Nontoxigenic protein A vaccine for methicillin-resistant Staphylococcus aureus infections in mice

The contribution of the spa gene toward lethal S. aureus challenge has thus far not been appreciated. To address this, we generated the isogenic spa deletion variant S. aureus Newman Δspa. After intraperitoneal challenge with 2 × 10⁸ CFU of wild-type S. aureus Newman, 60% of animals succumbed to challenge. In contrast, animals infected with the isogenic mutant resulted in only 25% mortality (Fig. S1 A). In addition, the spa mutant displayed a consistent survival defect when examined in naive mouse blood (see Fig. 3 D). These results suggest that SpA is a crucial virulence factor for lethal infections of S. aureus in mice.

Guided by amino acid homology, the triple α-helical bundle structure of Ig binding domains (Deisenhofer, 1981), and their atomic interactions with Fab V_H3 (Graille et al., 2000) or Fcγ (Gouda et al., 1998), we selected glutamine 9 and 10, as well as aspartate 36 and 37, as critical for the association of SpA with immunoglobulin (Fig. 1, A and B; and Fig. S2, A and B). To test this, substitutions Gln⁹Lys, Gln¹⁰Lys, Asp³⁶Ala, and Asp³⁷Ala were introduced into the D domain to generate SpA-D_KKAA (Fig. 1 B). The ability of isolated SpA-D or SpA-D_KKAA to bind human IgG or IgM was analyzed by affinity chromatography and ELISA (Fig. 1, C and D). Polyhistidine-tagged SpA-D, as well as full-length SpA, retained human IgG on Ni-NTA, whereas SpA-D_KKAA or a negative control (sortase A; Mazmanian et al., 1999) did not (Fig. 1, C and D). A similar result was observed with vWF (Hartleib et al., 2000), which, along with TNFR1 (Gómez et al., 2004), can also bind SpA via glutamines 9 and 10 (Gómez et al., 2006; Fig. 1 D). Human Ig encompasses ∼50% V_H3-type IgG (Cook and Tomlinson, 1995). Human Fcγ and F(ab)₂ fragments, as well as IgM, all bound to full-length SpA or SpA-D but not to SpA-D_KKAA (Fig. 1 D). Injection of SpA-D into the peritoneal cavity of mice resulted in B cell expansion followed by apoptotic collapse of CD19⁺ (or CD45R⁺) lymphocytes in the spleen tissue of BALB/c or C57BL/6 mice (Goodyear and Silverman, 2003; Fig. 1 E). B cell superantigen activity was not observed after injection with SpA-D_KKAA, and TUNEL-staining of splenic tissue failed to detect the increase in apoptotic cells that follows injection of SpA or SpA-D (Fig. 1 E; and Fig. S2, E–G).

Naive BALB/c mice were injected with 50 µg each of purified SpA, SpA-D, or SpA-D_KKAA emulsified in CFA and boosted with the same antigen emulsified in IFA. IgG responses to immunization were examined by ELISA with SpA-D_GGSS and SpA-D_KKAA. SpA-D_GGSS harbors amino acid substitutions at the same positions as SpA-D_KKAA; however, glutamines 9 and 10 were each replaced with glycine and aspartic acids 36 and 37 with serine. Similar to SpA-D_KKAA, SpA-D_GGSS does not interact with human IgG (unpublished data). It is of note that similar antibody titers were measured with SpA-D_GGSS and SpA-D_KKAA antigen, indicating that the four amino acid substitutions do not diminish the reactivity of antibodies raised with heterologous antigens (IgG titers against SpA-D_KKAA vs. SpA-D_GGSS, P = 0.8315). After immunization of mice with either SpA-D or SpA-D_KKAA, we observed a 10-fold higher titer of SpA-specific antibodies for the nontoxigenic variant as compared with the B cell superantigen (P < 0.0001; Table I). Antibody titers raised by immunization with full-length SpA were higher than those elicited by SpA-D (P = 0.0022), which is likely a result of the larger size and reiterative domain structure of this antigen (Table I). Nevertheless, even SpA elicited lower antibody titers than SpA-D_KKAA (P = 0.0003), which encompasses only 50 aa of the mature 520-residue protein. Immunized mice were challenged by intravenous inoculation with S. aureus Newman, and the ability of staphylococci to seed abscesses in renal tissues was examined by necropsy 4 d after challenge (Cheng et al., 2009). In homogenized renal tissue of mock (PBS/adjuvant) immunized mice, a mean staphylococcal load of 6.46 log₁₀ CFU g⁻¹ was enumerated (Table I). Immunization of mice with SpA or SpA-D led to a reduction in staphylococcal load; however, SpA-D_KKAA–vaccinated animals displayed an even greater 3.07 log₁₀ CFU g⁻¹ reduction of S. aureus Newman in renal tissues (P < 0.0001; Table I). Abscess formation in kidneys was analyzed by histopathology (Fig. S3 A-H). Mock immunized animals harbored a mean of 3.7 (±1.2) abscesses per kidney (Table I). Vaccination with SpA-D_KKAA reduced the mean number of abscesses to 0.5 (±0.4; P = 0.0204), whereas immunization with SpA or SpA-D did not cause a significant reduction in the number of abscess lesions (Table I). Lesions from SpA-D_KKAA–vaccinated animals were smaller in size, with fewer infiltrating PMNs, and characteristically lacked staphylococcal abscess communities (Cheng et al., 2009; Fig. S3, A–H). Abscesses in animals that had been immunized with SpA or SpA-D displayed the same overall structure of lesions in mock immunized animals (Fig. S3, A–H).

