A Synthetic M Protein Peptide Synergizes with a CXC Chemokine Protease To Induce Vaccine-Mediated Protection against Virulent Streptococcal Pyoderma and Bacteremia

Streptococcus pyogenes (group A Streptococcus [GAS]) is one of the most prevalent human pathogens. Impetigo lesions serve as a major primary site of infection and reservoir for transmission. Skin infections result in >100 million annual cases of pyoderma (1). Pyoderma has been linked with outbreaks of acute poststreptococcal glomerulonephritis and with very high rates of rheumatic heart disease (2), as well as severe deep tissue sepsis (3). In Australia’s Indigenous populations, the skin is the major site of infection (4) with prevalence rates in children approaching 70% in remote communities (5). A vaccine is desperately needed.

Vaccine candidates based on the conserved region of the M protein of GAS were efficacious in protection against intranasal infection with GAS (6, 7). We previously described a vaccine candidate peptide, J8, based on a minimal epitope from the conserved C3 repeat of the GAS surface M protein (8, 9). When linked to the carrier protein, diphtheria toxoid (DT), and administered with Alhydrogel, J8 induces opsonic Abs that protect mice from a systemic i.p. challenge with multiple GAS strains (10). Other vaccine candidates are being evaluated (reviewed in Ref. 11), but none have been tested for their ability to protect against pyoderma. In this article, we define a model of pyoderma involving minor scarification of mouse skin, followed by topical application of GAS. We describe the pathological features of GAS pyoderma and demonstrate that vaccination with J8-DT can provide profound protection against pyoderma and bacteremia; however, to our surprise, protection against cluster of virulence responder/sensor (CovR/S) mutant strains was severely limited.

The CovR/S regulon is a major virulence-determining region of the streptococcal genome, which controls ∼10–15% of the bacterial genome (12–14). Bacteria isolated from deep tissue infections often have mutations or deletions within this regulon, leading to upregulation of a number of virulence factor genes (15, 16). A major factor that is upregulated in mutants is S. pyogenes cell envelope proteinase (SpyCEP), a CXC chemokine protease. SpyCEP can cleave the human chemokine IL-8 (17), leading to the blockade of neutrophil chemotaxis to the site of infection. Mutation within the CovR/S regulon is thought to occur postinfection. Although any strain can give rise to invasive disease, this is often linked to dissemination worldwide of the M1T1 clone that has a high propensity to CovR/S mutation as a result of expression of the phageborne DNase gene sda1 (18, 19). Hypervirulent strains of GAS pose a particular challenge to vaccine development because many of the upregulated virulence factors directly affect immune function (14, 20).

Although J8-DT vaccine efficacy against CovR/S mutants was greatly impaired, it could be restored completely by covaccination with a recombinant fragment of SpyCEP, with Abs induced by the SpyCEP fragment protecting CXC chemokines from degradation, thus allowing neutrophils to work with the anti-J8 Abs to kill the bacteria. To our knowledge, this is a unique example of a vaccine requiring the induction of two Ab specificities that act synergistically to destroy an organism.

BALB/c, Swiss, and B10.BR mice (female, 4–6 wk old) were sourced from the Animal Resource Centre (Perth, WA, Australia). All protocols were approved by Griffith University’s Animal Ethics Committee in accordance with the National Health and Medical Research Council of Australia guidelines.

A number of GAS isolates obtained from various sources were used in the study. S. pyogenes 2031 (emm1), 88/30 (emm97), BSA10 (emm124), NS27 (emm91), NS1 (emm100), and 90/31 (emm57) were obtained from The Menzies School of Health Research (Darwin, NT, Australia). All strains (except for 90/31) were passaged in mice and made streptomycin resistant (200 μg/ml) by continually replating them on increasing concentrations of streptomycin. GAS strain 5448AP (emm1) was obtained from Prof. Mark Walker’s laboratory (University of Queensland). One of the main rationales for strain selection was to cover a diverse range of clinically relevant emm types (as well as emm clusters) representing various tissue sites of origin. However, because the study focused on GAS skin infection, the emphasis was given to isolates of skin origin. To prepare challenge inoculum, the GAS strains were grown for 16–18 h at 37°C in liquid medium containing Todd–Hewitt broth (THB; Oxoid, Adelaide, SA, Australia) supplemented with 1% Neopeptone (Difco). For CFU enumeration, 10-fold serial dilutions of bacterial cultures were plated in replicates on blood agar plates consisting of the medium described above, 2% agar, 200 μg/ml streptomycin, and 2% defibrinated horse blood. The broth culture inoculum was adjusted to obtain the intended challenge dose. For bacterial burden determination following infection, the samples were diluted and plated on blood agar plates, as described above.

