Vaccine 30 (2012) 5099–5109
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journal homepage: www.elsevier.com/locate/vaccine
Immunity to Staphylococcus aureus secreted proteins protects rabbits from
Adam R. Spauldinga,c, Ying-Chi Linb,1, Joseph A. Merrimana,c, Amanda J. Brosnahana,
Marnie L. Petersonb, Patrick M. Schlieverta,c,∗
aDepartment of Microbiology, Medical School, University of Minnesota, Minneapolis, MN 55455, USA
bDepartment of Experimental and Clinical Pharmacology, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455, USA
cDepartment of Microbiology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA
a r t i c l e i n f o
Received 22 January 2012
Received in revised form 23 May 2012
Accepted 25 May 2012
Available online 9 June 2012
a b s t r a c t
Staphylococcus aureus causes significant illnesses throughout the world, including toxic shock syndrome
(TSS), pneumonia, and infective endocarditis. Major contributors to S. aureus illnesses are secreted vir-
ulence factors it produces, including superantigens and cytolysins. This study investigates the use of
superantigens and cytolysins as staphylococcal vaccine candidates. Importantly, 20% of humans and 50%
of rabbits in our TSS model cannot generate antibody responses to native superantigens. We generated
three TSST-1 mutants; G31S/S32P, H135A, and Q136A. All rabbits administered these TSST-1 toxoids
generated strong antibody responses (titers>10,000) that neutralized native TSST-1 in TSS models, both
in vitro and in vivo. These TSST-1 mutants lacked detectable residual toxicity. Additionally, the TSST-1
mutants exhibited intrinsic adjuvant activity, increasing antibody responses to a second staphylococ-
cal antigen (?-toxin). This effect may be due to TSST-1 mutants binding to the immune co-stimulatory
molecule CD40. The superantigens TSST-1 and SEC and the cytolysin ?-toxin are known to contribute to
strains are common causes of staphylococcal infections. The same three exotoxins plus the cytolysins ?-
against these five exotoxins protected rabbits from infective endocarditis and lethal sepsis. These data
suggest that immunization against toxoid proteins of S. aureus exotoxins protects from serious illnesses,
and concurrently superantigen toxoid mutants provide endogenous adjuvant activity.
© 2012 Elsevier Ltd. All rights reserved.
Staphylococcus aureus is a major pathogen worldwide, respon-
sible for significant illnesses, many of which are life threatening
major histocompatibility complex; MRSA, methicillin-resistant S. aureus; PBS,
phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel
electrophoresis; SE, staphylococcal enterotoxin; TSS, toxic shock syndrome; TSST-
1, TSS toxin-1; V?-TCR, variable part of the ?-chain of the T cell receptor; HVECs,
human vaginal epithelial cells; KSFM, keratinocyte serum-free medium.
Medicine, 51 Newton Road, 3-403 BSB, Iowa City, IA 52252, USA.
Tel.: +1 319 335 7807; fax: +1 319 335 9006.
E-mail addresses: email@example.com (A.R. Spaulding),
firstname.lastname@example.org (Y.-C. Lin), email@example.com (J.A. Merriman),
1Present address: School of Pharmacy, College of Pharmacy, Kaohsiung Medical
University, N612, 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan.
Tel.: +886 7 3121101x2012.
CFUs, colony-forming units; LPS, lipopolysaccharide; MHC,
Corresponding author at: Department of Microbiology, Carver College of
such as toxic shock syndrome (TSS), infective endocarditis, sepsis,
and pneumonia [1,2]. S. aureus has the ability to cause a wide vari-
ety of infections by production of numerous virulence factors, both
cell-surface and secreted exoproteins [1,2]. Treatment of S. aureus
infections can be challenging and expensive, especially with the
high occurrence of antibiotic resistant infections, such as caused
by methicillin-resistant S. aureus (MRSA) .
Infective endocarditis is a life threatening infection of the
heart endothelium caused by many organisms [4,5]. In the past
decade, S. aureus has emerged as a primary cause of infective
endocarditis throughout the world, largely in elderly patients and
tion of large “cauliflower-like” vegetations on the endothelium of
the heart. These vegetations are composed of host factors (tissue
factor, fibronectin, and fibrinogen) and host cells, as well as micro-
bial colonies. Infective endocarditis is difficult to treat, and there
are many risks associated with the illness, including cardiac fail-
ure, embolisms, renal dysfunction, and mycotic aneurysms [4,5].
