One Dose of Staphylococcus aureus 4C-Staph Vaccine Formulated with a Novel TLR7-Dependent Adjuvant Rapidly Protects Mice through Antibodies, Effector CD4+ T Cells, and IL-17A

A rapidly acting, single dose vaccine against Staphylococcus aureus would be highly beneficial for patients scheduled for major surgeries or in intensive care units. Here we show that one immunization with a multicomponent S. aureus candidate vaccine, 4C-Staph, formulated with a novel TLR7-dependent adjuvant, T7-alum, readily protected mice from death and from bacterial dissemination, both in kidney abscess and peritonitis models, outperforming alum-formulated vaccine. This increased efficacy was paralleled by higher vaccine-specific and α-hemolysin-neutralizing antibody titers and Th1/Th17 cell responses. Antibodies played a crucial protective role, as shown by the lack of protection of 4C-Staph/T7-alum vaccine in B-cell-deficient mice and by serum transfer experiments. Depletion of effector CD4+ T cells not only reduced survival but also increased S. aureus load in kidneys of mice immunized with 4C-Staph/T7-alum. The role of IL-17A in the control of bacterial dissemination in 4C-Staph/T7-alum vaccinated mice was indicated by in vivo neutralization experiments. We conclude that single dose 4C-Staph/T7-alum vaccine promptly and efficiently protected mice against S. aureus through the combined actions of antibodies, CD4+ effector T cells, and IL-17A. These data suggest that inclusion of an adjuvant that induces not only fast antibody responses but also IL-17-producing cell-mediated effector responses could efficaciously protect patients scheduled for major surgeries or in intensive care units.


Introduction
Staphylococcus aureus infections of the bloodstream or deep wound are a serious complication of major surgeries, including cardiothoracic and orthopedic surgery, resulting in significant morbidity and mortality [1,2]. S. aureus is also the most commonly isolated microorganism from patients in intensive care units, which have mortality rates that reach 60% [3]. Due to methicillin-resistant S. aureus infections, up to one third of patients diagnosed with S. aureus bacteremia succumb even when treated with appropriate antibiotic therapy [4]. Therefore, a vaccine that provides rapid protection against S. aureus during the post-operative or intensive care period would address an important unmet medical need.
We recently developed a four-component S. aureus vaccine (4C-Staph) consisting of Hla H35L , EsxAB, FhuD2, and Csa1A recombinant proteins [5]. Hla H35L is a genetically detoxified mutant of α-hemolysin (Hla) [6], a highly conserved exotoxin that plays a prominent role in early stages of invasive infections disrupting epithelial and endothelial barriers, contributing to pathogen-associated mortality [6,7]. Immunization with Hla H35L partially protected mice against staphylococcal pneumonia, peritonitis, and skin infections inducing functional antibodies neutralizing the lytic activity of native Hla [8][9][10]. Remarkably, however, Hla neutralization was not sufficient to eradicate S. aureus infection, suggesting that additional antigens are required for an efficacious vaccine [9]. EsxAB is a fusion of the two ESAT-6-like secreted virulence factors EsxA and EsxB associated to abscess formation, which may facilitate persistence and spread of the pathogen in the infected host [11][12][13]. FhuD2 is a lipoprotein involved in iron uptake and in early stages of invasive S. aureus infection [14][15][16], while Csa1A is a putative lipoprotein whose role in pathogenesis is under investigation [17]. We have recently shown that two doses of 4C-Staph vaccine formulated with alum protected against a panel of epidemiologically relevant S. aureus strains in kidney abscess, peritonitis, skin, and pneumonia mouse models of S. aureus infection [5].