Peptides used in this study were synthesized in-house or synthesized commercially by Auspep (Tullamarine, VIC, Australia). Peptide J8 was conjugated to DT as described elsewhere (10). All peptides were stored lyophilized or in solution at −20°C. The recombinant SpyCEP (recSpyCEP) encompassing amino acid residues 35–587 (GenBank No. DQ413032) (21) was expressed and purified at GenScript (Piscataway, NJ).

To develop a superficial skin infection model for GAS, inbred female BALB/c and outbred Swiss mice (4–6 wk old) were used. Mice were anesthetized with an i.p. injection (100 μl/10 g mouse) of ketamine (100 mg/ml stock)/xylazil-20 (20 mg/ml stock)/water in a ratio of 1:1:10. The fur from the nape of the neck of mice was removed using clippers and a shaver, and the skin was wiped clean with an ethanol swab. To optimize a method that would result in reproducible superficial skin damage, different methods of mechanical scarification of skin were attempted. These included scratching with a needle, gentle cuts with a scalpel, or using a metal file. Following a skin abrasion, the mice were infected with GAS. An inoculum (20 μl) of GAS containing 1 × 10⁶ CFU counts was topically applied. Once the inoculum had completely absorbed on the skin, a temporary cover was applied on the wounded site, and mice were housed in individual cages. Mice were given streptomycin (200 μg/ml) water 24 h prior to infection and remained on that throughout the course of the study. A parallel cohort of air sac–infected mice was included as a control. These mice were infected following the method of Raeder and Boyle (22). Mice were monitored daily for infected lesions, as well as signs of illness, as per the score sheet approved by Griffith University Institutional Biosafety Committee. The wounded site was monitored closely to evaluate the status of infection.

BALB/c mice were immunized s.c. at the tail base on day 0 with 30 μg J8-DT formulated in Alhydrogel aluminum hydroxide wet gel (Alum). For the combination vaccine, J8-DT and recSpyCEP were admixed in a ratio of 1:1 and formulated in Alum, as described previously. Each mouse received 60 μg total vaccine preparation. Mice were boosted on days 21 and 28, as described earlier (23). Control mice received recSpyCEP or adjuvant alone. Two weeks after the final immunization, mice were challenged with GAS, as described above.

At various time points postinfection (days 3, 6, and 9), a defined number of mice from each group was sacrificed. Blood samples were collected via cardiac puncture, spleens were removed, and skin biopsy samples (measuring 2–3 mm²) from the infected lesion at the nape of the neck were obtained. The skin and spleen samples were mechanically homogenized, and appropriate dilutions were plated in replicates on streptomycin–blood agar plates.

To characterize the histopathology of the model, biopsy specimens were taken from naive and GAS-infected mice. On day 3 postinfection, mice were sacrificed and biopsy specimens of excised skin were taken. The samples were immediately fixed in buffered formalin and embedded in paraffin for H&E staining. Five-micron-thick tissue sections were sliced and stained with H&E, as well as with Giemsa and Gram stains, to visualize Gram-positive organisms. For immunohistochemistry, the samples were frozen in OCT. Histology was also performed at various time points following macrophage and neutrophil depletion. Sections were scanned and read at high magnification using ImageScope software. Positive cells were counted in five areas of scanned slides and expressed as the average number of positive cells/high-powered field using ImageJ (National Institutes of Health, Bethesda, MD).