We wondered whether SpA-D_KKAA immunization could protect mice against MRSA strains and selected the USA300 LAC isolate for animal challenge (Diep et al., 2006). This highly virulent CA-MRSA strain spread rapidly throughout the United States, causing significant human morbidity and mortality. Compared with adjuvant control mice, SpA-D_KKAA–immunized animals harbored a 1.20 log₁₀ CFU g⁻¹ reduction in bacterial load of infected kidney tissues. Histopathology examination of renal tissue after S. aureus USA300 challenge revealed that the mean number of abscesses was reduced from 4.0 (±0.8) to 1.6 (±0.6; P = 0.0277). In contrast, SpA or SpA-D immunization did not cause a significant reduction in bacterial load or abscess formation (Table I; and Fig. S3, I–L and N–Q).

Rabbits were immunized with SpA-D_KKAA, and specific antibodies were purified on SpA-D_KKAA affinity column and analyzed by SDS-PAGE (Fig. 2 A, lane 1). SpA-D_KKAA–specific IgG was cleaved with pepsin to generate Fcγ and F(ab)₂ fragments (Fig. 2 A, lane 2), the latter of which were repurified by affinity chromatography on SpA-D_KKAA column (Fig. 2 A, lane 3). Binding of human IgG or vWF to SpA or SpA-D was perturbed by SpA-D_KKAA–specific F(ab)₂, indicating that SpA-D_KKAA–derived antibodies can block the Fcγ- and vWF-binding properties of SpA (Fig. 2, B and C).

To further improve the vaccine properties for nontoxigenic SpA, we generated SpA_KKAA, which includes all five Ig binding domains with four amino acid substitutions—Gln⁹Lys, Gln¹⁰Lys, Asp³⁶Ala, and Asp³⁷Ala—in each of its five domains (E, D, A, B, and C; Sjödahl, 1977). Polyhistidine-tagged SpA_KKAA was purified by affinity chromatography and analyzed by Coomassie blue–stained SDS-PAGE (Fig. 3 A). Unlike full-length SpA, SpA_KKAA did not bind human IgG, Fc and F(ab)₂, IgM, or vWF (Fig. 3 B). SpA_KKAA failed to display B cell superantigen activity, as injection of the variant into BALB/c or C57BL/6 mice did not cause a depletion of both CD19⁺ and CD45R⁺ B cells in splenic tissue (Fig. 3 C; Fig. S2, C and D; and not depicted). SpA_KKAA immunization generated higher specific antibody titers than SpA-D_KKAA and provided mice with elevated protection against S. aureus USA300 (Table I) or Mu50 challenge (Fig. S3 U; Mu50 is a Japanese MRSA isolate). 4 d after challenge, SpA_KKAA-vaccinated animals harbored 3.54 log₁₀ CFU g⁻¹ fewer staphylococci in renal tissues (P = 0.0001) and also caused a greater reduction in the number of abscess lesions (P = 0.0109; Table I; and Fig. S3, I–R). Furthermore, SpA_KKAA immunization reduced the mortality of mice that received a lethal S. aureus challenge dose (Fig. S1 B).