Treatment of S. aureus infective endocarditis typically requires
0264-410X/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
A.R. Spaulding et al. / Vaccine 30 (2012) 5099–5109
extensive antibiotic regimens, often lasting ≥6 weeks, and many
times surgery is required [4,5,7,8].
Although cell-surface virulence factors are critical for S. aureus
attachment and vegetation initiation, recent research has also
implicated secreted virulence factors as major contributors to
infective endocarditis progression with S. aureus. Pragman et al.
tant for infective endocarditis vegetation formation caused by
strains that produce the superantigen . In a rabbit model, the
researchers showed that strains producing native TSST-1 had sig-
nificantly larger vegetation sizes and increases of nearly 7logs
colony-forming units (CFUs)/vegetations compared to isogenic
strains lacking TSST-1. We have further noted that strains lack-
ing superantigens do not induce infective endocarditis in rabbits
. A research group recently published a study examining the
genotype of strains isolated from infective endocarditis patients
with persistent bacteremia and observed that the majority of them
are pulsed-field gel electrophoresis type USA200 and carried the
tstH gene that encodes TSST-1 ; there is a one:one correla-
tion between the presence of tstH and TSST-1 protein production.
Additionally, it has been published that 90% of infective endocardi-
tis cases are associated with USA200 strains and production of
TSST-1 . These studies collectively suggest that TSST-1 is highly
important for S. aureus in its ability to cause infective endocarditis.
Recent studies from Mattis et al. showed that another superanti-
gen, staphylococcal enterotoxin (SE) C, is highly important for
infective endocarditis caused by strains that produce that super-
antigen (Mattis DM, Spaulding AR, Chuang-Smith ON, Sundberg
EJ, Schlievert PM, Kranz DM. Enterotoxin C contributes to USA400
methicillin-resistant S. aureus infective endocarditis in rabbits.
treated rabbits with a specific SEC inhibitor after challenge with a
strain known to cause infective endocarditis at a high level in the
rabbit model, the microbes were significantly reduced in ability
to cause disease. Studies have also shown that secreted cytolysins
contribute to infective endocarditis. Huseby et al. recently pub-
lished that the cytolysin ?-toxin facilitates infective endocarditis
progression . Cheung et al. showed that a S. aureus mutant
that no longer produced ?-toxin, ?-toxin, ?-toxin, and ?-toxin
was drastically reduced in its ability to cause infective endocarditis
, although because these studies used a regulatory mutant for
their studies, numerous other factors may also have contributed to
reduced ability to cause illness.
tant information on the role of exoproteins in lethal sepsis, since
S. aureus is administered intravenously in high concentrations. Our
prior studies strongly suggest that superantigens are important in
lethal sepsis .
We and others have shown that superantigens and cytolysins
are critical determinants of staphylococcal pneumonia [15–18].
Rabbits actively immunized against TSST-1 and SEC, and animals
pulmonary S. aureus challenge . In addition, mice immunized
against ?-toxin are protected from lethal pneumonia .
These data led us to consider the possibility of a vaccine against
serious S. aureus infections using the major secreted virulence fac-
tors (cytolysins and superantigens) as immunizing agents. Here we
report our studies related to TSST-1, SEC, ?-toxin, ?-toxin, and
?-toxin, alone and in combination for protection against staphy-
lococcal pneumonia, infective endocarditis, and sepsis. Our studies
show that vaccines containing these important secreted virulence
factors lead to immunity that protects against illness and increases
survival. Additionally, TSST-1 mutant toxoids have endogenous
adjuvant activities, dependent on interaction with the immune co-
stimulatory molecule CD40 that amplifies immune responses to
2. Materials and methods
2.1. Bacterial strains and growth
S. aureus strain RN4220 containing plasmids encoding TSST-1,
TSST-1 mutants, or SEC were used as sources of TSST-1, TSST-1
mutants, or SEC, respectively [19–21]. Strain RN4220 does not pro-
duce detectable endogenous superantigens. RN4220 was also used
as the source of ?-toxin . S. aureus strain MNPE was the source
of native ?-toxin . Escherichia coli clones were the sources
of mutant ?-toxin (H35L), as provided by Dr. Juliane Bubeck-
Wardenburg, University of Chicago, and ?-toxin as expressed from
a pET vector . S. aureus strain MNPE was used in microbial chal-
lenge studies; this organism caused a fatal case of post-influenza
TSS in Minnesota . This organism is USA200; these organ-
isms cause the majority of TSS cases . MNPE has the following
secreted virulence factor phenotype of importance for our studies:
TSST-1high+, SEChigh+, ?-toxinhigh+, ?-toxinhigh+, and ?-toxin+.