Depletion of macrophages with carrageenan (CGN) was performed as previously described (24). Subcutaneous CGN administration was used for depletion of skin macrophages, whereas i.p. CGN administration was used for depletion of systemic macrophages. The dose and time course for CGN injection were optimized using flow cytometric analysis of spleen cells after double staining with FITC-conjugated anti-mouse Mac-1 or CD11b and allophycocyanin-conjugated F4/80 (BD Biosciences, Franklin Lakes, NJ). To deplete neutrophils, anti-Ly6G mAb (clone 1A8) was used as previously described (25, 26). The depletion of neutrophils was verified by flow cytometric analysis of blood, bone marrow, and spleen cells using CD11b-PerCP-Cy5.5 and Gr-1–allophycocyanin mAbs (BD Biosciences).

Splenocytes and memory B and T cells from J8-DT/Alum- and PBS/Alum-immunized rested mice were purified and adoptively transferred to naive syngeneic mice, as previously described (23). Briefly, following RBC lysis of splenocytes, CD19 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) were used for positive selection of B cells. After positively selecting B cells, the T cells were purified by negative selection using a Pan T Cell Isolation Kit (Miltenyi Biotec), as per the manufacturer’s instruction. For in vivo studies, each mouse received one spleen equivalent of splenocytes or splenic B or T cells.

IL-8, MIP-2, and KC degradation was performed and quantified by ELISA using the Quantikine kit (R&D Systems, Minneapolis, MN) as described previously (27). Using this method, the amounts of undegraded chemokines (IL-8, MIP-2, and KC) postincubation with GAS culture supernatants (S/Ns) were measured. Briefly, to collect culture S/Ns, various GAS strains were grown to mid-log phase (OD₆₀₀ 0.5), reinoculated into fresh THB, and grown overnight at 37°C. Cell-free GAS culture S/Ns from each strain were incubated at 37°C with a known concentration of recombinant chemokine (IL-8, MIP-2, and KC). Samples were collected at 2, 4, 8, or 24 h, and the amount of undegraded chemokine was determined by ELISA (R&D Systems) as described above.

Neutrophils were isolated from mouse bone marrow using a neutrophil isolation kit (Miltenyi Biotec). Neutrophils (2.5 × 10⁵ in 100 μl media) were added to the upper chamber of the Transwell system (Costar 24-well Transwell; Corning, Corning, NY), which was then placed in the lower chamber containing media alone or intact or degraded chemokines. As a positive control, wells containing a known concentration of each recombinant chemokine were used. Following 2 h of incubation at 37°C, the cells were collected from the upper and lower chambers, and the number of viable neutrophils transmigrated was determined using trypan blue exclusion. The percentage of migrating neutrophils was calculated by dividing the number of migrating neutrophils by the total number of neutrophils present.

GAS were incubated with a 1 in 50 dilution of J8-DT, J8–DT–SpyCEP, SpyCEP, or PBS antisera for 1 h at 4°C with rotation. Following opsonization, the cells were collected by centrifugation, washed, and resuspended in fresh THB. A bacterial inoculum containing 3.5 × 10⁴ GAS CFU in 400 μl media was injected i.p. into SCID mice. The mice were culled post 48 h of challenge, blood was harvested, and serial dilutions were plated on blood agar plates to determine bacterial burden.

GAS strains including BSA10 and 5448AP were grown to stationary phase. The cell-free GAS culture S/Ns were coincubated with recombinant chemokines and either 1 in 50 dilution of PBS or anti-SpyCEP serum for 16 h at 37°C. The S/N without any serum was used as a negative control. Uncleaved IL-8 was measured using a Quantikine ELISA Kit (R&D Systems), and neutralization of IL-8–cleaving activity due to SpyCEP antiserum was calculated in comparison with the controls (IL-8 with normal serum or in media alone).

Data were analyzed using GraphPad Prism version 6.00 for Mac. Statistical differences between the two groups were determined using two-tailed t tests corrected for multiple comparisons using the Holm–Sidak method. ANOVA with the Tukey or Dunnett post hoc method for multiple comparisons was used for pairwise comparisons. The p values <0.05 were considered significant.