Staphylococci persist in mouse organ tissues (Cheng et al., 2009). Over time, abscesses harboring communities of the infectious agent increase in size and eventually rupture, thereby releasing staphylococci into circulation. This initiates the formation of new abscesses and precipitates lethal outcomes 30–60 d after challenge (Cheng et al., 2009). We asked whether SpA-D_KKAA or SpA_KKAA immunization prevents staphylococcal replication over a longer period of time. 15 d after challenge with S. aureus USA300, immunization with SpA did not generate significant protection of animals compared with the mock control (P = 0.5817; Table S1). In contrast, immunization with SpA-D_KKAA caused a 1.56 log₁₀ CFU g⁻¹ reduction (P = 0.0183), whereas SpA_KKAA vaccination reduced the load by 2.7 log₁₀ CFU g⁻¹ (P = 0.0059). These data suggest that antibodies against SpA, generated via active immunization using nontoxigenic SpA_KKAA, can interfere with bacterial persistence in host tissues.

SpA_KKAA was used to immunize rabbits. Rabbit antibodies specific for SpA-D_KKAA or SpA_KKAA were affinity purified on matrices with immobilized cognate antigen and injected at a concentration of 5 mg × kg⁻¹ body weight into the peritoneal cavity of BALB/c mice (Table II). 24 h later, antibody titers specific for SpA-D_KKAA/SpA_KKAA were determined in serum and animals challenged by intravenous inoculation with S. aureus Newman. Passive transfer reduced the staphylococcal load in kidney tissues for SpA-D_KKAA- (P = 0.0016) or SpA_KKAA (P = 0.0005)-specific antibodies. On histopathology examination, both antibodies reduced the abundance of abscess lesions in kidneys of mice challenged with S. aureus Newman (Table II). Compared with control cohorts treated with nonspecific antibody (α-V10), animals that had been injected with SpA-specific antibodies were protected against the B cell superantigen activity of SpA (Fig. S2, C and D). In addition, SpA-specific antibodies (2 µg × ml⁻¹) induced opsonophagocytic clearance of S. aureus Newman inoculated into naive mouse blood (Fig. 3 D) and reduced the mortality associated with lethal staphylococcal challenge (Fig. S1 C). Together these data reveal that disease protection after immunization with SpA-D_KKAA or SpA_KKAA is conferred by antibodies that bind SpA and neutralize its ability to bind Ig.

After infection with virulent S. aureus Newman and clearance of the pathogen with antibiotic treatment, mice do not develop protective immunity against subsequent infection with the same strain (Fig. S3 S). The mean abundance of SpA-D_KKAA–specific IgG in these animals was determined by dot blot as 0.20 µg ml⁻¹ (±0.04) and 0.14 µg ml⁻¹ (±0.01) for infections caused by S. aureus strains Newman and USA300 LAC, respectively (Fig. 4 A). A concentration of 4.05 µg ml⁻¹ (±0.88) for SpA-specific IgG was estimated to confer disease protection in SpA_KKAA-or SpA-D_KKAA–immunized mice (P ≤ 0.05 log₁₀ reduction in staphylococcal CFU g⁻¹ renal tissue; unpublished data). The mean serum concentration of SpA-specific IgG in adult healthy human volunteers (n = 16) was 0.21 µg ml⁻¹ (±0.02). Such antibody concentration may not be sufficient to generate protection against staphylococcal infection. By comparison, the mean serum concentration of IgG specific for diphtheria toxin in human volunteers, 0.68 µg ml⁻¹ (± 0.20), is thought to be within range for protective immunity against diphtheria (Lagergård et al., 1992).

These results are in agreement with a model of immune evasion during S. aureus infection. Cell wall–anchored or secreted SpA (e.g., 20% of peptidoglycan and attached surface protein is released during bacterial division; Ton-That et al., 1999) activate B cells via IgM receptor cross-linking. Without stimuli from specific antigens, activated B cells undergo apoptosis, thereby hindering the production of antibody against staphylococcal antigens. If so, neutralizing antibodies directed against SpA may enable humoral immune responses against many different staphylococcal antigens. This was tested by immunizing BALB/c mice with SpA_KKAA or an adjuvant (aluminum hydroxide) control, followed by intravenous challenge with a sublethal dose of MRSA strain USA300. Serum samples were withdrawn 30 d after MRSA challenge and then analyzed by immunoblotting with 27 staphylococcal antigens immobilized on a membrane filter (Fig. 4 B). Naive mice, which had not been infected with the MRSA strain USA300 LAC, did not harbor antibodies against staphylococcal antigens (unpublished data). Mock immunized mice (adjuvant only) that had been subjected to USA300 infection developed high-titer antibodies against the Eap protein as well as low-titer antibodies against Hla, IsdA, IsdB, LukD, LukE, and LukF (Fig. 4 B). In response to USA300 challenge, animals that had been immunized with SpA_KKAA (IgG titer 2,907 [±357]; P < 0.001, SpA_KKAA vs. mock) mounted humoral immune responses against every antigen examined (Fig. 4 B). With the exception of Eap, IsdA, and IsdB antibodies, the serum of SpA_KKAA-immunized animals harbored higher antibody titers against staphylococcal antigens as compared with mice that had been naive at the time of challenge (Fig. 4 B).