For use in pneumonia and infective endocarditis/sepsis studies, the
oratories, Detroit, MI) broth at 37◦C with shaking at 200rpm under
standard air conditions . The organism was washed one time
with phosphate-buffered saline (PBS; 0.005M sodium phosphate,
pH 7.2; 0.15M NaCl) through centrifugation at 14,000×g, 5min,
and then resuspended in Todd Hewitt medium at 2×109/0.2ml
volume for high-dose injection in pneumonia studies , and in
PBS at 1×108/ml, with 2ml being injected intravenously for infec-
tive endocarditis/sepsis studies .
2.2. Secreted virulence factor purification
All reagents used in preparation of superantigens were main-
tained pyrogen-free. For production of TSST-1, TSST-1 toxoids, SEC,
native ?-toxin, and native ?-toxin, the organisms were grown
overnight in dialyzed beef-heart media . TSST-1, TSST-1 tox-
oids, SEC and ?-toxin were precipitated from culture fluids with
4 volumes of absolute ethanol for two days (80% final concen-
tration), resolubilized in distilled water, and then purified by
thin-layer isoelectric focusing. Isoelectric focusing pH gradients
were pH 3.5–10 for initial separation, followed by gradients of
pH 6–8 for TSST-1, TSST-1 toxoids, and ?-toxin and 7–9 for SEC
and ?-toxin . Native ?-toxin was produced comparably from S.
aureus MNPE, except the toxin was precipitated from culture flu-
ids with 80% final saturation of ammonium sulfate, followed by
solubilization in distilled water and three days dialysis, and then
followed by isoelectric focusing. The biologically inactive mutant
of ?-toxin (H35L) and an enriched preparation of ?-toxin were
produced from E. coli clones in pET vectors and purified on nickel
columns . TSST-1, TSST-1 mutants, and SEC were homoge-
neous when tested by sodium dodecyl sulfate polyacrylamide gel
ative for contaminating lipopolysaccharide (LPS), peptidoglycan,
cytolysins, lipase, and proteases. Native ?-toxin was further puri-
fied by reversed-phase high-performance liquid chromatography
and was homogeneous . The ?-toxin mutant H35L and ?-toxin,
as produced in E. coli contained minor E. coli contaminates that did
not affect experimentation. Purified toxins were quantified using
the BioRad protein assay .
2.3. Production of TSST-1 mutants
Three site-specific mutants of TSST-1 were prepared through
use of the Quikchange method (Stratagene, La Jolla, CA). The initial
plasmid was native tstH, on a shuttle plasmid pCE104, cloned into
E. coli . After performing mutagenesis, the resultant plasmids
A.R. Spaulding et al. / Vaccine 30 (2012) 5099–5109
were cloned first into E. coli, verified to have the correct TSST-
1 mutations by sequencing the entire structural genes, and then
cloned into S. aureus RN4220 for production and purification. The
mutants were TSST-1 (G31S/S32P) that fails to interact with major
histocompatibility complex (MHC) class II molecules [20,29,30],
H135A that fails to bind the variable part of the ? chain of the T
cell receptor (V?-TCR) [20,29,30], and Q136A that also fails to bind
to V?-TCR [20,29,30].
by the University of Minnesota Institutional Animal Care and Use
bits, male and female, weighing 1–2kg were used for pneumonia
studies. New Zealand white rabbits, male and female, weighing
2–3kg were used for endocarditis/sepsis studies. All rabbits were
purchased from Bakkom Rabbitry, Red Wing, MN.
nized against biologically inactivated proteins (TSST-1 and ?-toxin
mutants) by emulsifying 25?g of each alone or in combination in
PBS with an equal volume of Freund’s incomplete adjuvant. Immu-
nizations were in multiple subcutaneous sites in the nape of the
necks. Native toxins (TSST-1, SEC, ?-toxin, ?-toxin, and ?-toxin)
were used at a dose of approximately 10?g/ml for immunization
following the same protocol. Immunizations for all experiments
were every-other-week (days 0, 14, and 28) for three injections.
