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Generation of a Novel Staphylococcus aureus Ghost Vaccine andExamination of Its Immunogenicity against Virulent Challenge in Rats
Nagarajan Vinod, Sung Oh, Hyun Jung Park, Jung Mo Koo, Chang Won Choi, Sei Chang Kim
Department of Biology & Medicinal Science, Pai Chai University, Daejeon, South Korea
Staphylococcus aureus is a Gram-positive pathogen that causes a wide range of infections in humans and animals. Bacterialghosts are nonliving, empty cell envelopes and are well represented as novel vaccine candidates. In this study, we examined theimmunogenicity and protective efficacy of S. aureus ghosts (SAGs) against a virulent challenge in rats. Nonliving SAGs were gen-erated by using the MIC of sodium hydroxide. The formation of a transmembrane lysis tunnel structure in SAGs was visualizedby scanning electron microscopy. To investigate these SAGs as a vaccine candidate, rats were divided into four groups, A (non-immunized control), B (orally immunized), C (subcutaneously immunized), and D (intravenously immunized). The IgG anti-body responses were significantly stronger in the SAG-immunized groups than in the nonimmunized control group (P < 0.05).Moreover, a significant increase in the populations of CD4� and CD8� T cells was observed in all three immunized groups (P <0.05). We also found that serum bactericidal antibodies were significantly elicited in the SAG-immunized groups (P < 0.05).Most importantly, the bacterial loads in the immunized groups were significantly lower than those in the nonimmunized controlgroup (P < 0.01). These results suggest that immunization with SAGs induces immune responses and provides protectionagainst a virulent S. aureus challenge.
Staphylococcus aureus is a Gram-positive bacterium that is re-sponsible for a wide range of infections in both hospitals and
communities (1). S. aureus pathogenicity can range from skininfections to life-threatening diseases such as osteomyelitis, endo-carditis, pneumonia, bacteremia, and septicemia (2, 3). Moreover,it is a common food-borne pathogen that can cause gastroenteritisfrom the consumption of contaminated food (4). In most cases, S.aureus enters the human body through the skin and mucousmembrane and spreads via the bloodstream. It can infect everytissue and organ of the human body (5). Over the past decades,multidrug-resistant S. aureus strains have emerged rapidly, andparticularly, methicillin-resistant S. aureus (MRSA) is a majorpublic health problem. MRSA can cause greater morbidity, mor-tality, length of hospital stay, and hospitalization costs than me-thicillin-susceptible S. aureus (6). Regrettably, treatment optionsfor multidrug-resistant S. aureus strains are limited (7). Therefore,vaccination remains the best way to prevent and control S. aureusinfections.
In recent years, it has become well known that bacterial ghosts(BGs) offer a promising and innovative approach in nonlivingvaccine technology (8). BGs are nonliving, empty cell envelopes,and the most common method used to produce BGs from Gram-negative bacteria is controlled expression of the lysis E gene. Pro-tein E leads to the formation of transmembrane structures on thecell surface, which results in empty cell envelopes. The resultingBGs induce strong immune responses and protect against specificinfections in experimental animal models (9, 10). However, themajor drawback of the protein E-induced inactivation method isthat it is restricted to Gram-negative bacteria only (11). Interest-ingly, Ra et al. (12) have demonstrated that Escherichia coli/Strep-tococcus iniae glyceraldehyde 3-phosphate dehydrogenase ghosts,produced with a double-cassette vector system, protected aquaticfish from streptococcal diseases. Exceptionally, S. iniae ghosts pro-duced by E gene-mediated lysis were being suggested as a potentialvaccine candidate (13). However, a number of studies have dem-onstrated that the lysis efficiency of genetically inactivated BGs
was 99.9% (10, 14), suggesting a potential risk of their use as avaccine. Alternatively, the new approach used to generate E. colighosts by using the MICs and minimum growth concentrations ofsodium hydroxide (NaOH), sodium dodecyl sulfate, and calciumcarbonate (CaCO3) has been described in detail (15–17). More-over, we have recently shown that chemically induced Salmonellaenterica serovar Enteritidis BGs induce immune responses andstrong protective immunity to Salmonella infection in a rat model(18). This new strategy of BG induction with NaOH was fasterthan the protein E-mediated lysis system.