BALB/c and Swiss mice were assessed for their ability to develop a skin infection at the nape of the neck following minor mechanical scarification achieved by one of three methods: scratching with a needle, superficial cuts with a scalpel, or mild scarification with a metal file. After the skin was abraded, a bacterial cell suspension containing 1 × 10⁶ CFU of 2031 GAS in 20 μl volume was applied with a pipette to the open wound, as described in 2Materials and Methods. Scarification with a file resulted in the most reproducible results, with infection lasting for ≥9 d (Supplemental Fig. 1A). Following scarification, the skin became visibly damaged, as characterized by reddening and glistening but without bleeding. This method was used for all subsequent experiments. Histological observations at 72 h postinfection revealed damage to the epidermal and dermal layers (Fig. 1Ai, Aii, Aiii), and GAS organisms were visible within various dermal layers (Fig. 1Aiv). A large influx of neutrophils (polymorphonuclear cells [PMNs]) to the site of infection was noted (Fig. 1Aiii); however, this was not observed in uninfected mice whose skin was scarified but GAS was not applied (Fig. 1Aii). PMNs were observed to undergo degranulation (Fig. 1Av). F4/80⁺ macrophages were also present (Fig. 1Avi). A comparison of this GAS-infection method with the existing air sac model (28) demonstrated that it generated reproducible and similar outcomes in terms of bacterial burden of skin and blood (Supplemental Fig. 1B, 1C). However, we adopted the scarification method to investigate vaccine-induced protection because it mimicked more closely the human situation of superficial skin infection (29).

Mice vaccinated with J8-DT and control mice were challenged with 2031 GAS, and histological examinations were performed. Vaccinated and control mice demonstrated influx of PMNs and macrophages as early as 24 h postinfection; however, the intensity of both cell populations was significantly higher in the nonvaccinated cohort compared with the vaccinated group (p < 0.01) (Fig. 1B, 1C). The number of PMNs dropped in vaccinated mice by day 1 postinfection, whereas many PMNs were observed in nonvaccinated mice at day 9 postinfection when the experiment was terminated (Fig. 1B, 1D). Similarly, macrophages appeared in both the vaccinated and nonvaccinated cohorts on day 1, but their numbers were significantly lower in vaccinated mice (p < 0.01) and remained lower in this group (Fig. 1C, 1E).

To assess the protective efficacy of J8-DT against pyoderma, immunized mice were challenged with various GAS strains, including 2031, 88/30, and NS1, all belonging to different emm types and different emm clusters (30). Supplemental Table I lists the strains, their clinical origin, and their phenotype. At different time points postinfection, mice were euthanized, and the total GAS bacterial burdens in excised skin and blood were determined by plating. The extent of infection with different strains varied, suggesting a difference in virulence. However, in all experiments, the vaccinated mice were able to reduce the bacterial burden at the site of infection significantly more so than the nonvaccinated group (p < 0.05–0.001) (Fig. 2A, 2C, 2E). To determine whether vaccination protected against systemic infection/bacteremia, the bacterial burden in blood was measured for the same mice at the same time points. Vaccinated mice did not develop a systemic infection with GAS 2031 (a reference strain) (Fig. 2B) and cleared GAS 88/30 (a clinical skin isolate) completely by day 6 (Fig. 2D). However, following infection with a clinical blood isolate (NS1), bacteria were still detectable until day 6, although the bacterial burden was significantly reduced in the vaccinated group (p < 0.05) (Fig. 2F). Similar observations were made with other strains used in this study (experiments described below); vaccinated mice either did not develop a systemic infection or rapidly cleared the infection.

Vaccine-mediated memory responses are critical for long-lived protection. To assess the role of vaccine-induced memory responses in protection against skin infection, J8-DT–immunized and control B10.BR mice were rested for 10–12 wk, after which time the protective capacity of splenic lymphocytes was assessed. Following the resting period, the vaccinated cohort showed a significant drop in their Ab levels (data not shown). To assess the efficacy of memory lymphocytes independent of circulating Abs, the splenocytes (Fig. 3A, 3B) or purified B or T cells (Fig. 3C–F) from these mice were adoptively transferred to naive B10.BR mice, which were then challenged. The recipient mice had no detectable Abs at 24 h postadoptive transfer at the time of infection with GAS NS27 (emm91) via the skin (data not shown). Adoptive transfer of memory splenocytes (Fig. 3A, 3B) and B cells (Fig. 3C, 3D) resulted in significantly reduced bacterial burden in the skin (p < 0.05 to p < 0.001) (Fig. 3A, 3C) and the blood (Fig. 3B, 3D) of recipient mice. Adoptive transfer of memory T cells alone did not transfer protection (Fig. 3E, 3F). These data demonstrated a critical protective role for vaccine-specific memory B cells and suggest that protection will still be evident after Ab titers have waned.

