasmid (Qiagen, Valencia, USA) with ClfA40 or FnbpA-BCD as inserts, respectively, were kindly provided by Timothy Foster (Trinity College, Dublin, Ireland). Both recombinant proteins contained an N-terminal 6-His tag and were purified from E. coli lysates as described elsewhere [24]. Purified rClfA was dialyzed against 50 mM NaCl, 2 mM EDTA, 20 mM Tris–HCl (pH 7.4) and the purified rFnbpA against 25 mM Tris–HCl (pH 8.0) [26] overnight at 4 ◦ C. 2.3. Production of anti-recombinant proteins immune antisera in rats Immune antisera against rClfA and rFnbpA were produced by Eurogentec (Liege, Belgium), by injecting rats with 60 g of rClfA or rFnbpA, at days 0, 7, 10 and 18, by using a Speedy-28 days immunization protocol. Blood samples were taken at days 0 (before
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immunization) and 28 (final bleed) and antisera prepared therewith. 2.4. Animal studies All animal protocols were reviewed and approved by the Cantonal Committee on Animal Experiments of the State of Vaud, Switzerland (Permit Number: 879.8). A mixture of ketamine (75 mg/kg) and midazolam (5 mg/kg) anesthetics was administered to the animals before any surgical procedure. 2.4.1. Immunization studies Six weeks old female Wistar rats (75–100 g) were used in the immunization experiments. L. lactis strains were cultured overnight, harvested by centrifugation, resuspended in PBS and adjusted to 1 × 108 CFU/ml. Bacteria were then killed by ultraviolet (U.V.) exposure for 1 h at 1 J/cm2 . U.V-induced killing was confirmed by the absence of viable organisms. L. lactis pIL253, L. lactis ClfA, L. lactis FnbpA, and L. lactis ClfA/FnbpA were emulsified with Freund’s adjuvant and administered at days 1, 14 and 28. At day 1 emulsification was in a 1:1 ratio with complete Freund’s adjuvant (CFA; Sigma Aldrich, Buchs, Switzerland). At days 14 and 28 emulsification was in a 1:1 ratio with incomplete Freund’s adjuvant (IFA; Sigma). Control groups of rats were injected with phosphate buffer saline (PBS), CFA or IFA (mixed with PBS in a 1:1 ratio). Blood samples were collected at days 0 (before immunization), 7, 21 and 41, the antisera were prepared therewith and stored at −80 ◦ C. 2.4.2. Animal model of endocarditis The production of catheter-induced aortic vegetations and the installation of an intravenous (i.v.) line to deliver the inocula, were performed in rats on day 41, i.e., two weeks after the last immunization dose, as described [18]. Twenty-four hours later, rats were inoculated by continuous infusion (0.0017 ml/min over 10 h) of 104 to 105 CFU of S. aureus Newman or S. aureus P8 [18]. Rats were euthanized 24 h after the end of inoculation. The cardiac vegetations and the spleen were removed, weighed, homogenized, serially diluted and plated on blood agar plates. Plates were incubated for 48 h at 37 ◦ C to determine the number of viable organisms in the tissues. 2.5. Enzyme-linked immunosorbent assay (ELISA) for detection of antibodies ELISA assays were performed as described by Hawkins et al. [27], to detect the presence of polyclonal antibodies specific for rClfA, rFnbpA, ClfA and FnbpA in rat antisera. The assays were performed on two independent occasions. The ELISA titers were expressed as Relative Fluorescence Units (RFU). 2.6. Characterization of functional antibodies in serum The presence of functional antibodies was determined by testing the ability of serum from immunized rats to inhibit the adherence of S. aureus to immobilized fibrinogen and fibronectin. Microtiter plates were coated with 10 g/ml of human fibrinogen or human fibronectin (Sigma) overnight at 4 ◦ C, incubated for 1 h with blocking solution (PBS-0.01% bovine serum albumin) to reduce non-specific binding, and washed with PBS. S. aureus Newman and S. aureus P8 (109 CFU/ml) were preincubated for 30 min at 37 ◦ C with the test antisera (diluted 1:100 in PBS-0.05% Tween 20). Samples were washed and resuspended in PBS, and 50 l were added in triplicate to the wells of fibrinogenor fibronectin-coated plates. Controls with only bacteria and buffer were included. The binding assays were performed as described
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by Ythier et al. [28]. At least three independent experiments were performed. 2.7. Interleukin-17A (IL-17A) production induced by immunization Levels of IL-17A, considered as a surrogate marker of cellmediated response, were measured in the plasma of animals at day 41 of the immunization protocol by the Bio-Plex Rat Cytokine assay (Bio-Rad, Hercules, CA) according to the manufacturer’s indications. 2.8. Statistical analysis Data of antibody titers, protein adherence inhibition assays, IL-17A production, and mean bacterial densities in vegetations and spleens were evaluated by the one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison tests. The incidences of valve infection were compared by the Chi-squared test. A value of P < 0.05 was considered significant by using two-tailed significance levels. All statistical analyses were performed with the GraphPad Prism 6.0 program (www.graphpad.com). 3. Results
Titers of anti-ClfA and anti-FnbpA in rat sera are shown in Fig. 1. In animals immunized with L. lactis expressing ClfA the titer of anti-ClfA antibodies increased by almost three times (14,308 ± 13,501 RFU) as compared with values in animals before immunization (∼5000 RFU). A similar outcome was observed in animals immunized with L. lactis ClfA/FnbpA (Fig. 1A). Anti-ClfA levels in rats receiving PBS, the adjuvant, L. lactis pIL253 or L. lactis FnbpA were comparable to the levels detected in the sera before immunization. Anti-FnbpA titers (Fig. 1B) in animals immunized with L. lactis FnbpA or L. lactis ClfA/FnbpA were approximately six (28,878 ± 12,840 RFU) and three times (16,246 ± 10,051) higher than the levels detected in sera before immunization (∼5,000 RFU). Anti-FnbpA titers in rats receiving PBS, the adjuvant, the L. lactis pIL253 or L. lactis ClfA did not significantly differ from the levels detected in the sera before immunization. 3.2. Fibrinogen- and fibronectin-binding inhibition assays 3.2.1. S. aureus Newman As shown in Fig. 2A, sera taken from L. lactis ClfA-immunized animals at day 41 (see Fig. 1A) significantly inhibited the adhesion capacity of S. aureus Newman to fibrinogen while sera of non-immunized control groups did not. The inhibition pattern was similar to that observed with the rClfA protein, used as control.