The bacterial envelope of S. aureus is composed of peptidogly-can, teichoic acid, and proteins. Several studies have suggestedthat S. aureus envelope components are potential vaccine candi-dates in animal models (19, 20). Recently, immunization with S.aureus peptidoglycan has been found to induce protective immu-nity to a lethal challenge in experimental animals (21). Vaccina-tion of rats with iron-responsive surface determinant A (IsdA) orIsdH protected against nasal carriage (22). Earlier studies havealso shown that protein A (PA), a cell wall component of S. aureus,is highly immunogenic (23). Stranger-Jones et al. (24) demon-strated that immunization with a combination of four surfaceantigens (IsdA, IsdB, serine-aspartate repeat A [SdrA], and SdrE)
Received 6 January 2015 Returned for modification 8 March 2015Accepted 2 May 2015
Accepted manuscript posted online 11 May 2015
Citation Vinod N, Oh S, Park HJ, Koo JM, Choi CW, Kim SC. 2015. Generation of anovel Staphylococcus aureus ghost vaccine and examination of itsimmunogenicity against virulent challenge in rats. Infect Immun 83:2957–2965.doi:10.1128/IAI.00009-15.
Editor: A. Camilli
Address correspondence to Chang Won Choi, [email protected], orSei Chang Kim, [email protected].
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
doi:10.1128/IAI.00009-15
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induced opsonophagocytic antibodies and provided 100% pro-tection from clinical S. aureus isolates in experimental animals. Ithas been reported that S. aureus cell wall components are able toinduce both humoral and cell-mediated immunity (25). Alto-gether, these whole-cell envelope components of S. aureus repre-sent an attractive vaccine candidate.
In the present study, we generated novel S. aureus ghosts(SAGs) by using the MIC of NaOH. Additionally, we demon-strated that immunization with SAG vaccine via the oral, subcu-taneous, and intravenous routes could induce both humoral andcellular immune responses in rats. Furthermore, these immuneresponses provided protective immunity to a challenge with viru-lent S. aureus.
MATERIALS AND METHODSBacterial strain, medium, and culture condition. Virulent S. aureusstrain KCCM12256 was kindly provided by Ki-Sung Lee, Department ofBiology and Medicinal Science, Pai Chai University, Daejeon, South Ko-rea. S. aureus was cultured in tryptic soy broth (TSB; Difco) at 37°C in ashaking incubator at 200 rpm. Growth and lysis rates were measuredspectrophotometrically by determination of optical density at 600 nm(OD600).
Determination of MIC. Determination of the MIC of NaOH for S.aureus was performed by the 2-fold broth dilution method as describedpreviously (26, 27), with some modifications. Briefly, a virulent culture ofS. aureus was grown in TSB and adjusted to 1 � 108 CFU/ml. The initialconcentration of NaOH was 60 mg/ml. Two-fold dilutions of NaOH wereadded to samples of the virulent bacterial culture, and they were incubatedat 37°C for 18 h. After incubation, the turbidity of each individual tubewas assessed visually and the MIC was determined as the lowest concen-tration of NaOH that completely killed the bacterial growth. Further, todetermine viability, the culture that showed no visible bacterial growthwas verified by spreading 100 �l of the culture onto tryptic soy agar (TSA)plates and incubating them at 37°C for 24 h. The MIC was determined inthree independent experiments.
Production of SAGs. SAGs were produced by using the MIC of NaOHas described previously (18). In brief, the biomass of 72-h-old S. aureuscells was collected by centrifugation (10,000 � g for 10 min at 4°C) andwashed three times with phosphate-buffered saline (PBS; 3.2 mMNa2HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl, pH 7.4). Onemilliliter of the MIC of NaOH was added to 2 ml of the bacterial suspen-sion (1 � 108 CFU/ml) and incubated at 37°C for 75 min. To determinethe lysis rate, samples of cells treated with the MIC of NaOH and non-treated control cells were collected at 15-min intervals (15, 30, 45, 60, and75 min) after treatment and spread onto TSA plates. After incubation at37°C for 24 h, viable colonies were enumerated and results were expressedin numbers of CFU/ml. The efficiency of lysis was determined by using thefollowing formula: lysis rate � [1 � (CFU of treated cells/CFU of non-treated control cells)] � 100%. Lysis efficiency was determined in threeindependent experiments. At the end of the lysis process, SAGs were har-vested by centrifugation (10,000 � g for 10 min at 4°C) and washed threetimes with PBS. The final pellet was resuspended in sterile PBS and storedat 4°C until further use.