Accessory immune cells are known to play a critical role in protection against streptococci (24, 31, 32). We show that skin-resident macrophages contribute to the control of bacterial burden in the skin, whereas systemic macrophages contribute to the control of systemic infection. To assess the role of macrophages in vaccine-mediated immunity, they were depleted postvaccination, prior to challenge infection, and after challenge using CGN (Supplemental Fig. 2A). Although CGN injected i.p. depleted splenic macrophages, the effect on the dermal macrophage population was minimal (Supplemental Fig. 2B–E). We observed that following infection with GAS NS27, the skin of vaccinated mice, irrespective of whether their systemic macrophages were depleted, was better protected than was the skin of control mice (Fig. 4A). By day 6 postinfection, a significant difference in the skin bacterial burden of the vaccinated cohorts was noted. However, we observed that vaccinated macrophage-deficient mice were more susceptible to a systemic infection compared with their macrophage-sufficient counterparts (Fig. 4B). This difference was observed during the early phase of infection (days 3 and 6). Following from these and other observations where macrophages were shown to be important during the early phase of infection, samples from only days 3 and 6 were tested in the subsequent macrophage-depletion–challenge experiments.

To explore the role of skin-resident macrophages, we either left mice untreated or injected them s.c. with CGN into the nape of the neck every 3 d after vaccination and prior to and following challenge. Subcutaneous administration of CGN did not deplete splenic macrophages (Supplemental Fig. 2B, 2C) but did deplete dermal macrophages (Supplemental Fig. 2D, 2E). We observed that, on day 3 postchallenge, the skin bacterial burden was significantly higher in vaccinated skin macrophage-deficient mice compared with the vaccinated macrophage-sufficient cohort (p < 0.05) and was comparable to that of the nonvaccinated cohort (Fig. 4C). However, by day 6, the effect of macrophage depletion had faded, and the vaccinated cohort, whether macrophage sufficient or deficient, had skin bacterial burdens that were significantly reduced compared with the nonvaccinated cohort (p < 0.001) (Fig. 4C).

We also observed that the absence of dermal macrophages did not increase susceptibility to systemic infection in vaccinated mice (Fig. 4D); rather, as shown above, it was the absence of systemic macrophages that increased susceptibility to systemic infection in these mice (Fig. 4B). We then depleted both skin-resident and systemic macrophages by injecting CGN via the i.p. and s.c. routes. This resulted in severe local and systemic infections in both vaccinated and control mice (Fig. 4E, 4F).

We next assessed the role of neutrophils in vaccine-mediated protection using an optimized protocol (Fig. 5A). Treatment with 1A8 mAb resulted in >95% depletion, as confirmed by histology and quantification (Supplemental Fig. 3A, 3B) (25). Bacterial burdens in skin and blood were monitored following infection with GAS skin isolate 90/31 (emm57). Strain 90/31 was selected on the basis of its ability to grow rigorously in an in vitro mouse blood-growth assay. In the absence of PMNs, vaccinated mice suffered bacterial burdens in skin and blood that were significantly higher than their PMN-sufficient counterparts (p < 0.01 to p < 0.001) (Fig. 5B, 5C). Gross pathology at the site of infection demonstrated that the skin lesion in vaccinated PMN-deficient mice did not heal (Supplemental Fig. 3C), whereas the lesions in vaccinated PMN-sufficient counterparts recovered by day 3 (Supplemental Fig. 3D). For the nonvaccinated mice, the PMN-depleted group showed an enhanced bacterial burden in the blood at day 9, at which time the PMN-sufficient mice had cleared the infection from the blood (p < 0.001). We observed that nonvaccinated PMN-sufficient mice were able to control their bacterial levels in the skin and blood significantly better than were vaccinated PMN-deficient mice by day 9 (p < 0.001) (Fig. 5B. 5C), possibly reflecting the development of Abs as a result of infection (M. Pandey. V. Ozberk and M.F. Good, unpublished observations).