3.1. Titers of anti-ClfA and anti-FnbpA antibody Antibody titers in rats immunized with purified rClfA and rFnbpA were, respectively more than six (30,560 ± 3237 RFU) and four (35,100 ± 1733 RFU) times higher than those before immunization (4631 ± 2164 and 7854 ± 2333 RFU, respectively).
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3.2.2. S. aureus P8 Sera taken from animals immunized with L. lactis ClfA or L. lactis FnbpA at day 41 (see Fig. 1B) did not inhibit binding of S. aureus P8 to fibrinogen (Fig. 2B, upper panel). Immunization with L. lactis ClfA/FnbpA provided some inhibition, although it was
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Fig. 1. Anti-ClfA (A) and anti-FnbpA (B) antibody titers of immunized rats. Animals were immunized with PBS, adjuvant, strain L. lactis pIL253 or recombinant L. lactis ClfA, L. lactis FnbpA or L. lactis ClfA/FnbpA as described in Section 2. Antibodies were quantified by ELISA. Each bar represents the mean ± standard deviation of six serum samples per group, which were obtained in two independent experiments. P values for selected comparisons were determined by ANOVA with Dunnett’s multiple comparisons post-test.
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Fig. 2. Effect of anti-ClfA sera on S. aureus Newman (A), and S. aureus P8 ((B) upper panel) binding to immobilized fibrinogen. S. aureus P8 binding to immobilized fibronectin ((B) lower panel) was also tested in presence of anti-FnbpA sera. Bacteria were pre-incubated for 30 min with sera (diluted 1:100) and added to fibrinogen- or fibronectincoated microtiter plate wells. Results are expressed as means ± standard deviations of six serum samples per group, which were obtained in two independent experiments. * P < 0.05 by ANOVA with Dunnett’s multiple comparisons post-test compared to both BPS and adjuvant groups.
relatively marginal. Moreover, sera from L. lactis FnbpA or L. lactis ClfA/FnbpA-immunized animals did not reduce binding of this strain to fibronectin (Fig. 2B, lower panel). S. aureus P8 even exhibited increased adherence to fibronectin in sera from L. lactis FnbpAand rFnbpA immunized animals.
in both the vegetations and spleen tissues were similar to those of control animals.
3.3. IL-17A production induced by recombinant L. lactis immunization
This study showed that vaccination of rats with L. lactis expressing S. aureus ClfA protected them against endocarditis induced by S. aureus Newman, a strain expressing ClfA but no other important factors of S. aureus IE, such as FnbpA. The protective effect correlated with the production of blocking anti-ClfA antibodies as well as IL-17A. These findings are in accord with a previous work carried out by Josefsson et al. [29], who showed that immunization with the recombinant A domain of ClfA induced protective humoral immunity and prevented S. aureus Newman-induced arthritis in mice, as well as with those of Narita et al. [30], who showed that immunization with the fibrinogen-binding domain of ClfA induced substantial production of IL-17A, suggesting a role of the cellularmediated immunity in the protection against S. aureus infection in mice. Other immunization studies using a Candida albicans surface protein with structural similarity to ClfA or nanoparticules loaded with ClfA also induced protection against S. aureus infection which is supported by a IL-17A-enhanced response [31,32]. However, when S. aureus P8 (which carries both ClfA and FnbpA) was tested instead of S. aureus Newman, neither the monovalent (L. lactis ClfA and L. lactis FnbpA) nor the bivalent (L. lactis ClfA/FnbpA) vaccines afforded binding inhibition in vitro or protection against endocarditis in rats. Yet, they all elicited anti-ClfA and -FnbpA antibodies as determined by ELISA. In the case of vaccination with monovalent L. lactis ClfA, one could postulate that in vitro binding and in vivo infection were mediated by the FnbpA adhesin of S. aureus P8, which was not included in the vaccine. However, the results became intriguing when FnbpA was delivered as an antigen alone (i.e., in L. lactis FnbpA). Indeed, rather than inhibiting adherence, it slightly increased in vitro binding of S. aureus P8 to both fibrinogen and fibronectin. The addition of ClfA to the immunization protocol (in L. lactis ClfA/FnbpA) could somewhat modulate this effect, but could not entirely revert it. This suggested that
As shown in Fig. 3, control animals (PBS, adjuvant and L. lactis pIL253) exhibited similar IL-17A values than in plasma collected on day 0. Only plasma of rats immunized with L. lactis ClfA contained IL-17A levels substantially higher than control animals, while those of rats immunized with L. lactis FnbpA and L. lactis ClfA/FnbpA were unchanged. 3.4. Experimental endocarditis 3.4.1. S. aureus Newman As shown in Fig. 4A, rats immunized with L. lactis ClfA and subsequently challenged with S. aureus Newman, a strain expressing ClfA but non-functional FnbpA due to gene truncation, had significantly (P < 0.05) less infected vegetations (6 of 19; 32%) as compared to controls receiving PBS (9 of 12; 75%), the adjuvant alone (8 of 11; 73%) or L. lactis pIL253 (6 of 8; 75%). L. lactis ClfA-immunized animals also showed reduced bacterial loads in infected vegetations (4.4 log10 CFU/g) as compared to control animals (5.2 to 6.0 log10 CFU/g), although the difference was not statistically significant. In contrast, immunization with L. lactis ClfA did not decrease the rate or the bacterial load of spleen infection (data not shown). 3.4.2. S. aureus P8 The protection efficacy of this strategy was also investigated toward experimental endocarditis induced by the S. aureus isolate P8, a strain carrying both functional ClfA and FnbpA. Rats were immunized with L. lactis ClfA, L. lactis FnbpA, or L. lactis ClfA/FnbpA, but none of the immunizations protected the rats from IE (Fig. 4B) or spleen infection (data not shown). Furthermore, bacterial loads
4. Discussion
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IL-17A concentration (pg/ml)
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Fig. 3. IL-17A levels in the plasma of immunized rats. Titers of IL-17a were determined after the last booster immunization dose, i.e., at day 41. Results are expressed as means ± standard deviation of three to six individual animals per group.