DNA-free SAG analysis by real-time PCR. To analyze the degrada-tion of DNA in S. aureus cells, samples of bacterial cells treated with theMIC of NaOH and nontreated control cells were collected at 15, 30, 45, 60,and 75 min, respectively. Their genomic DNA was prepared with a bacte-rial genomic DNA isolation kit (iNtRON Biotechnology), according tothe manufacturer’s instructions. The extracted genomic DNA was ana-lyzed by electrophoresis in 1% agarose gel stained with ethidium bromide.The gel was examined with the UV gel documentation system and photo-graphed (Canon PowerShot A620). Quantitative real-time PCR assayswere performed with the Sybr green detection system. The genomic DNAextracted after various lysis times was used as the template for real-time
PCR assays. The S. aureus 16S rRNA was amplified with specific primers(forward, 5=-AGTGTCAAGTGTTAGGGGGTTTC-3=; reverse, 5=-ATTCCTTTGAGTTTCAACCTTGCGGT-3=). The total volume of each tubewas 20 �l containing 1 �l of 1:100 template, 1 �l of each primer (10pmol/�l), 10 �l of 2� Sybr green quantitative PCR master mix (AgilentTechnologies), and 7 �l of sterilized distilled water. The reaction wasinitiated at 95°C for 15 min, which was followed by 40 cycles of 95°C for 10s, 60°C for 40 s, and 72°C for 60 s. Real-time PCR was carried out in aStratagene Mx3000P real-time PCR machine (Agilent Technologies).Each sample, including a no-template control, was analyzed in triplicate.
SEM. Morphological features of SAGs were analyzed by scanning elec-tron microscopy (SEM) as described previously (10). SAGs and non-treated control bacterial cells were fixed in 2.5% glutaraldehyde in 0.1 Mphosphate buffer (pH 7.0) for 2 h at 4°C, washed three times with the samebuffer, and then postfixed in 1% osmium tetroxide for 1.5 h at 4°C. Afteranother washing, cells were dehydrated in a graded series of ethanol con-centrations (10, 30, 50, 70, and 100%). After the samples were criticalpoint dried with liquid CO2, they were mounted on SEM stubs, subse-quently sputter coated with gold-palladium, and examined with a scan-ning electron microscope (Leo 1455VP; Korea Basic Science Institute,Daejeon, South Korea) at an accelerating voltage of 20 kV and a magnifi-cation of �20,000.
Experimental animals. Ten-week-old male Sprague-Dawley rats weremaintained at 23 to 25°C with a 12-h light/dark cycle and had access to astandard pellet diet and water ad libitum. All animal experimental proce-dures were approved by the Pai Chai University institutional animal careand use committee.
Immunization and challenge experiment. In order to assess the im-munogenicity and protective efficacy of SAGs, a group of 32 rats wasdivided into four groups of 8, named A, B, C, and D. Group A rats (non-immunized control group) received sterilized PBS by the subcutaneousroute. Group B, C, and D rats were immunized with SAGs (1 � 106
cells/ml) in sterile PBS by the oral, subcutaneous, and intravenous routes,respectively. Rats from all four groups were immunized three times at2-week intervals (weeks 1, 3, and 5). Two weeks after the last immuniza-tion (week 7), all rats were challenged intravenously with virulent S. au-reus at 2 � 108 CFU/ml of PBS. To determine the immune response, bloodsamples were taken from the tail veins of individual rats at 2-week inter-vals during immunization and after the challenge.