Adjuvants enhance and accelerate adaptive immune responses toward a co-administered antigen, while also directing the quality of the immune response [18,19]. Three major types of cell-mediated effector immunity meant to optimally respond to distinct threats have been identified: type 1 protects against intracellular pathogens and comprises IFN-γ-producing cells (e.g. Th1 cells), type 2 protects against helminths and comprises IL-4/IL-13-producing cells (e.g. Th2), while type 3 protects against extracellular bacteria and fungi and comprises IL-17-producing cells (e.g. Th17) [20]. Aluminum salts-based adjuvants, which are included in many licensed vaccines, preferentially induce type 2 responses while agonists of Toll-like receptors (TLRs), a family of receptors that recognize pathogen-associated molecular patterns [21,22], induce mainly type 1 responses [18]. The potential of small molecule immune potentiators (SMIPs) targeting TLR7, an endosomal TLR that recognizes single-stranded RNAs, as vaccine adjuvants has been shown in pre-clinical settings, especially for Imiquimod. However, systemic activation induced by these SMIPs has posed safety issues [23]. The rational design of SMIP.7-10, a novel TLR7 agonist that can be stably adsorbed to alum (T7-alum), minimized systemic exposure and inflammation while retained in vivo potency [24]. Therefore, in this study, we investigated whether single immunization with 4C-Staph/ T7-alum conferred prompt protection against S. aureus infection. We found that this was indeed the case and we elucidated the immune mechanisms involved.
Five-week old female BALB/c (Charles River Laboratories) or J H (Taconic) mice were used. For active immunization, mice were immunized i.m. (50 μl/hind leg quadriceps). Control mice received equal amounts of alum, or T7-alum. For passive immunization, pools of sera were prepared from BALB/c mice immunized once with 4C-Staph/T7-alum (immune serum) or T7-alum (control serum) by 32 days. Five-week-old naïve BALB/c mice were injected with 150 μl of immune or control serum into the tail vein the day before challenge with S. aureus.

Bacterial inoculum and challenge
S. aureus Newman strain (kindly provided by Professor Schneewind, University of Chicago) was grown in tryptic soy broth (Difco Laboratories) at 37°C shaking at 250 rpm to an optical density at 600 nm (OD 600 ) of 2. After centrifugation at 3,320 x g for 10 min., bacteria were washed with 50 ml of phosphate-buffered saline (PBS, pH 7.4; Invitrogen) and then diluted in PBS to obtain the required infectious dose/mouse. Inocula were verified experimentally by plating on tryptic soy agar and enumeration of the colony forming units (CFU) the day after.
Kidney abscess model: mice were challenged i.v. with 100 μl of a sublethal dose of bacteria (~2×10 7 CFU). After 4 days, mice were euthanized; both kidneys were removed, homogenized in pool in 2 ml PBS, 2-fold serially diluted, and plated in duplicate for CFU counts.
Peritonitis model: mice were challenged i.p. with 100 μl of a LD 80 of bacteria (~5 x 10 8 CFU). Mice were monitored daily for 15 or 30 days, as indicated. Mice that survived were euthanized and CFU in kidneys were enumerated as described above.

Quantification of 4C-Staph-specific and Hla-neutralizing antibodies
Vaccine-specific antibody titers were measured by Luminex (Luminex 1 200 TM) at a fixed serum dilution. Hla H35L , FhuD2, EsxAB and Csa1A purified proteins were covalently conjugated to the free carboxyl groups of xMAP multi-analyte microspheres (Luminex Corporation). Antigen-specific antibodies were revealed using PE-labeled secondary antibodies. For total IgG, median fluorescence intensity (MFI) values were converted into relative Luminex units (RLU)/ml using a mouse serum as standard.
Hla neutralizing antibodies were quantified with a rabbit red blood cells (RBCs)-based functional assay. Serial 2-fold dilutions of pooled mouse sera (n = 8/pool) were incubated with 12.5 nM Hla (Sigma) in triplicates. After 30 min at 37°C in agitation (350 rpm), RBCs (Emozoo Snc.) were added and incubation prolonged for 30 min without shaking. Then plates were centrifuged for 5 min at 1,000 x g and the OD 540 of supernatants was determined with a SpectraMax 1 340PC384 Absorbance Microplate Reader (Molecular Devices). Percent hemolysis was calculated using as denominator the OD 540 obtained following lysis of rabbit RBCs without mouse serum. Hla neutralizing titers were expressed as the reciprocal of the serum dilution inhibiting 50% of Hla hemolysis (ED 50 ) given by native Hla, calculated using a "log (agonist) vs. normalized response" nonlinear regression curve (GraphPad Software, Inc.).