anti-FnbpA vaccination conveyed some kind of binding-enhancing rather than blocking properties. A similar outcome was observed in previous works conducted by Rindi et al. and Casolini et al., whereby pre-incubation of S. aureus with sera of patients with IE containing high titers of anti FnbpA affected only slightly the ability of S. aureus to bind fibronectin [33,34]. Speziale et al. demonstrated that a monoclonal antibody generated from a mouse immunized against Streptococcus disgalactiae fibronectin binding protein FnbA cooperated to stabilize the binding of the protein to the ligand fibronectin [35]. These observations and the present results suggest that during the process of binding to fibronectin, FnbpA may dislodge potential neutralizing antibodies – may be due to a greater affinity for its natural ligand – or may constantly hide sensitive domains from blocking antibodies. Thus, blockage of FnbpA with natural antibodies might not be achieved unless we have a better comprehension of its dynamic interaction with its target. Such interference extended even to the cellular immune response. IL-17A was specifically induced by vaccination with L. lactis ClfA, but not with the control vaccine and especially not with L.
lactis FnbpA. Moreover, the presence of FnbpA in the immunization protocol prevented IL-17A induction when both ClfA and FnbpA were present in the vaccine. Fibronectin has been shown to promote the proliferation of T cells through interaction with its cell surface ligands [36]. One may postulate that anti-FnbpA antibodies may interfere with T cell proliferation by blocking fibronectin and subsequent fibronectin-T cell-ligand interactions. The present results with S. aureus P8 may help explain weak prevention in earlier experimental immunization studies using only anti-ClfA immunoglobulins [13] or the domains of FnbpA responsible for binding to fibronectin [12] against S. aureus isolates other than Newman. Moreover, they help dissect out the role of individual vaccine proteins in a specific disease. In case of vaccination with L. lactis ClfA protection was afforded only against valve infection, not spleen infection. This suggests that ClfA was either not required for infection of this organ or that other virulence determinants were at work [37,38]. Several immunizations studies using a multivalent vaccine consisting of several S. aureus antigens proved to be more effective than single antigen vaccines against S. aureus infections in
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Fig. 4. Results of immunization with recombinant L. lactis expressing ClfA or/and FnbpA against experimental IE induced by S. aureus Newman (A) and S. aureus P8 (B). Values on top of each column are the mean ± standard deviation of bacterial densities (log10 CFU) recovered from infected vegetations. * P < 0.05 by Chi-square test compared to all the other groups.
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mice [5,39,40]. One drawback of multivalent antigen vaccination is represented by the complex interactions and the difficulty to understand them. Although it imitates spontaneous immunization following S. aureus infection, it does not help understand why natural immunization does not confer prolonged immunity. One potential advantage of displaying antigens (especially surface proteins) on the surface of bacterial vectors is to present them in a more natural context, which may influence their conformation. This may help both display a more natural set of epitopes, and understand why there might be immune escape phenomena, such in the case of FnbpA. In conclusion, the present results using L. lactis as an in vivo vector suggest that ClfA is a good candidate for vaccination against S. aureus IE, but cautious should be raised toward FnbpA. Conflict of interest statement The authors have no financial or personal relationships with any persons or organizations that might influence the content of this paper. The University of Lausanne has submitted a patent application and named as inventors the authors TRV, YAQ, PM and JME. All authors have approved the final article. Funding sources This work was funded by grant 310030-143799 from the Swiss National Science Foundation. References [1] Que YA, Moreillon P. Staphylococcus aureus (including Staphylococcal toxic shock). In: Bennett JE, Dolin R, Blaser MJ, editors. Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases. 8th ed. Philapelphia: Elsevier/Saunders; 2015. p. 2237–71. [2] Verkaik NJ, van Wamel WJ, van Belkum A. Immunotherapeutic approaches against Staphylococcus aureus. Immunotherapy 2011;3:1063–73. [3] Jansen KU, Girgenti DQ, Scully IL, Anderson AS. Vaccine review: Staphyloccocus aureus vaccines: problems and prospects. Vaccine 2013;31:2723–30. [4] Proctor RA. Challenges for a universal Staphylococcus aureus vaccine. Clin Infect Dis 2012;54:1179–86. [5] Rauch S, Gough P, Kim HK, Schneewind O, Missiakas D. Vaccine protection of leukopenic mice against Staphylococcus aureus bloodstream infection. Infect Immun 2014;82:4889–98. [6] Spellberg B, Ibrahim AS, Yeaman MR, Lin L, Fu Y, Avanesian V, et al. The antifungal vaccine derived from the recombinant N terminus of Als3p protects mice against the bacterium Staphylococcus aureus. Infect Immun 2008;76: 4574–80. [7] Schmidt CS, White CJ, Ibrahim AS, Filler SG, Fu Y, Yeaman MR, et al. NDV-3, a recombinant alum-adjuvanted vaccine for Candida and Staphylococcus aureus, is safe and immunogenic in healthy adults. Vaccine 2012;30:7594–600. [8] Fowler Jr VG, Proctor RA. Where does a Staphylococcus aureus vaccine stand? Clin Microbiol Infect 2014;20(Suppl. 5):66–75. [9] Que YA, Haefliger JA, Piroth L, Francois P, Widmer E, Entenza JM, et al. Fibrinogen and fibronectin binding cooperate for valve infection and invasion in Staphylococcus aureus experimental endocarditis. J Exp Med 2005;201:1627–35. [10] Widmer E, Que YA, Entenza JM, Moreillon P. New concepts in the pathophysiology of infective endocarditis. Curr Infect Dis Rep 2006;8:271–9. [11] Veloso TR, Chaouch A, Roger T, Giddey M, Vouillamoz J, Majcherczyk P, et al. Use of a human-like low-grade bacteremia model of experimental endocarditis to study the role of Staphylococcus aureus adhesins and platelet aggregation in early endocarditis. Infect Immun 2013;81:697–703. [12] Schennings T, Heimdahl A, Coster K, Flock JI. Immunization with fibronectin binding protein from Staphylococcus aureus protects against experimental endocarditis in rats. Microb Pathog 1993;15:227–36. [13] Vernachio J, Bayer AS, Le T, Chai YL, Prater B, Schneider A, et al. Anti-clumping factor A immunoglobulin reduces the duration of methicillin-resistant Staphylococcus aureus bacteremia in an experimental model of infective endocarditis. Antimicrob Agents Chemother 2003;47:3400–6. [14] Villatoro-Hernandez J, Montes-de-Oca-Luna R, Kuipers OP. Targeting diseases with genetically engineered Lactococcus lactis and its course towards medical translation. Expert Opin Biol Ther 2011;11:261–7. [15] Bahey-El-Din M. Lactococcus lactis-based vaccines from laboratory bench to human use: an overview. Vaccine 2012;30:685–90.