Measurement of antibody response by ELISA. Sera from immunizedand nonimmunized control rats were examined for the presence of spe-cific immunoglobulin G (IgG) by indirect enzyme linked immunosorbentassay (ELISA). Briefly, microtiter plates were coated with 100 �l of antigen(1 � 106 cells/ml) in coating buffer (pH 9.6) and incubated for 2 h at roomtemperature. The plates were then washed three times with PBS contain-ing Tween 20 (PBS-T) and then blocked with 1% bovine serum albumin(BSA) in PBS-T for 2 h at room temperature. After washing, 100 �l ofserially diluted serum was added and the mixture was incubated for 2 h atroom temperature. Plates were washed three times with PBS-T, and then100 �l of alkaline phosphatase-conjugated goat anti-rat IgG (1:30,000;Sigma-Aldrich) in PBS-T with 1% BSA was added and the mixture wasincubated for 2 h at room temperature. Plates were then washed threetimes with PBS-T, and the color was developed by using 100 �l of p-nitrophenylphosphate substrate (Sigma-Aldrich, St. Louis, MO) and in-cubating the plates for 30 min at room temperature in the dark. Thereaction was stopped by adding 100 �l of 3 M NaOH, and the absorbanceat 405 nm of the plates was read with a microplate reader (Bio-Rad).
Flow cytometric analysis. To study the cellular immune response,T-cell markers were examined by collecting blood samples from immu-nized and nonimmunized control rats on the 7th day (week 6) after thefinal immunization. Peripheral blood mononuclear cells (PBMCs) wereprepared with Histopaque-1077 (Sigma-Aldrich, St. Louis, MO) accord-ing to the manufacturer’s protocol. The PBMCs were washed three timeswith cold PBS and stained with appropriately diluted fluorescein isothio-cyanate-labeled anti-CD4 or -CD8 monoclonal antibodies at 4°C for 30
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min in the dark. After incubation, all samples were washed three timeswith PBS and resuspended in 0.5 ml of PBS. Data were collected by fluo-rescence-activated cell sorter (FACS) analysis with a BD FACSCanto IIflow cytometer (BD Biosciences) and BD FACSDiva software (BD Biosci-ences).
Determination of SBA. Serum bactericidal activity (SBA) was deter-mined as described previously (13, 18), with some modifications. In brief,100 �l of a bacterial suspension (1 � 106 CFU/ml) was added to 25 �l ofserum and incubated at room temperature for 1 h. After incubation, themixture was spread onto selective medium and incubated at 37°C for 48 h.For the control, serum was replaced with PBS. The percentage of SBA wasdetermined by using the following formula: SBA � [1 � (number ofviable bacteria after serum treatment/number of viable bacteria after PBStreatment)] � 100%.
Bacteriological analysis. To evaluate the protective efficacy of SAGvaccine against a virulent challenge, all experimental rats were sacrificed at2 weeks (week 9) postchallenge. Their livers, lungs, spleens, and kidneyswere collected and aseptically homogenized in 5 ml of sterile cold PBS byusing a tissue homogenizer (Sun MI Technology). Tenfold serial dilutionsof these tissue homogenates were plated onto selective medium, and thenumbers of CFU were determined.
Statistical analysis. Statistical analyses were performed by one-wayanalysis of variance or the Kaplan-Meier log rank test with SPSS software(version 14.0). All data are expressed as the mean � the standard error ofthe mean. Differences were considered statistically significant at P � 0.05.
RESULTS AND DISCUSSIONProduction and characterization of SAGs. To produce SAGs,we first determined the MIC of NaOH for S. aureus strainKCCM12256 by the 2-fold broth dilution method. The MIC ofNaOH for S. aureus was 7.50 mg/ml, and this specific concentra-tion was used to produce SAGs. It is well known that NaOH hasthe ability to create transmembrane lysis tunnels on the bacterialcell surface, degrade DNA, and turn bacteria into empty cell en-
FIG 1 Curves of S. aureus cell lysis at different time points (15, 30, 45, 60, and75 min). The graph shows the lysis of bacterial cells treated with the MIC ofNaOH and nontreated control cells. Lysis was measured by the number of CFUat 15-min intervals after treatment.
FIG 2 Analysis of DNA-free SAGs. (a) Total DNA extracted from nontreated control S. aureus cells (lanes 1 to 5) and S. aureus cells treated with the MIC ofNaOH (lanes 6 to 10). Lane M, 1-kb marker ladder. (b) DNA contents of nontreated control cells and cells treated with the MIC of NaOH at various time pointsduring lysis, as quantified by real-time PCR with the Sybr green detection system. For practical reasons, the DNA concentrations that fell below the detection limit(d.l.; gray line) are illustrated as corresponding to the limit of detection and the specific concentrations are not shown.