Statistical analysis
Log-rank test was used to evaluate differences in survival of infected mice. One-way ANOVA and Sidak's multiple comparisons test were used to evaluate differences in the bacterial load in kidneys of infected mice and differences in CD4 + T-cell responses to vaccination when more than two groups were compared. Comparisons between frequencies of cytokine-producing CD4 + T cells in response to 4C-Staph/alum or 4C-Staph/T7-alum vaccination were performed using a Student's t test and a partial permutation test. For total IgG titers, Kruskall-Wallis and Dunn's multiple comparisons tests were used to evaluate differences in vaccine-specific antibody titers. Statistical analyses were performed using GraphPad Prism 6 software (GraphPad Software, Inc.) and SPICE version 5.1 (http://exon.niaid.nih.gov).

Ethics statement
All animal studies were carried out in compliance with current Italian legislation on the care and use of animals in experimentation (Legislative Decree 26/2014, authorizations 185/2011-B and 249/2011-B approved by the Italian Ministry of Health) and with the Novartis Animal Welfare Policy and Standards (authorizations AWB 201105 and AWB 201106). After infection, mice were monitored daily using a dedicated clinical score system based on the following parameters: piloerection, hunched posture, decreased mobility, rolling/twisting movements and weight loss. Mice were euthanized by cervical dislocation when they exhibited either one of the following humane endpoints: a loss of 20% bodyweight and/or moderate to severe signs of rolling/twisting movements that were pre-established in agreement with Novartis Animal Welfare Policies. All animals were provided environmental enrichment and food and water were offered ad libitum.

One dose of 4C-Staph/T7-alum vaccine confers rapid and efficacious protection against S. aureus
We tested the efficacy of one dose of adjuvanted 4C-Staph vaccines in the mouse kidney abscess and peritonitis models. For this purpose, BALB/c mice were vaccinated i.m. with 4C-Staph/T7-alum or 4C-Staph/alum vaccines. Control groups of mice received T7-alum or alum. Twelve days after immunization, mice were challenged with S. aureus Newman strain.
In the peritonitis model, which can be used to study the spread of bacteria into the bloodstream [9], mice were challenged i.p. (5x10 8 CFU) with a lethal dose of S. aureus and their survival was monitored for 30 days. One month after infection, 75% of mice immunized with 4C-Staph/T7-alum survived S. aureus challenge compared to 41% of mice immunized with 4C-Staph/alum (p = 0.0058, Fig 1B). Furthermore, mice vaccinated with 4C-Staph/T7-alum that survived had significantly less bacteria in kidneys (1.7 log 10 reduction, p = 0.0091) and 91% of them had no detectable bacteria in kidneys as compared to survivors in the 4C-Staph/ alum group (54%, Fig 1C).
Taken together, these data showed that single dose of 4C-Staph/T7-alum vaccine considerably reduced bacterial load in kidneys upon S. aureus i.v. challenge and not only allowed the vast majority of mice to survive after i.p. challenge, but also promoted efficient control of bacteria in kidneys of survivors.

4C-Staph/T7-alum vaccine rapidly induces functional and protective antibodies
The induction of antibodies specific for each component of the vaccine by 4C-Staph/T7-alum and 4C-Staph/alum was evaluated 12 and 32 days after vaccination. IgG specific for Hla H35L , EsxAB, FhuD2, and Csa1A were detected in sera from all mice vaccinated with 4C-Staph/ T7-alum by day 12 and further increased by day 32, with the exception of one mouse that had no detectable anti-Csa1A IgG (Fig 2A). In contrast, only Hla H35L -specific IgG were detected in all sera from mice immunized with 4C-Staph/alum 12 days after vaccination, while EsxABand FhuD2-specific IgG were detected only in some sera, and Csa1A-specific IgG were not found. By day 32, Hla H35L -and EsxAB-specific IgG were found in sera from all 4C-Staph/alum vaccinated mice, while FhuD2-and Csa1A-specific IgG were found only in some sera. Higher levels of IgG1 and IgG2a specific for each vaccine component were found in sera of mice vaccinated with 4C-Staph/T7-alum as compared to sera of mice vaccinated with 4C-Staph/alum (Fig 2C), while no differences in vaccine-specific IgM levels were observed (Fig 2D). In addition, Hla neutralizing titers were measurable in sera from mice vaccinated with 4C-Staph/ T7-alum by day 12 (ED 50 = 41) and increased by 5-fold at day 32 (ED 50 = 215), while Hlaneutralizing activity was detected in sera from mice vaccinated with 4C-Staph/alum at d32 (ED 50 = 39) but not at day 12 (ED 50 < 6, Fig 2B). Altogether, these data show that single dose of 4C-Staph/T7-alum vaccine readily induced not only seroconversion of vaccinated mice against each 4C-Staph component but also Hla-neutralizing antibodies.