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[16] Trombert A. Recombinant lactic acid bacteria as delivery vectors of heterologous antigens: the future of vaccination? Benefic Microbes 2015;6:1–12. [17] Ythier M, Resch G, Waridel P, Panchaud A, Gfeller A, Majcherczyk P, et al. Proteomic and transcriptomic profiling of Staphylococcus aureus surface LPXTGproteins: correlation with agr genotypes and adherence phenotypes. Mol Cell Proteomics 2012;11:1123–39. [18] Veloso TR, Amiguet M, Rousson V, Giddey M, Vouillamoz J, Moreillon P, et al. Induction of experimental endocarditis by continuous low-grade bacteremia mimicking spontaneous bacteremia in humans. Infect Immun 2011;79:2006–11. [19] Grundmeier M, Hussain M, Becker P, Heilmann C, Peters G, Sinha B. Truncation of fibronectin-binding proteins in Staphylococcus aureus strain Newman leads to deficient adherence and host cell invasion due to loss of the cell wall anchor function. Infect Immun 2004;72:7155–63. [20] Que YA, Haefliger JA, Francioli P, Moreillon P. Expression of Staphylococcus aureus clumping factor A in Lactococcus lactis subsp. cremoris using a new shuttle vector. Infect Immun 2000;68:3516–22. [21] Que YA, Francois P, Haefliger JA, Entenza JM, Vaudaux P, Moreillon P. Reassessing the role of Staphylococcus aureus clumping factor and fibronectin-binding protein by expression in Lactococcus lactis. Infect Immun 2001;69:6296–302. [22] Moreillon P, Entenza JM, Francioli P, McDevitt D, Foster TJ, Francois P, et al. Role of Staphylococcus aureus coagulase and clumping factor in pathogenesis of experimental endocarditis. Infect Immun 1995;63:4738–43. [23] Entenza JM, Drugeon H, Glauser MP, Moreillon P. Treatment of experimental endocarditis due to erythromycin-susceptible or -resistant methicillinresistant Staphylococcus aureus with RP 59500. Antimicrob Agents Chemother 1995;39:1419–24. [24] O’Connell DP, Nanavaty T, McDevitt D, Gurusiddappa S, Hook M, Foster TJ. The fibrinogen-binding MSCRAMM (clumping factor) of Staphylococcus aureus has a Ca2+ -dependent inhibitory site. J Biol Chem 1998;273:6821–9. [25] Fitzgerald JR, Loughman A, Keane F, Brennan M, Knobel M, Higgins J, et al. Fibronectin-binding proteins of Staphylococcus aureus mediate activation of human platelets via fibrinogen and fibronectin bridges to integrin GPIIb/IIIa and IgG binding to the FcgammaRIIa receptor. Mol Microbiol 2006;59:212–30. [26] Wann ER, Gurusiddappa S, Hook M. The fibronectin-binding MSCRAMM FnbpA of Staphylococcus aureus is a bifunctional protein that also binds to fibrinogen. J Biol Chem 2000;275:13863–71. [27] Hawkins J, Kodali S, Matsuka YV, McNeil LK, Mininni T, Scully IL, et al. A recombinant clumping factor A-containing vaccine induces functional antibodies to Staphylococcus aureus that are not observed after natural exposure. Clin Vaccine Immunol 2012;19:1641–50. [28] Ythier M, Entenza JM, Bille J, Vandenesch F, Bes M, Moreillon P, et al. Natural variability of in vitro adherence to fibrinogen and fibronectin does not correlate with in vivo infectivity of Staphylococcus aureus. Infect Immun 2010;78:1711–6. [29] Josefsson E, Hartford O, O’Brien L, Patti JM, Foster T. Protection against experimental Staphylococcus aureus arthritis by vaccination with clumping factor A, a novel virulence determinant. J Infect Dis 2001;184:1572–80. [30] Narita K, Hu DL, Mori F, Wakabayashi K, Iwakura Y, Nakane A. Role of interleukin-17A in cell-mediated protection against Staphylococcus aureus infection in mice immunized with the fibrinogen-binding domain of clumping factor A. Infect Immun 2010;78:4234–42. [31] Lin L, Ibrahim AS, Xu X, Farber JM, Avanesian V, Baquir B, et al. Th1–Th17 cells mediate protective adaptive immunity against Staphylococcus aureus and Candida albicans infection in mice. PLoS Pathog 2009;5:e1000703. [32] Misstear K, McNeela EA, Murphy AG, Geoghegan JA, O’Keeffe KM, Fox J, et al. Targeted nasal vaccination provides antibody-independent protection against Staphylococcus aureus. J Infect Dis 2014;209:1479–84. [33] Rindi S, Cicalini S, Pietrocola G, Venditti M, Festa A, Foster TJ, et al. Antibody response in patients with endocarditis caused by Staphylococcus aureus. Eur J Clin Invest 2006;36:536–43. [34] Casolini F, Visai L, Joh D, Conaldi PG, Toniolo A, Hook M, et al. Antibody response to fibronectin-binding adhesin FnbpA in patients with Staphylococcus aureus infections. Infect Immun 1998;66:5433–42. [35] Speziale P, Joh D, Visai L, Bozzini S, House-Pompeo K, Lindberg M, et al. A monoclonal antibody enhances ligand binding of fibronectin MSCRAMM (adhesin) from Streptococcus dysgalactiae. J Biol Chem 1996;271:1371–8. [36] Davis LS, Oppenheimer-Marks N, Bednarczyk JL, McIntyre BW, Lipsky PE. Fibronectin promotes proliferation of naive and memory T cells by signaling through both the VLA-4 and VLA-5 integrin molecules. J Immunol 1990;145:785–93. [37] Cheng AG, McAdow M, Kim HK, Bae T, Missiakas DM, Schneewind O. Contribution of coagulases towards Staphylococcus aureus disease and protective immunity. PLoS Pathog 2010;6:e1001036. [38] Guggenberger C, Wolz C, Morrissey JA, Heesemann J. Two distinct coagulasedependent barriers protect Staphylococcus aureus from neutrophils in a three dimensional in vitro infection model. PLoS Pathog 2012;8:e1002434. [39] Arrecubieta C, Matsunaga I, Asai T, Naka Y, Deng MC, Lowy FD. Vaccination with clumping factor A and fibronectin binding protein A to prevent Staphylococcus aureus infection of an aortic patch in mice. J Infect Dis 2008;198:571–5. [40] Stranger-Jones YK, Bae T, Schneewind O. Vaccine assembly from surface proteins of Staphylococcus aureus. Proc Natl Acad Sci USA 2006;103: 16942–7.