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velopes (15, 18, 28). In our recent study, we demonstrated that S.Enteritidis ghosts could be produced within 45 min by using theMIC of NaOH (18). In the present study, S. aureus cells treatedwith the MIC of NaOH showed effective lysis and a rapid decreasein the number of viable cells. Most significantly, the lysis timecourse curve presented in Fig. 1 shows 100% lysis efficiency in S.aureus cells treated with the MIC of NaOH and no viable cells weredetected after 60 min of lysis. These data suggest that the MIC ofNaOH would be sufficient to produce inactivated S. aureus bacte-ria. Generation of BGs from S. aureus has not been reported pre-viously. However, this new strategy has been used with a numberof Gram-negative bacterial strains, such as E. coli BL21 (15), E. coliJM109 (16, 17), and S. Enteritidis (18). Therefore, it is interesting
that this method might successfully be used to produce BGs fromboth Gram-positive and Gram-negative bacteria.
As shown in Fig. 2a, the total DNA content was well compara-ble to that of the nontreated control (Fig. 2a, lanes 1 to 5) and cellstreated with the MIC of NaOH (Fig. 2a, lanes 6 to 10) at variouslysis time points. Subsequently, we found that S. aureus cellstreated with the MIC of NaOH showed gradual degradation ofDNA content at successive time points during lysis, and completeabsence of detectable DNA was observed after 60 min of lysis, asshown by real-time PCR analysis (Fig. 2b). This result indicatesthat cells treated with the MIC of NaOH degraded the geneticcontent of bacteria during the lysis process. Several reports havedemonstrated that the synthesis of virulence and colonizing fac-
FIG 3 SEM images of S. aureus (a) and SAGs (b). Arrows indicate transmembrane lysis tunnels.
FIG 4 The IgG antibody response levels in rats immunized with SAGs were determined by indirect ELISA. Sera were obtained from rats in groups A(nonimmunized control), B (orally immunized), C (subcutaneously immunized), and D (intravenously immunized) at weeks 2, 4, 6, and 9. Results are expressedas means � the standard errors of the means. The asterisks indicate significant differences between the antibody responses of the immunized and nonimmunizedgroups (**, P � 0.01; *, P � 0.05).
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tors in S. aureus is controlled by several regulatory loci, such as anaccessory gene regulator (agr), a staphylococcal accessory regula-tor (sarA), and an autolysis-related locus (arl) (29–31). In addi-tion, the agr locus is mainly responsible for causing virulence andcolonization in experimental animals (32, 33). In this regard, theempty cell envelope of S. aureus would not be expected to causeany problem because of the total loss of DNA content in SAGs. Wealso examined the formation of transmembrane lysis tunnel struc-tures in SAGs by SEM. Electron microscopic images, as shown inFig. 3, indicate that the MIC of NaOH induced transmembranelysis tunnels in SAGs (Fig. 3b; arrowheads) but not in unlysedcontrol cells (Fig. 3a). Furthermore, the MIC of NaOH did notcause any cellular morphology damage except for the lysis pore.
Humoral immune responses. Serum IgG antibodies were in-duced in rats after immunization and a challenge, as shown in Fig.4. In week 2, the serum IgG antibody responses in SAG-immu-nized group C rats were significantly higher than those in nonim-
munized control group A rats (P � 0.05). However, no significantdifference was observed between groups B and D and group C.Interestingly, the IgG antibody responses in groups B (P � 0.05),C (P � 0.01), and D (P � 0.01) were statistically significantlydifferent from that in control group A in week 4. In week 6, themaximum IgG antibody levels were detected in immunizedgroups compared to group A (P � 0.01) during the immunizationperiod. Moreover, each booster immunization with SAGs greatlyincreased the IgG antibody levels in the immunized groups (B, C,and D), with group C rats showing the maximum IgG antibodyresponse. Most significantly, after a challenge with virulent S. au-reus, serum IgG antibody levels increased robustly in all of theSAG-immunized groups but not in the nonimmunized group (P� 0.01). Furthermore, no significant differences in IgG antibodieswere observed among the immunized groups (B, C, and D) post-challenge. This indicates that immunization with SAGs via theoral, subcutaneous, and intravenous routes was able to induce
FIG 5 Assessment of CD4� and CD8� T cells by FACS analysis. Shown are populations of CD4� (a) and CD8� (b) T cells and the corresponding analysis ofsignificant differences (c and d) between immunized and nonimmunized groups 1 week (week 6) after the final immunization with SAGs. Groups: A, nonim-munized control; B, orally immunized; C, subcutaneously immunized; D, intravenously immunized. The values shown are means � the standard errors of themeans. The asterisks indicate significant differences between the T-cell populations of the immunized and nonimmunized groups (*, P � 0.05).