To determine if antibodies induced by 4C-Staph/T7-alum vaccination were protective against a lethal challenge with S. aureus, sera collected from mice 32 days after immunization with 4C-Staph/T7-alum (or T7-alum, as control) were passively transferred into naïve mice one day before i.p. infection. All mice that received immune serum were protected from death during the first 3 days after infection (Fig 3A). Thereafter, the survival gradually declined to become 28% at day 15, when all survivors had bacteria in kidneys (Fig 3B). In contrast, 100% of mice that received control sera died by day 6 after infection. Finally, we showed that 4C-Staph/T7-alum vaccination required B cells to protect mice against S. aureus infection by using J H mice, which lack mature B cells and antibodies [25]. Within two days after i.p. challenge, 96% of J H mice vaccinated with 4C-Staph/T7-alum succumbed to the infection while 88% of BALB/c mice were still alive 15 days after infection (p < 0.0001, Fig 3C).
Overall these data suggest that antibodies induced by 4C-Staph/T7-alum vaccine are necessary and sufficient to protect against early death caused by S. aureus i.p. infection.
These results demonstrated that one dose of 4C-Staph/T7-alum induced higher frequencies of vaccine-specific cytokine-producing CD4 + T cells and polarized the CD4 + T cells more towards a Th1/Th17 phenotype as compared to 4C-Staph/alum.

CD4 + T cells induced by 4C-Staph/T7-alum contribute to the protection against S. aureus
To assess if vaccine-specific CD4 + T cells play a role in protection, mice were treated with an anti-CD4 mAb 6 and 8 days after immunization with 4C-Staph/alum or 4C-Staph/T7-alum to allow T cell-dependent antibody production, but to eliminate CD4 + T cells with effector function before S. aureus challenge (S2 Fig). In the kidney abscess model, the depletion of CD4 + T cells abolished the protection conferred by 4C-Staph/T7-alum, as evidenced by the numbers of CFU found in kidneys that was not different in the T7-alum vaccine group (p = 0.145, Fig 5A). In contrast, CD4 + T cell depletion did not impact the efficacy of 4C-Staph/alum (statistically significant decrease in CFU in kidneys vs. mice vaccinated with alum, p = 0.002). In the peritonitis model, depletion of CD4 + T cells significantly decreased the survival of mice vaccinated with 4C-Staph/T7-alum (58% survival vs. 79% of mice treated with isotype control antibody, p = 0.034), but not of mice vaccinated with 4C-Staph/alum (13% survival vs. 31% of mice treated with isotype control antibody, p = 0.207, Fig 5B). In addition, depletion of CD4 + T cells in mice vaccinated with 4C-Staph/T7-alum caused an increase of 1.74 log 10 in the CFU/kidneys of survivors as compared to isotype control antibody-treated mice (p = 0.0372, Fig 5C).