The data suggested that vaccine-induced immunity could be compromised against strains that could prevent neutrophil ingress. GAS express on their surface and shed the CXC chemokine protease SpyCEP, which can block neutrophil chemoattraction (33). Thus, we tested the various strains to document the extent of inhibition of neutrophil migration mediated by culture S/Ns in a Transwell assay. We observed that S/Ns from all strains inhibited migration (Fig. 5D); however, the greatest inhibition was observed using S/Ns from the CovR/S mutant strain 5448AP. The mutation in CovR/S leads to overexpression of SpyCEP, as well as a number of other virulence factors. Therefore, we measured the degradation of mouse and human CXC chemokines KC, MIP-2, and IL-8 by S/Ns from these strains and observed that all S/Ns could degrade chemokines (Fig. 5E–G); however, the greatest activity was observed with S/Ns from 5448AP.

To test whether this might affect immunity, vaccinated and control mice were infected with two CovR/S mutant strains: 5448AP (emm1) and BSA10 (emm124). 5448AP is derived from the M1T1 strain responsible for the current global epidemic. BSA10 is a skin isolate of a different emm type isolated from a patient in the Northern Territory of Australia. We observed that J8-DT had diminished protective effect on the skin against these strains except for at day 9, when the difference between the vaccinated and control cohorts was found to be significant (p < 0.05 to p < 0.01) (Fig. 6A, 6C). Similarly, the blood bacterial burden, although reduced in vaccinated mice, was not significantly different from control mice at a number of time points with the exception of day 9, when a significant reduction in bacterial burden in blood of vaccinated mice was observed for BSA10 (Fig. 6D). This observation correlated with the strain’s moderate ability to inhibit neutrophil migration (Fig. 5C) and to degrade CXC chemokines (Fig. 5E–G), suggesting its lesser virulence compared with 5448AP. Examination of gross pathology demonstrated severely compromised wound healing in vaccinated and control mice, and histopathology revealed a paucity of neutrophils at the infection site (data not shown). Collectively, these data suggested that vaccine-mediated protection requires recruitment of neutrophils for Ab-mediated phagocytosis and protection, and GAS that prevent neutrophil chemoattraction are less susceptible to vaccine-mediated killing.

Because the data showed that neutrophils are critical to J8-DT–mediated immunity, we asked whether a combination vaccine that induced both Abs to J8 and Abs that could protect neutrophil-attracting CXC chemokines from degradation would protect mice. In previous studies, vaccination with a recombinant fragment of the CXC chemokine protease SpyCEP afforded modest protection (21, 34). We initially asked whether Abs raised to this fragment could protect the human CXC chemokine IL-8 from degradation mediated by S/Ns from the two CovR/S mutant strains. As shown in Fig. 7A, the antiserum completely protected IL-8, indicating that SpyCEP mediated all IL-8 degradation caused by these strains. We then combined the SpyCEP fragment (recSpyCEP; amino acid residues 35–587) with J8-DT in a ratio of 1:1 by weight and formulated the combination with Alum. Mice were vaccinated with J8-DT alone, recSpyCEP alone, a combination of both, or PBS and then challenged with the most virulent strain, 5448AP. Mice vaccinated with J8-DT+recSpyCEP displayed Ag-specific Ab titers (>10⁶) to both J8 and recSpyCEP (data not shown). Vaccinated mice were then challenged with 5448AP, and data from two repeat experiments are shown (Fig. 7B–E). Again, we observed that mice vaccinated with J8-DT alone demonstrated limited protection from pyoderma or from bacteremia. However, the combination vaccine afforded significantly enhanced protection against pyoderma (Fig. 7B, 7D) and bacteremia (Fig. 7C, 7E). On day 3 postchallenge, the skin bacterial burden in mice receiving the combination vaccine was comparable to the bacterial burden in mice receiving J8-DT alone; however, by day 6 it was significantly reduced (p < 0.01) compared with the bacterial burden in mice vaccinated with J8-DT, SpyCEP alone, or PBS (Fig. 7 B). Although by day 9 there was significant reduction (p < 0.05) in skin bacterial burden of J8-DT–vaccinated mice, the enhanced protective efficacy (p < 0.01) of the combination vaccine in comparison with J8-DT was clearly evident (Fig. 7B). Similar observations were made in the repeat experiments in which J8–DT+SpyCEP demonstrated significantly enhanced protective efficacy against pyoderma in comparison with J8-DT (Fig. 7D).