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Vaccine journal homepage: www.elsevier.com/locate/vaccine
Vaccination against Staphylococcus aureus experimental endocarditis using recombinant Lactococcus lactis expressing ClfA or FnbpA Tiago Rafael Veloso a,1 , Stefano Mancini a , Marlyse Giddey a , Jacques Vouillamoz a , Yok-Ai Que b , Philippe Moreillon a , José Manuel Entenza a,∗ a b
Department of Fundamental Microbiology, University of Lausanne, CH-1015 Lausanne, Switzerland Department of Intensive Care Medicine, University Hospital Medical Centre (CHUV) and University of Lausanne, CH-1011 Lausanne, Switzerland
a r t i c l e
i n f o
Article history: Received 3 March 2015 Received in revised form 6 May 2015 Accepted 22 May 2015 Available online 3 June 2015 Keywords: Staphylococcus aureus Vaccine Lactococcus lactis immunization ClfA FnbpA Endocarditis Rat
a b s t r a c t Staphylococcus aureus is a major cause of serious infections in humans and animals and a vaccine is becoming a necessity. Lactococcus lactis is a non-pathogenic bacterium that can be used as a vector for the delivery of antigens. We investigated the ability of non-living L. lactis heterologously expressing S. aureus clumping factor A (ClfA) and fibronectin-binding protein A (FnbpA), alone or together, to elicit an immune response in rats and protect them from S. aureus experimental infective endocarditis (IE). L. lactis ClfA was used for immunization against S. aureus Newman (expressing ClfA but not FnbpA), while L. lactis ClfA, L. lactis FnbpA, as well as L. lactis ClfA/FnbpA, were used against S. aureus P8 (expressing ClfA and FnbpA). Vaccination of rats with L. lactis ClfA elicited antibodies that inhibited binding of S. aureus Newman to fibrinogen, triggered the production of IL-17A and conferred protection to 13/19 (68%) of the animals from IE (P < 0.05). Immunization with L. lactis ClfA, L. lactis FnbpA or L. lactis ClfA/FnbpA also produced antibodies against the target proteins, but these did not prevent binding of S. aureus P8 to fibrinogen or fibronectin and did not protect animals against S. aureus P8 IE. Moreover, immunization with constructs containing FnbpA did not increase IL-17A production. These results indicate that L. lactis is a valuable antigen delivery system able to elicit efficient humoral and cellular responses. However, the most appropriate antigens affording protection against S. aureus IE are yet to be elucidated. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Staphylococcus aureus is a major pathogen responsible for a variety of diseases, ranging from benign skin infections to lifethreatening sepsis, pneumonia and infective endocarditis (IE) [1]. Given the high morbidity and mortality rates associated with S. aureus infections and the limited therapeutic options, the development of a safe and effective vaccine against S. aureus represents a genuine need for the medical community. Several capsular polysaccharides components, cell-wall associated proteins, secreted clotting factors and secreted proteins have been tested as vaccine candidates for S. aureus infections [2–5]. However, despite promising results obtained in pre-clinical and
∗ Corresponding author. Tel.: +41 21 6925612. E-mail address: [email protected] (J.M. Entenza). 1 Present address: Cardiovascular Developmental Biology, Department of Cardiovascular Sciences, Katholieke Universiteit Leuven, 3000 Leuven, Belgium. http://dx.doi.org/10.1016/j.vaccine.2015.05.060 0264-410X/© 2015 Elsevier Ltd. All rights reserved.
initial clinical studies, no licensed S. aureus vaccines are available so far. A novel recombinant alum-adjuvanted vaccine (NDV-3) is currently under human clinical evaluation [6,7]. S. aureus produces numerous infections that imply different pathogenic mechanisms and different types of host defences. Therefore, one approach to immunize against S. aureus would be to design vaccines targeting specific diseases [8]. Some of them have very specific features, as for instance IE, where initial adherence of circulating bacteria to damaged heart valves is pathognomonic of disease establishment. S. aureus ClfA (fibrinogen/fibrin-binding protein clumping factor A) and FnbpA (fibronectin binding protein A) are critical virulence factors for IE [9–11]. Thus, they represent intuitively logical targets for vaccine development against this disease. However, previous immunization studies showed that they were not totally satisfactory in animal models [12,13]. These failures could relate to several reasons including the nature of the presented antigens, the plethora of S. aureus surface proteins, or even the inoculation method used in the in vivo infection model.
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Lactococcus lactis has recently emerged as a promising candidate to be used as a vehicle for the delivery of antigens against bacterial infections [14–16]. A momentous advantage conferred by this system is the capability to safely deliver the antigens to the immune system, possibly in their native conformation [17]. We therefore explored the capacity of non-living recombinant L. lactis, heterologously expressing on its surface ClfA, FnbpA, or ClfA together with FnbpA, to induce both humoral and cellular responses and to protect against S. aureus IE. These immunization studies were conducted in a rat model of endocarditis induced by low-grade bacteremia, a setting that better mimics the infection process of S. aureus in humans [18]. We first tested the concept by immunizing rats with L. lactis ClfA against S. aureus Newman. This organism lacks FnbpA [19], which carries both fibronectin- and fibrinogen-binding domains. We then evaluated the protective effect of immunizing with L. lactis ClfA, L. lactis FnbpA, and of L. lactis ClfA/FnpbA against S. aureus P8, an endocarditis isolate that expresses both ClfA and FnbpA on its surface. The results indicate that L. lactis was an efficient delivery system of S. aureus ClfA and FnbpA antigens and elicited both humoral and cellular responses. However, protection efficacies were different against S. aureus strains carrying only ClfA or ClfA plus FnbpA.