In summary, S. aureus isolates express SpA, an essential virulence factor whose B cell superantigen activity and evasive attributes toward opsonophagocytic clearance are required for staphylococcal abscess formation and the establishment of lethal disease (Cheng et al., 2009). SpA can be thought of as a toxin that is essential for pathogenesis and whose molecular attributes must be neutralized to achieve protective immunity. By generating nontoxigenic variants unable to bind Igs via Fcγ or V_H3-Fab domains, we identified SpA-neutralizing immune responses as a correlate for protective immunity against S. aureus infection. In contrast to many methicillin-sensitive strains, the CA-MRSA isolate USA300 LAC is significantly more virulent (Cheng et al., 2009). For example, immunization of experimental animals with the surface protein IsdB (Kuklin et al., 2006) raises antibodies that confer protection against S. aureus Newman (Kuklin et al., 2006) but not against USA300 challenge (Fig. S3 T). In contrast, neutralizing SpA antibody responses generate protection against strains of the current MRSA epidemic. These antibodies exert at least two functions. As shown in Fig. 3, the SpA antibodies enable phagocytic killing of staphylococci in blood. Moreover, by neutralizing B cell superantigen activity, SpA antibodies enable the development of humoral immune responses to many different antigens that, assuming synergism, may together contribute toward the establishment of immunity. In agreement with this, the SpA_KKAA vaccine elicited greater protection against abscess formation, which monitors infected animals over a prolonged period of time (Fig. 4), as compared with the lethal challenge, when most animals die within 1–2 d (Fig. S1).

5 mg of protein was covalently linked to HiTrap NHS-activated HP and loaded with rabbit serum. Antibodies were eluted with 1 M glycine, pH 2.5, and 0.5 M NaCl, neutralized with 1 M Tris-HCl, pH 8.5, and dialyzed against PBS. Affinity-purified antibodies were mixed with 3 mg pepsin at 37°C for 30 min and quenched with 1 M Tris-HCl, pH 8.5. F(ab)₂ fragments were again affinity purified, dialyzed against PBS at 4°C, separated by 15% SDS-PAGE, and visualized with Coomassie Blue.

The coding sequence for SpA was PCR amplified with two primers, 5′-GCTGCACATA­TGGCGCAACAC­GATGAAGCTCAAC-3′ and 5′-AGTGGATCCT­TATGCTTGAGC­TTTGTTAGCAT­CTGC-3′, using S. aureus Newman DNA. SpA-D was PCR amplified with two primers: 5′-AACATATGTTCAACAAAGATCAACAAAGC-3′ and 5′-AAGGATCCAGATTCGTTTAATTTTTTAGC-3′. The sequence for SpA-D_KKAA was mutagenized with two sets of primers: 5′-CATATGTTCA­ACAAAGATAAA­AAAAGCGCCTT­CTATGAAATC-3′ and 5′-GATTTCATAG­AAGGCGCTTTT­TTTATCTTTGT­TGAACATATG-3′ for Q9K and Q10K, and 5′-CTTCATTCAA­AGTCTTAAAGC­CGCCCCAAGCC­AAAGCACTAAC-3′ and 5′-GTTAGTGCTT­TGGCTTGGGGC­GGCTTTAAGAC­TTTGAATGAAG-3′ for D36A and D37A. The sequence of SpA_KKAA was synthesized by Integrated DNA Technologies, Inc. PCR products were cloned into pET-15b generating N-terminal His₆-tagged recombinant protein. BALB/c mice were immunized by intramuscular injection and boosted with the same antigen after 11 d. On day 20, mice were bled to obtain serum for specific antibody titers. Affinity-purified antibodies were injected into the peritoneal cavity of BALB/c mice at 4–24 h, and blood was collected for specific antibody titers before challenge.