Overall, these data show that effector CD4 + T cells contribute to the protection conferred by one dose of 4C-Staph vaccine adjuvanted with T7-alum both in the kidney abscess and peritonitis models, although other mechanisms of protection were involved, in agreement with the role of humoral immunity that we have shown in Fig 3. Neutralization of IL-17A increases the bacterial load in kidneys of mice vaccinated with 4C-Staph/T7-alum upon i.p. S. aureus infection Depletion of effector CD4 + T cells had no effect on protection conferred by vaccination with 4C-Staph/alum in either the kidney abscess or the peritonitis models (Fig 5A and 5B) indicating that the quality of effector CD4 + T cells induced by 4C-Staph/T7-alum vaccination is important. Since 4C-Staph/T7-alum induced a vaccine-specific CD4 + T cell response more polarized towards Th1 and Th17 cells as compared to 4C-Staph/alum (Fig 4B), we neutralized IL-17A and IFN-γ, separately or together, immediately before and repeatedly after i.p. challenge to block the effects of these cytokines produced in response to S. aureus. Treatment with neutralizing mAb specific for IL-17A and/or IFN-γ had no effect on survival compared to treatment with isotype-matched control antibody (Fig 6A-6C, left panels). However, neutralization of IL-17A alone or in combination with IFN-γ caused a 1.85 log 10 (p = 0.019) and a 1.66 log 10 (p = 0.0002) CFU increase, respectively, in kidneys of mice that survived the infection compared to survivors injected with isotype-matched control antibody (Fig 6A and 6C, right panels). Neutralization of IFN-γ alone did not cause a significant increase in CFU/kidneys (p = 0.253, Fig 6B, right panel). These data indicated that IL-17A produced in mice vaccinated with 4C-Staph/T7-alum in response to S. aureus i.p. infection is important to control dissemination of staphylococci to organs distant from the infection site.

Discussion
A vaccine that elicits a fast protective response against S. aureus infection would be highly desirable for patients with planned surgery or in intensive care units [1][2][3]. Appropriately adjuvanted 4C-Staph vaccine might achieve this goal. Therefore, we formulated 4C-Staph with a novel small molecule targeting TLR7 adsorbed to alum [24] to give 4C-Staph/T7-alum, and tested this vaccine formulation as single dose in S. aureus kidney abscess and peritonitis mouse models.
We showed that 4C-Staph/T7-alum vaccine reduced by more than 100-fold the bacterial burden in kidneys of mice infected i.v. and protected roughly 80% of vaccinated mice from the lethal outcome associated with an i.p. challenge, outperforming 4C-Staph/alum formulation. Remarkably, 91% of 4C-Staph/T7-alum vaccinated mice that survived the i.p. infection had no detectable staphylococci in kidneys as compared to 54% of 4C-Staph/alum vaccinated mice.  These observations on one hand showed that 4C-Staph/T7-alum promoted a more efficient control of bacteria than 4C-Staph/alum, and on the other hand highlighted that considering just survival rates as an index of S. aureus vaccine efficacy can lead to overestimated results. Indeed, the dichotomy between survival and bacterial burden has been recently highlighted by an elegant study by Maurer et al. [28].
The immunologic correlates of protection induced by 4C-Staph/T7-alum vaccine consisted in faster: (i) seroconversion of nearly 100% of vaccinated mice; (ii) production of antibodies specific for each of the vaccine antigens; (iii) induction of Hla-neutralizing antibodies; (iv) induction of vaccine-specific CD4 + T cells, a higher percentage of which produced IFN-γ, IL-17A, and TNF than those induced by 4C-Staph/alum.
Protection conferred by 4C-Staph/T7-alum was mediated by antibodies, as demonstrated by the lack of efficacy of the vaccine in B cell-deficient mice and the passive protection against S. aureus challenge achieved by transferring immune sera. Furthermore, depletion of effector CD4 + T cells in mice vaccinated with 4C-Staph/T7-alum, but not 4C-Staph/alum, resulted in an increase in renal bacterial burden upon i.v. or i.p. challenge as well as in reduction of survival rate upon i.p. challenge, indicating that effector CD4 + T cells' polarization towards Th1 and Th17 induced by 4C-Staph/T7-alum (but not 4C-Staph/alum) vaccination is likely to be crucial. Indeed, neutralization of IL-17A, alone or together with IFN-γ (but not IFN-γ alone) resulted in a 100-fold increase in bacterial load in kidneys of 4C-Staph/T7-alum-vaccinated mice. While not statistically significant, there was a trend for an increase in CFU in IFN-γ-neutralizing antibody-treated mice and a more marked increase in CFU in mice treated with the combination of IL-17A-and IFN-γ-neutralizing antibodies compared to IL-17A-treated mice, suggesting that IFN-γ might also play a role in protection. Neutralization of either cytokine, alone or in combination, had no effect on survival. This result together with the partial effect on survival of effector CD4 + T cell depletion as opposed to the dramatic effect of the lack of B cells/antibodies in J H mice indicated that antibodies are required at the moment of intraperitoneal infection to control bacterial growth and toxicity, giving time to effector CD4 + T cells to expand and exert their protective role.