2. Materials and methods 2.1. Bacterial strains and growth conditions L. lactis heterologously expressing S. aureus ClfA and FnbpA on their surface via the pIL253-based expression plasmid, either individually (L. lactis ClfA and L. lactis FnbpA) or together (L. lactis ClfA/FnbpA) [9,20,21], were used for immunization. L. lactis carrying the empty vector pIL253 was used as a control. S. aureus Newman [22] and S. aureus P8 [23] were used to induce experimental endocarditis. All L. lactis strains were grown at 30 ◦ C in M17 broth medium (Difco; Becton Dickinson, Sparks, MD) or on M17 agar plates supplemented with 0.5% glucose and 5 g/ml erythromycin. S. aureus isolates were grown at 37 ◦ C in tryptic soy broth (Difco) or on tryptic soy agar plates (Difco). 2.2. Recombinant proteins The S. aureus recombinant proteins ClfA40 (rClfA), corresponding to the A domain (amino acid 40 to 559) of the S. aureus Newman ClfA [24], and FnbpA-BCD (rFnbpA), corresponding to the BCD domains (amino acid 476 to 838) of the FnbpA of S. aureus 83254 [25] were used to generate polyclonal antibodies in rats. The Escherichia coli TOPP3 (rClfA) and XL1-Blue (rFnbpA) strains containing the pQE30 plasmid (Qiagen, Valencia, USA) with ClfA40 or FnbpA-BCD as inserts, respectively, were kindly provided by Timothy Foster (Trinity College, Dublin, Ireland). Both recombinant proteins contained an N-terminal 6-His tag and were purified from E. coli lysates as described elsewhere [24]. Purified rClfA was dialyzed against 50 mM NaCl, 2 mM EDTA, 20 mM Tris–HCl (pH 7.4) and the purified rFnbpA against 25 mM Tris–HCl (pH 8.0) [26] overnight at 4 ◦ C. 2.3. Production of anti-recombinant proteins immune antisera in rats Immune antisera against rClfA and rFnbpA were produced by Eurogentec (Liege, Belgium), by injecting rats with 60 g of rClfA or rFnbpA, at days 0, 7, 10 and 18, by using a Speedy-28 days immunization protocol. Blood samples were taken at days 0 (before
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immunization) and 28 (final bleed) and antisera prepared therewith. 2.4. Animal studies All animal protocols were reviewed and approved by the Cantonal Committee on Animal Experiments of the State of Vaud, Switzerland (Permit Number: 879.8). A mixture of ketamine (75 mg/kg) and midazolam (5 mg/kg) anesthetics was administered to the animals before any surgical procedure. 2.4.1. Immunization studies Six weeks old female Wistar rats (75–100 g) were used in the immunization experiments. L. lactis strains were cultured overnight, harvested by centrifugation, resuspended in PBS and adjusted to 1 × 108 CFU/ml. Bacteria were then killed by ultraviolet (U.V.) exposure for 1 h at 1 J/cm2 . U.V-induced killing was confirmed by the absence of viable organisms. L. lactis pIL253, L. lactis ClfA, L. lactis FnbpA, and L. lactis ClfA/FnbpA were emulsified with Freund’s adjuvant and administered at days 1, 14 and 28. At day 1 emulsification was in a 1:1 ratio with complete Freund’s adjuvant (CFA; Sigma Aldrich, Buchs, Switzerland). At days 14 and 28 emulsification was in a 1:1 ratio with incomplete Freund’s adjuvant (IFA; Sigma). Control groups of rats were injected with phosphate buffer saline (PBS), CFA or IFA (mixed with PBS in a 1:1 ratio). Blood samples were collected at days 0 (before immunization), 7, 21 and 41, the antisera were prepared therewith and stored at −80 ◦ C. 2.4.2. Animal model of endocarditis The production of catheter-induced aortic vegetations and the installation of an intravenous (i.v.) line to deliver the inocula, were performed in rats on day 41, i.e., two weeks after the last immunization dose, as described [18]. Twenty-four hours later, rats were inoculated by continuous infusion (0.0017 ml/min over 10 h) of 104 to 105 CFU of S. aureus Newman or S. aureus P8 [18]. Rats were euthanized 24 h after the end of inoculation. The cardiac vegetations and the spleen were removed, weighed, homogenized, serially diluted and plated on blood agar plates. Plates were incubated for 48 h at 37 ◦ C to determine the number of viable organisms in the tissues. 2.5. Enzyme-linked immunosorbent assay (ELISA) for detection of antibodies ELISA assays were performed as described by Hawkins et al. [27], to detect the presence of polyclonal antibodies specific for rClfA, rFnbpA, ClfA and FnbpA in rat antisera. The assays were performed on two independent occasions. The ELISA titers were expressed as Relative Fluorescence Units (RFU). 2.6. Characterization of functional antibodies in serum The presence of functional antibodies was determined by testing the ability of serum from immunized rats to inhibit the adherence of S. aureus to immobilized fibrinogen and fibronectin. Microtiter plates were coated with 10 g/ml of human fibrinogen or human fibronectin (Sigma) overnight at 4 ◦ C, incubated for 1 h with blocking solution (PBS-0.01% bovine serum albumin) to reduce non-specific binding, and washed with PBS. S. aureus Newman and S. aureus P8 (109 CFU/ml) were preincubated for 30 min at 37 ◦ C with the test antisera (diluted 1:100 in PBS-0.05% Tween 20). Samples were washed and resuspended in PBS, and 50 l were added in triplicate to the wells of fibrinogenor fibronectin-coated plates. Controls with only bacteria and buffer were included. The binding assays were performed as described
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by Ythier et al. [28]. At least three independent experiments were performed. 2.7. Interleukin-17A (IL-17A) production induced by immunization Levels of IL-17A, considered as a surrogate marker of cellmediated response, were measured in the plasma of animals at day 41 of the immunization protocol by the Bio-Plex Rat Cytokine assay (Bio-Rad, Hercules, CA) according to the manufacturer’s indications. 2.8. Statistical analysis Data of antibody titers, protein adherence inhibition assays, IL-17A production, and mean bacterial densities in vegetations and spleens were evaluated by the one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison tests. The incidences of valve infection were compared by the Chi-squared test. A value of P < 0.05 was considered significant by using two-tailed significance levels. All statistical analyses were performed with the GraphPad Prism 6.0 program (www.graphpad.com). 3. Results
Titers of anti-ClfA and anti-FnbpA in rat sera are shown in Fig. 1. In animals immunized with L. lactis expressing ClfA the titer of anti-ClfA antibodies increased by almost three times (14,308 ± 13,501 RFU) as compared with values in animals before immunization (∼5000 RFU). A similar outcome was observed in animals immunized with L. lactis ClfA/FnbpA (Fig. 1A). Anti-ClfA levels in rats receiving PBS, the adjuvant, L. lactis pIL253 or L. lactis FnbpA were comparable to the levels detected in the sera before immunization. Anti-FnbpA titers (Fig. 1B) in animals immunized with L. lactis FnbpA or L. lactis ClfA/FnbpA were approximately six (28,878 ± 12,840 RFU) and three times (16,246 ± 10,051) higher than the levels detected in sera before immunization (∼5,000 RFU). Anti-FnbpA titers in rats receiving PBS, the adjuvant, the L. lactis pIL253 or L. lactis ClfA did not significantly differ from the levels detected in the sera before immunization. 3.2. Fibrinogen- and fibronectin-binding inhibition assays 3.2.1. S. aureus Newman As shown in Fig. 2A, sera taken from L. lactis ClfA-immunized animals at day 41 (see Fig. 1A) significantly inhibited the adhesion capacity of S. aureus Newman to fibrinogen while sera of non-immunized control groups did not. The inhibition pattern was similar to that observed with the rClfA protein, used as control.