Bacteremia was also monitored. Five of six mice that received the combination vaccine had no detectable bacteremia by day 9, compared with zero of six mice vaccinated with J8-DT alone and zero of six mice vaccinated with recSpyCEP alone. In terms of bacterial load, there was an ∼4-log reduction in bacterial burden in the blood of mice that had received the combination vaccine by day 6 compared with mice receiving either vaccine component alone (Fig. 7C). Significantly (p < 0.01) enhanced protective efficacy of the combination vaccine was confirmed in a repeat experiment in which J8–DT+SpyCEP–vaccinated mice demonstrated a complete clearance of the blood bacterial burden by day 6 (Fig. 7E). To further confirm these findings, we also assessed the efficacy of combination vaccine against another CovR/S mutant strain, NS88.2 (emm98.1). Again, the combination vaccine demonstrated significantly improved protection against pyoderma and bacteremia (data not shown).

To confirm the synergistic effect of combining J8-DT and the recSpyCEP fragment in protection against 5448AP, another set of mice was vaccinated with the individual components or the combination, and the bacteria were incubated in vitro with sera from the vaccinated mice. Bacteria were washed and injected into immunodeficient SCID mice. After 48 h, the numbers of GAS colonies present in their blood were determined. This assay showed that J8-DT and SpyCEP Abs alone were protective, but that the combination protected significantly better (Fig. 7D). The protection mediated by SpyCEP Abs alone suggested that they were active at the bacterial surface, prior to shedding of SpyCEP.

The combination vaccine also was tested for efficacy against a CovR/S wild-type strain (SN1, emm89) and demonstrated significant protection, with vaccinated mice not developing a blood bacteremia (data not shown). However, as expected, the mice vaccinated with J8-DT alone also were strongly protected and did not develop a blood bacteremia in comparison with mice given a PBS vaccine. Mice receiving the SpyCEP vaccine alone were not protected.

Skin infections caused by GAS are a significant global health concern. Acute infections lead to ∼110,000 deaths each year, and >350,000 deaths are due directly or indirectly to rheumatic heart disease (1). A vaccine would be a critically important public health tool to alleviate this burden of disease. We describe an animal model for GAS pyoderma that closely resembles the human situation and used this to assess, analyze, and significantly improve the efficacy of a leading M protein–derived candidate vaccine, J8-DT. Although demonstrating profound efficacy of J8-DT alone against strains of different emm types and different emm clusters (30), it was ineffective against a CovR/S mutant of the global epidemic strain M1T1, as well as an endemic CovR/S mutant from the Northern Territory of Australia. The CovR/S mutants were shown to degrade the neutrophil chemoattractants IL-8, KC, and MIP-2 and to greatly reduce neutrophil chemotaxis. To overcome this, we combined J8-DT with a recombinant fragment of the streptococcal CXC protease SpyCEP to induce Abs that both target the conserved domain of the M protein (anti-J8) and could protect the human and mouse neutrophil-attractant CXC chemokines from streptococcal SpyCEP-mediated degradation. We demonstrated that the addition of recSpyCEP rendered the combination vaccine highly effective.

We also assessed the capacity of a vaccine-induced memory response to protect against infection. Previously, we demonstrated that vaccination with J8-DT generates memory B cells that were protective against systemic infection initiated by i.v. inoculation of GAS (23). In this study, we show that memory B cells also can mediate protection against superficial skin infection and subsequent bacteremia. Adoptive transfer of vaccine-specific splenocytes or purified B or T cells taken during the memory phase of the immune response confirmed that protection afforded by J8-DT vaccination is mediated by memory B cells. Furthermore, we demonstrated that, even if mice did not have vaccine-specific Ab at the time of challenge, their memory B cells were sufficient to render them protected. These data, together with our previously published data (23), suggest that Ab production from memory B cells is rapidly induced upon exposure to GAS, thus affording rapid protection. These findings have significant implications for a vaccine in that not only would protection against streptococcal skin infection be evident after Ab levels had waned, but also that infection would boost immunity.