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protective immunity to a virulent challenge. It has been estab-lished that BG vaccines derived from Gram-positive bacteria in-duced significant immune responses and protective efficacyagainst infections in immunized animals (12, 13). Clarke et al.(22) reported that surface proteins of S. aureus (IsdA or IsdH)stimulated greater IgG immune responses and protected rats froma virulent challenge, suggesting that the humoral immune re-sponse is essential for protection against S. aureus. It has previ-ously been demonstrated that peptidoglycan from S. aureus is theimportant component of protection (34). In another study, im-munization with INH-A21 (human IgG antibodies developedfrom the surface proteins clumping factor A [ClfA] and SdrG)induced phagocytic antibodies and protective immunity to staph-ylococcal infection in rats and rabbits (35). Further, the adminis-tration of various microbial surface components recognizing ad-hesive matrix molecules from S. aureus has been found to increaseIgG specific antibody levels significantly, suggesting that the sur-face components can induce humoral immune responses (24).
Cellular immune responses. We examined changes in CD4�
and CD8� T-cell populations in immunized groups by FACS (Fig.5). The CD4� and CD8� T cells of rats in all of the immunizedgroups differed significantly from those of nonimmunized controlgroup A (P � 0.05). The highest percentage of CD4� and CD8�
T-cell populations were observed in group C rats. However, thenumbers of CD4� and CD8� T cells did not differ significantlybetween groups B and D or between groups C and D. In general,CD4� (Th1 and Th2) T-helper cells activate opsonic IgG antibod-ies and cell-mediated immune responses, and cytotoxic CD8� Tcells destroy the intracellular bacterial pathogens (36, 37). It haspreviously been suggested that CD4� T cells are essential for the S.aureus vaccine to be effective (38). Moreover, the S. aureus enve-lope component of PA-immunized animals elicited higher levelsof CD4� and CD8� T cells than that of control animals (39).Furthermore, CD4� and CD8� T cells were found to be involved
in protection against S. aureus infections (40). These findings sug-gest that SAG-induced CD4� and CD8� T cells are capable ofresponding to staphylococcal antigen during a virulent challenge.Earlier attempts to develop whole-cell live or killed S. aureus vac-cines had failed to induce protection against S. aureus infection(41). In most cases, administration of live-attenuated vaccines didnot induce humoral immunity and protection. Furthermore, vac-cination with heat-killed S. aureus was not successful in reducingbacterial loads in organs (42). Compared with other inactivatedvaccines, the main advantage of BG vaccines is the ability to pre-serve their surface antigenic components, which themselves canprovide excellent natural intrinsic adjuvant properties (43). Moreimportantly, the empty cell envelope of SAGs contains pathogen-associated molecular patterns (PAMPs) such as peptidoglycan,lipoteichoic acid, and lipoproteins. A number of studies have sug-gested that these PAMPs can be recognized by Toll-like receptors(TLRs), which induce an innate immune response (44, 45). Inparticular, TLR2 has been widely thought to play a crucial role inthe host immune response to S. aureus (46).
SBA. The bactericidal activities against virulent S. aureus weredetermined in both immunized and nonimmunized controlgroups (Fig. 6). In week 2, sera from groups C (P � 0.01) and D(P � 0.05) showed statistically significantly higher bactericidalactivity than those from group A. Interestingly, we found that thepercentage of SBA was significantly higher in all immunizedgroups (B, C, and D) than in control group A (P � 0.01) in weeks4 and 6. Moreover, In weeks 4 and 6, no statistically significantdifferences were observed among the three immunized groups.Most importantly, bactericidal percentages were also robustlyhigher in SAG-immunized groups than in group A (P � 0.01)postchallenge. Bactericidal activities were not statistically signifi-cantly different between SAG-immunized groups. Overall, eachbooster immunization increased the SBA in all three immunizedgroups (B, C, and D) during the immunization period and post-
FIG 6 Serum bactericidal activities of rats immunized with SAGs. Groups: A, nonimmunized control; B, orally immunized; C, subcutaneously immunized; D,intravenously immunized. Data are expressed as means � the standard errors of the means. The asterisks indicate significant differences between the serumbactericidal activities of the immunized and nonimmunized groups (**, P � 0.01; *, P � 0.05).