3.1. Titers of anti-ClfA and anti-FnbpA antibody Antibody titers in rats immunized with purified rClfA and rFnbpA were, respectively more than six (30,560 ± 3237 RFU) and four (35,100 ± 1733 RFU) times higher than those before immunization (4631 ± 2164 and 7854 ± 2333 RFU, respectively).
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3.2.2. S. aureus P8 Sera taken from animals immunized with L. lactis ClfA or L. lactis FnbpA at day 41 (see Fig. 1B) did not inhibit binding of S. aureus P8 to fibrinogen (Fig. 2B, upper panel). Immunization with L. lactis ClfA/FnbpA provided some inhibition, although it was
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Fig. 1. Anti-ClfA (A) and anti-FnbpA (B) antibody titers of immunized rats. Animals were immunized with PBS, adjuvant, strain L. lactis pIL253 or recombinant L. lactis ClfA, L. lactis FnbpA or L. lactis ClfA/FnbpA as described in Section 2. Antibodies were quantified by ELISA. Each bar represents the mean ± standard deviation of six serum samples per group, which were obtained in two independent experiments. P values for selected comparisons were determined by ANOVA with Dunnett’s multiple comparisons post-test.
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Fig. 2. Effect of anti-ClfA sera on S. aureus Newman (A), and S. aureus P8 ((B) upper panel) binding to immobilized fibrinogen. S. aureus P8 binding to immobilized fibronectin ((B) lower panel) was also tested in presence of anti-FnbpA sera. Bacteria were pre-incubated for 30 min with sera (diluted 1:100) and added to fibrinogen- or fibronectincoated microtiter plate wells. Results are expressed as means ± standard deviations of six serum samples per group, which were obtained in two independent experiments. * P < 0.05 by ANOVA with Dunnett’s multiple comparisons post-test compared to both BPS and adjuvant groups.
relatively marginal. Moreover, sera from L. lactis FnbpA or L. lactis ClfA/FnbpA-immunized animals did not reduce binding of this strain to fibronectin (Fig. 2B, lower panel). S. aureus P8 even exhibited increased adherence to fibronectin in sera from L. lactis FnbpAand rFnbpA immunized animals.
in both the vegetations and spleen tissues were similar to those of control animals.
3.3. IL-17A production induced by recombinant L. lactis immunization
This study showed that vaccination of rats with L. lactis expressing S. aureus ClfA protected them against endocarditis induced by S. aureus Newman, a strain expressing ClfA but no other important factors of S. aureus IE, such as FnbpA. The protective effect correlated with the production of blocking anti-ClfA antibodies as well as IL-17A. These findings are in accord with a previous work carried out by Josefsson et al. [29], who showed that immunization with the recombinant A domain of ClfA induced protective humoral immunity and prevented S. aureus Newman-induced arthritis in mice, as well as with those of Narita et al. [30], who showed that immunization with the fibrinogen-binding domain of ClfA induced substantial production of IL-17A, suggesting a role of the cellularmediated immunity in the protection against S. aureus infection in mice. Other immunization studies using a Candida albicans surface protein with structural similarity to ClfA or nanoparticules loaded with ClfA also induced protection against S. aureus infection which is supported by a IL-17A-enhanced response [31,32]. However, when S. aureus P8 (which carries both ClfA and FnbpA) was tested instead of S. aureus Newman, neither the monovalent (L. lactis ClfA and L. lactis FnbpA) nor the bivalent (L. lactis ClfA/FnbpA) vaccines afforded binding inhibition in vitro or protection against endocarditis in rats. Yet, they all elicited anti-ClfA and -FnbpA antibodies as determined by ELISA. In the case of vaccination with monovalent L. lactis ClfA, one could postulate that in vitro binding and in vivo infection were mediated by the FnbpA adhesin of S. aureus P8, which was not included in the vaccine. However, the results became intriguing when FnbpA was delivered as an antigen alone (i.e., in L. lactis FnbpA). Indeed, rather than inhibiting adherence, it slightly increased in vitro binding of S. aureus P8 to both fibrinogen and fibronectin. The addition of ClfA to the immunization protocol (in L. lactis ClfA/FnbpA) could somewhat modulate this effect, but could not entirely revert it. This suggested that
As shown in Fig. 3, control animals (PBS, adjuvant and L. lactis pIL253) exhibited similar IL-17A values than in plasma collected on day 0. Only plasma of rats immunized with L. lactis ClfA contained IL-17A levels substantially higher than control animals, while those of rats immunized with L. lactis FnbpA and L. lactis ClfA/FnbpA were unchanged. 3.4. Experimental endocarditis 3.4.1. S. aureus Newman As shown in Fig. 4A, rats immunized with L. lactis ClfA and subsequently challenged with S. aureus Newman, a strain expressing ClfA but non-functional FnbpA due to gene truncation, had significantly (P < 0.05) less infected vegetations (6 of 19; 32%) as compared to controls receiving PBS (9 of 12; 75%), the adjuvant alone (8 of 11; 73%) or L. lactis pIL253 (6 of 8; 75%). L. lactis ClfA-immunized animals also showed reduced bacterial loads in infected vegetations (4.4 log10 CFU/g) as compared to control animals (5.2 to 6.0 log10 CFU/g), although the difference was not statistically significant. In contrast, immunization with L. lactis ClfA did not decrease the rate or the bacterial load of spleen infection (data not shown). 3.4.2. S. aureus P8 The protection efficacy of this strategy was also investigated toward experimental endocarditis induced by the S. aureus isolate P8, a strain carrying both functional ClfA and FnbpA. Rats were immunized with L. lactis ClfA, L. lactis FnbpA, or L. lactis ClfA/FnbpA, but none of the immunizations protected the rats from IE (Fig. 4B) or spleen infection (data not shown). Furthermore, bacterial loads
4. Discussion
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IL-17A concentration (pg/ml)
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Fig. 3. IL-17A levels in the plasma of immunized rats. Titers of IL-17a were determined after the last booster immunization dose, i.e., at day 41. Results are expressed as means ± standard deviation of three to six individual animals per group.