One week after the last immunization of animals, blood was drawn
from the marginal ear veins, sera collected, and antibody titers
determined by ELISA. In one experiment, following the initial three
injections, animals were immunized monthly for up to six months.
One week after each immunization, blood was drawn from the
marginal ear veins, sera collected, and antibody titers determined
2.6. ELISA antibody quantification
Enzyme-linked immunosorbent assays (ELISAs) were used to
determine antibody titers of immunized animals as described pre-
Portsmouth, NH) were coated with 1.0?g/well of purified native
homologous superantigen or cytolysin and then washed. Rabbit
serum samples were serially diluted 2-fold beginning with a 1:10
perature, and then washed. Horseradish peroxidase-conjugated
anti-rabbit IgG antibodies (Sigma–Aldrich, St. Louis, MO) were
added to the wells. The plates were again incubated for a minimum
of 1.5h, and the wells were washed. The relative levels of IgG were
determined by 100?l/well addition of an o-phenylenediamine and
H2O2substrate. Colorimetric reactions were halted by the addition
of 50?l of a 12.5% sulfuric acid solution. Plates were scanned for
absorbance at 490nm wavelength using a spectrophotometer.
2.7. TSST-1 mutant toxicity studies
Three assays were used to assess residual toxicity of TSST-
1 mutants that are proposed for use as toxoids. Mutants
tested included TSST-1 (G31S/S32P), TSST-1 (H135A), and TSST-1
(Q136A). Three assays were used.
TSST-1 amplifies the lethal effects of LPS by as much as 106-fold
. Our prior studies in rabbits indicate the LD50of LPS alone in
Dutch-belted rabbits is 500?g/kg given intravenously . Addi-
tionally, bolus administration of as much as 2mg/kg of TSST-1
alone intravenously to rabbits is not lethal; TSST-1 is more lethal
when continuous exposure occurs over several days [32,33]. The
relationship between TSST-1 and LPS in this enhancement phe-
nomenon is log:log, such that for each 10-fold increase in TSST-1
pre-treatment, the amount of LPS administered 4h later to kill
the animals is reduced by 10-fold. Thus, we used 500?g/kg native
TSST-1 or the three TSST-1 mutants for pre-treatment of 5 Dutch-
belted rabbits/group intravenously, followed by 100?g/kg of LPS
laboratory by the hot-phenol method , at the 4h time-point.
Fevers were recorded at the 4h time-point, just prior to adminis-
tration of LPS compared to pre-injection of TSST-1 or mutants, and
deaths were recorded over a 48h time-period. If mutants lacked
lethality, this would indicate they were >500,000-fold inactivated
TSST-1 alone is lethal to rabbits when administered in subcuta-
model is 75?g/animal (11?g/day). Native TSST-1 and each TSST-
1 mutant (1000?g/animal; 143?g/day or 10× lethal dose) were
administered in miniosmotic pumps (Alza Corporation, Vacaville,
CA) to 5 rabbits/group [32,33]. Pumps were implanted while ani-
mals were anesthetized with ketamine (25mg/kg) and xylazine
(25mg/kg) (Phoenix Pharmaceuticals, Burlingame, CA). Rabbits
were monitored for 7 days for the development of TSS symp-
toms (fever, diarrhea, reddening of conjunctivae, and evidence of
hypotension) and lethal illness, defined as the point 100% pre-
dictive of impending death, including simultaneous failure of the
animals to remain upright and failure to exhibit flight responses.
Animals were euthanized with intravenous injection of 1ml/kg of
Beuthanasia-D (Shering-Plough, Westlake, TX). Surviving rabbits
were euthanized at the end of 7 days.