We also assessed the function of accessory immune cells. Although the roles of macrophages during GAS infection were addressed previously (24), their roles in vaccine-mediated immunity have not been studied. We examined the roles of both skin-resident and systemic macrophages. The absence of skin macrophages led to a significantly higher skin bacterial burden in the vaccinated cohort. However, the skin macrophage status did not affect susceptibility to systemic infection. By comparison, the absence of systemic macrophages increased susceptibility to systemic infection. These findings suggest that skin-resident macrophages play a role in controlling local infection and that systemic macrophages help to control dissemination of GAS from the dermis to other tissues. In addition to other functions performed by resident tissue macrophages, they are the source of KC and MIP-2, the major chemokines responsible for neutrophil chemoattraction (35, 36). Therefore, it is likely that, in their absence, neutrophil chemotaxis and, hence, vaccine efficacy are affected.

Neutrophils are widely considered the major responders to an infection. This recruitment is vital both for direct action against micro-organisms and for attracting lymphocytes (26). PMNs represent an important first line of effector cells in the control of GAS infections. Activated PMNs can phagocytose and kill GAS (37). Similarly, impaired PMN function, due to failure of PMN migration following CXC chemokine destruction or due to necrosis of PMNs by the pore-forming toxin streptolysin O, results in severe GAS infection (32). In this study, we observed that the efficacy of J8-DT vaccination was ablated in PMN-depleted mice. Consistent with this observation, vaccine efficacy was greatly reduced when the GAS strain responsible for the infection could block neutrophil migration. S/Ns from all strains tested were effective in destroying CXC chemokines and inhibiting chemokine-mediated neutrophil migration to some extent; however, S/Ns from a CovR/S mutant strain, 5448AP, demonstrated a dramatic effect, with a complete impediment to chemokine-mediated neutrophil migration. This strain has upregulated SpyCEP, which degrades CXC chemokines (14, 27, 38, 39), resulting in reduced CXC-induced neutrophil transmigration and, thus, conferring resistance to GAS killing by isolated neutrophils (38). The spycep gene is negatively regulated by CovR/S, and mutation within the regulator can enhance the expression of SpyCEP ≥40-fold (37). Our data show that 5448AP-derived SpyCEP is solely responsible for CXC chemokine degradation, in that anti-SpyCEP Abs can completely protect IL-8 from degradation by this strain. Thus, we asked whether inclusion of a recombinant fragment of SpyCEP, which has moderate efficacy as a vaccine candidate in its own right (21), would enhance the protective efficacy of J8-DT. We demonstrated a significant synergistic enhancement of efficacy against pyoderma and bacteremia following pyoderma. We showed that anti-SpyCEP Abs work by protecting CXC chemokines from shed SpyCEP (Fig. 7A) but also can act at the bacterial cell surface (Fig. 7D).

In conclusion, we demonstrated that a leading GAS vaccine candidate, J8-DT, can protect against GAS pyoderma and bacteremia. The skin scarification model described in this article enables an evaluation of pathogenesis and vaccine mode of action in a model that closely mimics the major route of human infection. We further show that inclusion of a recSpyCEP fragment with J8-DT significantly enhances protection against a hypervirulent GAS strain, 5448AP. Our data strongly suggest that this is due to the blocking of degradation of CXC chemokines by anti-SpyCEP Abs. This, together with opsonic Abs from J8-DT vaccination, resulted in a profound level of protection. To our knowledge, this is a unique example of a vaccine that requires the independent action of Abs of two specificities for effect.

We thank Prof. Sri Sriprakash for providing GAS strains endemic in the Northern Territory of Australia, Prof. Mark Walker and Dr. Jenny Robson for providing the 5448AP and SN1 GAS strains, respectively, and Clay Winterford (QIMR Berghofer Medical Research Institute) for histology. We thank Gus Nossal, Mark Walker, Sri Sriprakash, Stephanie Yanow, Andrew Steer, and Pierre Smeester for critical appraisal of the manuscript.

The authors have no financial conflicts of interest.