Staphylococci were used to infect anesthetized mice by retroorbital injection (1 × 10⁷ CFU S. aureus Newman, 5 × 10⁶ CFU S. aureus USA300, or 3 × 10⁷ CFU S. aureus Mu50). On day 4, 15, or 30, mice were killed, kidneys removed, and homogenized tissue spread on agar for colony formation. Animal experiments were performed in agreement with the institutional guidelines according to experimental protocol review and approval by the Institutional Biosafety Committee and the Institutional Animal Care and Use Committee at the University of Chicago.

As previously described (Thammavongsa et al., 2009), whole blood was collected from BALB/c mice with 5 µg ml⁻¹ of lepirudin anticoagulant. 50 µl of 5 × 10^4–6 CFU ml⁻¹ S. aureus Newman were mixed with 950 µl of mouse blood. Samples were incubated at 37°C with slow rotation for 30 min and then incubated on ice with 1% saponin/PBS. Dilutions of staphylococci were plated on agar for colony formation.

1 µg ml⁻¹ of purified SpA and its variants were coated onto ELISA plates in 0.1 M carbonate buffer, pH 9.5. Plates were incubated with peroxidase-conjugated human IgG, Fc, or F(ab)₂ fragments, IgM (The Jackson Laboratory), and vWF (Thermo Fisher Scientific) and developed using OptEIA reagent. For inhibition, plates were incubated with either naive rabbit F(ab)₂ fragments (The Jackson Laboratory) or 10 µg ml⁻¹ of affinity-purified F(ab)₂ fragments before ligand binding.

150 µg of purified protein was injected into the peritoneum of 6-wk-old BALB/c. 4 h after injection, animals were killed and spleens removed and homogenized. Red blood cells were lysed in ACK buffer. White blood cells were stained with R-PE–conjugated anti-CD19 (eBioscience). Cells were washed and fixed with formalin and analyzed by FACSCanto (BD).

Nitrocellulose membrane was blotted with human/mouse IgG (The Jackson Laboratory), SpA_KKAA, and CRM₁₉₇, blocked, and incubated with either human or mouse sera. IRDye 700DX–conjugated anti–human/mouse IgG (Rockland Immunochemicals, Inc.) was used to quantify signal intensities from healthy human volunteers or mice using the Odyssey infrared imaging system (LI-COR Biosciences). Experiments with blood from human volunteers involved protocols that were reviewed, approved, and performed under regulatory supervision of The University of Chicago’s Institutional Review Board. For the staphylococcal antigen matrix, nitrocellulose membrane was blotted with 2 µg of a collection of Ni-NTA affinity-purified recombinant His₆-tagged staphylococcal proteins. Signal intensities in mouse sera were quantified and normalized using anti-His₆ antibody with the Odyssey.

Unpaired two-tailed Student’s t tests were performed to analyze the statistical significance of renal abscess, ELISA, and B cell superantigen data.

Fig. S1 shows that SpA is a virulence factor for lethal infection after intraperitoneal injection of S. aureus Newman into BALB/c mice and that active or passive immunization with antibodies raised against SpA_KKAA can protect against this disease. Fig. S2 shows that SpA_KKAA, unlike wild-type SpA, does not induce B cell apoptosis in mice and that antibodies raised against SpA_KKAA neutralize the B cell superantigen attributes of SpA. Fig. S3 shows that immunization of mice with SpA_KKAA prevents multiple S. aureus strains from inducing renal abscess formation in mice and that prior infection with S. aureus does not elicit immunity to subsequent infection with the same strain.

We thank Matt Frankel and Vilasack Thammavongsa for experimental assistance and others members of our laboratory for discussion.

This work was supported by grants from the National Institute of Allergy and Infectious Diseases, Infectious Diseases Branch (AI52474 to O. Schneewind and AI75258 to D.M. Missiakas) and by Novartis Vaccines and Diagnostics (Siena, Italy). A.G. Cheng was a trainee of the National Institutes of Health (NIH) Medical Scientist Training Program at The University of Chicago (GM07281). D.M. Missiakas and O. Schneewind acknowledge membership within and support from the Region V Great Lakes Regional Center of Excellence in Biodefense and Emerging Infectious Diseases Consortium (NIH Award 1-U54-AI-057153).

The authors have no conflicting financial interests.