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challenge. These data indicated that immunization with SAGsorally, subcutaneously, or intravenously efficiently produced bac-terium-killing antibodies after a challenge. Recently, it has alsobeen reported that S. iniae ghost vaccine stimulated higher serum
bactericidal antibody levels than formalin-killed vaccine and pro-tected experimental animals from a subsequent challenge with S.iniae (13). An earlier report showed that peptidoglycan inducesstrong bactericidal antibodies capable of eradicating the bacterialpathogen (21).
Protective efficacy of SAGs against S. aureus challenge. Theprotective efficacy of SAG vaccine against a challenge with virulentS. aureus was evaluated (Fig. 7). Rats from the immunized andnonimmunized control groups were challenged with virulent S.aureus, and their livers, lungs, spleens, and kidneys were obtainedat 2 weeks postchallenge. In this study, we observed that the liver,lung, spleen, and kidney homogenates of groups B, C, and Dshowed bacterial loads dramatically lower than those of nonim-munized control group A (P � 0.01). Remarkably, immunizationwith SAGs by the oral route also efficiently stimulated protectiveimmunity to infection. Moreover, Kaplan-Meier survival analysis(Fig. 8) showed significant differences between SAG-immunizedgroups B, C, and D and nonimmunized control group A (P �0.05). These findings show that immunization with SAGs by theoral, subcutaneous, and intravenous routes elicited immune re-sponses and protected rats against a virulent S. aureus challenge.Similar findings were reported by Capparelli et al. (21), whereimmunization of mice with peptidoglycan via the intramuscular,intravenous, and aerosol routes reduced colonization of the liver,lungs, spleen, and kidneys and elicited significant protection froma lethal dose of S. aureus. Further, UV-irradiated genetically atten-
FIG 7 Bacterial loads in liver (a), lung (b), spleen (c), and kidney (d) homogenates after a virulent S. aureus challenge. Results are expressed as means � the standarderrors of the means. The asterisks indicate significant differences between the bacterial burdens of the immunized and nonimmunized groups (**, P � 0.01).
FIG 8 Kaplan-Meier analysis of the survival of rats immunized with SAGs(groups B, C, and D) or treated with PBS (group A) and challenged intrave-nously with S. aureus. Groups: A, nonimmunized control; B, orally immu-nized; C, subcutaneously immunized; D, intravenously immunized. Theasterisks indicate significant differences between the survival rates of theimmunized and nonimmunized groups (*, P � 0.05).
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uated vaccine decreased bacterial burdens and provided protec-tion from a subsequent challenge with S. aureus (47). A study of S.aureus surface proteins revealed that immunization with fourcombined surface proteins (IsdA, IsdB, SdrA, and SdrE) providedcomplete protection from a staphylococcal challenge (24). In thesame study, it was reported that bacterial clearance from the kid-neys was more effective than that achieved by immunization witha single surface protein, indicating that the multicomponent vac-cine is needed to control S. aureus infection. In addition, immu-nization with the multicomponent vaccine has the potential toprevent multiple organ infections, in contrast to a single-compo-nent vaccine. Specifically, recent studies suggest that a multicom-ponent vaccine rather than a single-component vaccine couldtrigger both humoral and cellular immunity and induce protectiveimmunity to staphylococcal diseases (48, 49). Therefore, ournovel strategy of using SAGs as a vaccine could be a new way toprevent S. aureus infections.
Conclusion. In conclusion, nonliving SAGs have been success-fully generated by using the MIC of NaOH and the approach ismore rapid and cost-effective than other methods. Interestingly,the present strategy may open the door to the production of BGsfrom Gram-positive bacteria. Most importantly, we have shownthat immunization with SAGs induced significant humoral andcellular immune responses and provided strong protectionagainst a virulent challenge in rats. Therefore, our present findingscould be useful in the future development of vaccines against S.aureus infections.
ACKNOWLEDGMENT
This work was supported by Business for Cooperative R&D between In-dustry, Academy, and Research Institute funded Korea Small and Me-dium Business Administration in 2014 (grant C0191740).
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