anti-FnbpA vaccination conveyed some kind of binding-enhancing rather than blocking properties. A similar outcome was observed in previous works conducted by Rindi et al. and Casolini et al., whereby pre-incubation of S. aureus with sera of patients with IE containing high titers of anti FnbpA affected only slightly the ability of S. aureus to bind fibronectin [33,34]. Speziale et al. demonstrated that a monoclonal antibody generated from a mouse immunized against Streptococcus disgalactiae fibronectin binding protein FnbA cooperated to stabilize the binding of the protein to the ligand fibronectin [35]. These observations and the present results suggest that during the process of binding to fibronectin, FnbpA may dislodge potential neutralizing antibodies – may be due to a greater affinity for its natural ligand – or may constantly hide sensitive domains from blocking antibodies. Thus, blockage of FnbpA with natural antibodies might not be achieved unless we have a better comprehension of its dynamic interaction with its target. Such interference extended even to the cellular immune response. IL-17A was specifically induced by vaccination with L. lactis ClfA, but not with the control vaccine and especially not with L.
lactis FnbpA. Moreover, the presence of FnbpA in the immunization protocol prevented IL-17A induction when both ClfA and FnbpA were present in the vaccine. Fibronectin has been shown to promote the proliferation of T cells through interaction with its cell surface ligands [36]. One may postulate that anti-FnbpA antibodies may interfere with T cell proliferation by blocking fibronectin and subsequent fibronectin-T cell-ligand interactions. The present results with S. aureus P8 may help explain weak prevention in earlier experimental immunization studies using only anti-ClfA immunoglobulins [13] or the domains of FnbpA responsible for binding to fibronectin [12] against S. aureus isolates other than Newman. Moreover, they help dissect out the role of individual vaccine proteins in a specific disease. In case of vaccination with L. lactis ClfA protection was afforded only against valve infection, not spleen infection. This suggests that ClfA was either not required for infection of this organ or that other virulence determinants were at work [37,38]. Several immunizations studies using a multivalent vaccine consisting of several S. aureus antigens proved to be more effective than single antigen vaccines against S. aureus infections in
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Fig. 4. Results of immunization with recombinant L. lactis expressing ClfA or/and FnbpA against experimental IE induced by S. aureus Newman (A) and S. aureus P8 (B). Values on top of each column are the mean ± standard deviation of bacterial densities (log10 CFU) recovered from infected vegetations. * P < 0.05 by Chi-square test compared to all the other groups.
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mice [5,39,40]. One drawback of multivalent antigen vaccination is represented by the complex interactions and the difficulty to understand them. Although it imitates spontaneous immunization following S. aureus infection, it does not help understand why natural immunization does not confer prolonged immunity. One potential advantage of displaying antigens (especially surface proteins) on the surface of bacterial vectors is to present them in a more natural context, which may influence their conformation. This may help both display a more natural set of epitopes, and understand why there might be immune escape phenomena, such in the case of FnbpA. In conclusion, the present results using L. lactis as an in vivo vector suggest that ClfA is a good candidate for vaccination against S. aureus IE, but cautious should be raised toward FnbpA. Conflict of interest statement The authors have no financial or personal relationships with any persons or organizations that might influence the content of this paper. The University of Lausanne has submitted a patent application and named as inventors the authors TRV, YAQ, PM and JME. All authors have approved the final article. Funding sources This work was funded by grant 310030-143799 from the Swiss National Science Foundation. References [1] Que YA, Moreillon P. Staphylococcus aureus (including Staphylococcal toxic shock). In: Bennett JE, Dolin R, Blaser MJ, editors. Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases. 8th ed. Philapelphia: Elsevier/Saunders; 2015. p. 2237–71. [2] Verkaik NJ, van Wamel WJ, van Belkum A. Immunotherapeutic approaches against Staphylococcus aureus. Immunotherapy 2011;3:1063–73. [3] Jansen KU, Girgenti DQ, Scully IL, Anderson AS. Vaccine review: Staphyloccocus aureus vaccines: problems and prospects. Vaccine 2013;31:2723–30. [4] Proctor RA. Challenges for a universal Staphylococcus aureus vaccine. Clin Infect Dis 2012;54:1179–86. [5] Rauch S, Gough P, Kim HK, Schneewind O, Missiakas D. Vaccine protection of leukopenic mice against Staphylococcus aureus bloodstream infection. Infect Immun 2014;82:4889–98. [6] Spellberg B, Ibrahim AS, Yeaman MR, Lin L, Fu Y, Avanesian V, et al. The antifungal vaccine derived from the recombinant N terminus of Als3p protects mice against the bacterium Staphylococcus aureus. Infect Immun 2008;76: 4574–80. [7] Schmidt CS, White CJ, Ibrahim AS, Filler SG, Fu Y, Yeaman MR, et al. NDV-3, a recombinant alum-adjuvanted vaccine for Candida and Staphylococcus aureus, is safe and immunogenic in healthy adults. Vaccine 2012;30:7594–600. [8] Fowler Jr VG, Proctor RA. Where does a Staphylococcus aureus vaccine stand? Clin Microbiol Infect 2014;20(Suppl. 5):66–75. [9] Que YA, Haefliger JA, Piroth L, Francois P, Widmer E, Entenza JM, et al. Fibrinogen and fibronectin binding cooperate for valve infection and invasion in Staphylococcus aureus experimental endocarditis. J Exp Med 2005;201:1627–35. [10] Widmer E, Que YA, Entenza JM, Moreillon P. New concepts in the pathophysiology of infective endocarditis. Curr Infect Dis Rep 2006;8:271–9. [11] Veloso TR, Chaouch A, Roger T, Giddey M, Vouillamoz J, Majcherczyk P, et al. Use of a human-like low-grade bacteremia model of experimental endocarditis to study the role of Staphylococcus aureus adhesins and platelet aggregation in early endocarditis. Infect Immun 2013;81:697–703. [12] Schennings T, Heimdahl A, Coster K, Flock JI. Immunization with fibronectin binding protein from Staphylococcus aureus protects against experimental endocarditis in rats. Microb Pathog 1993;15:227–36. [13] Vernachio J, Bayer AS, Le T, Chai YL, Prater B, Schneider A, et al. Anti-clumping factor A immunoglobulin reduces the duration of methicillin-resistant Staphylococcus aureus bacteremia in an experimental model of infective endocarditis. Antimicrob Agents Chemother 2003;47:3400–6. [14] Villatoro-Hernandez J, Montes-de-Oca-Luna R, Kuipers OP. Targeting diseases with genetically engineered Lactococcus lactis and its course towards medical translation. Expert Opin Biol Ther 2011;11:261–7. [15] Bahey-El-Din M. Lactococcus lactis-based vaccines from laboratory bench to human use: an overview. Vaccine 2012;30:685–90.
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