The importance of the humoral response in protection against S. aureus infection is shown by preclinical as well as clinical data (reviewed in [26]). Passive transfer of antibodies raised against different staphylococcal antigens conferred partial protection against S. aureus in mouse models [8,14,29]. In humans, circulating antibodies to several S. aureus antigens are commonly found, particularly in colonized subjects, which present milder disease outcomes to systemic infections as compared to non-colonized patients (reviewed in [26]). In this study, passive vaccination protected all animals from death in the first days after i.p. challenge, while survival rates decreased afterwards, suggesting that a continuous supply of functional antibodies is needed, and/or mechanisms other than antibodies contribute to 4C-Staph/T7-aluminduced protection. The concept that antibodies alone are insufficient to protect against S. aureus infection is supported by the lack of efficacy of several passive and active immunization strategies in phase 3 clinical trials, despite high antibody titers were achieved [26,30].
The role of cell-mediated immunity and in particular of IL-17-producing cells, belonging either to the innate immunity (e.g. γδ T cells) or to the adaptive immunity (i.e. Th17), in (A-C, left panels) Survival was monitored for 15 days after challenge. Data are the merge of two independent experiments. No statistically significant differences between mice vaccinated with 4C-Staph/T7-alum treated with neutr. Ab or isot. ctr. (Log-rank test). (A-C, right panels) Fifteen days after S. aureus inoculation, survivors (n = 7-8) were euthanized, both kidneys homogenized and CFU enumerated. Each symbol represents a mouse. Mean ± SEM. One representative experiment out of two is shown. The dotted line indicates the lower limit of CFU detection. *p < 0.01; ***p < 0.001 by unpaired Student t test, one-tailed.
In the peritonitis model, IL-17A neutralization and CD4 + effector T cell-depletion equally increased the bacterial load in kidneys of mice vaccinated with 4C-Staph/T7-alum, suggesting that Th17 are the main source of IL-17A in these settings. The contribution of IL-17-producing cells other than Th17 deserves further investigation. The fact that effector CD4 + T cell depletion, but not IL-17A (or IFN-γ) neutralization, affected also mice survival indicated that other cytokines produced by CD4 + effector T cells might be important. IL-17F, which was produced by vaccine-specific CD4 + T cells (data not shown) and was not neutralized by the anti-IL-17A antibody, could be such a cytokine. IL-17F contributes to control S. aureus infection since double il-17a -/il-17f -/deficient mice, but not single il-17a -/or il-17f -/deficient mice, were more susceptible to opportunistic S. aureus infections [43]. TNF could also be important since it can not only synergize with IL-17A and IL-17F, which on their own are poor activators of signaling [44,45], but also prime neutrophils, rendering them faster and more efficient against pathogens [46,47].
In summary, we showed that one dose of 4C-Staph/T7-alum vaccine elicits a fast and efficacious protection against S. aureus systemic as well as peripheral infection through the induction of vaccine-specific functional antibodies, CD4 + effector T cells, and IL-17A. Cooperation between humoral and cell-mediated immunity is likely to be required to achieve efficacious protection against the broad spectrum of pathologies induced by S. aureus infections.
Supporting Information S1 Fig. Gating-tree for phenotypic and functional characterization of vaccine-specific CD4 + T cells by polychromatic intracellular flow cytometry. Splenocytes from single mice immunized by 12 days were stimulated or not with vaccine antigens (10 μg/ml each) in vitro. Splenocytes were then stained and analyzed by intracellular cytokine staining. Live cells were identified based on Live/Dead staining. Lymphocytes were gated based on their forward side scatter (FSC) vs. side scatter (SSC) profile. Singlets were gated based on their SSC properties. CD4 + CD44 high T cells were identified based on CD3, CD4, and CD44 expression. Inside the CD4 + CD44 high T-cell population, cells producing IL-2, TNF, IL-4/IL-13, IFN-γ or IL-17A were identified setting gates on non-stimulated cells (not shown). The dot plots refer to splenocytes of a representative mouse immunized with 4C-Staph/T7-alum stimulated in vitro with vaccine proteins. (TIF)