The most sensitive measure of TSST-1 toxicity in vitro is tests of
superantigenicity in a 4-day assay . Native TSST-1 is superanti-
for superantigenicity with use of rabbit splenocytes in dose ranges
of 10–10−8?g/well. Proliferation was measured by incorporation
of3H-thymidine into DNA .
2.8. Immunization against TSST-1 mutant protein protection of
rabbits from native TSST-1
We examined the ability of TSST-1 (G31S/S32P), TSST-1
nized three times, every-other-week, with the individual TSST-1
mutants, and then these rabbits and control, non-immunized ani-
mals (5/group) were challenged with otherwise lethal doses of
native TSST-1, either (10?g/kg) plus LPS (10?g/kg) intravenously
(5000× LD50)  or alone (500?g/kg) in miniosmotic pumps
(71?g/day; 5.5× LD50) [32,33].
2.9. In vitro test of antibodies against TSST-1 mutants to
neutralize native TSST-1
Pre- and post-immunization sera from 10 rabbits per group
immunized three times with TSST-1 (G31S/S32P), TSST-1 (H135A),
and TSST-1 (Q136A) were pooled and tested for capacity to neu-
tralize superantigenicity of native TSST-1 (1?g/well) with use of
rabbit splenocytes in a standard 4 day assay .
2.10. Rabbit pulmonary illness model
Dutch-belted rabbits were administered MNPE (2×109colony-
forming units [CFUs] in 0.2ml volumes) via intra-tracheal
inoculation as described previously . Briefly, rabbits were
A.R. Spaulding et al. / Vaccine 30 (2012) 5099–5109
anesthetized with subcutaneous injections of ketamine (25mg/kg)
and xylazine (25mg/kg) (Phoenix Pharmaceuticals, Burlingame,
CA). Their necks were shaved, and small incisions were made to
expose the tracheas. Small (3mm) incisions were made into the
tracheas before inserting 1mm diameter polyethylene catheters
(Becton, Dickinson, and Co., Sparks, MD) and threading them into
the left bronchi. MNPE was administered through the catheters,
and then catheters removed and incision sites closed. Rabbits were
monitored for 7 days for the development of TSS symptoms (fever,
diarrhea, reddening of conjunctivae, and evidence of hypoten-
sion) and lethal illness, defined as the point 100% predictive of
impending death, including simultaneous failure of the animals to
euthanized with intravenous injection of 1ml/kg of Beuthanasia-D
(Shering-Plough, Westlake, TX). Surviving rabbits were euthanized
at the end of 7 days.
2.11. Rabbit infective endocarditis and sepsis model
New Zealand white rabbits were used for the rabbit model
of infective endocarditis and sepsis, as previously described .
Briefly, the rabbits were anesthetized with ketamine (25mg/kg)
and xylazine (25mg/kg). Incisions were made on the left side of the
necks to expose the left common carotid arteries. Catheters were
inserted into the left carotid arteries and threaded until against the
aortic valves, where they remained in place for 2h to induce dam-
age to the endothelia. After 2h, the catheters were removed and
the surgical sites closed. Doses of 2×108CFUs of S. aureus MNPE
were injected into the marginal ear veins. Because the animals
were injected intravenously, we were able to monitor progres-
sion to lethal sepsis as well as infective endocarditis. Rabbits were
monitored for 4 days for signs of illness and lethality, as described
above. At the time of impending death or after 4 days, rabbits
were euthanized. Hearts were removed and examined for vege-
tations. If vegetations were observed, they were excised, weighed,
were not present, scrapings of the aortic valves were taken, serially
diluted, and plated.
2.12. CD40 and antibodies
Purified CD40 and CD40 ligand (CD154) were purchased from R
and D Systems, Minneapolis, MN. Monoclonal antibodies that neu-
were also purchased from R and D Systems.
2.13. Human vaginal epithelial cells (HVECs)
HVECs from a pre-menopausal woman were described previ-
ously . A second HVEC line was purchased from ATCC. HVECs
were cultured in keratinocyte serum-free medium (KSFM) with
to lack MHC II molecules on their surfaces.
2.14. TSST-1 binding to CD40 by Western immunoblotting
CD40 (2?g/lane) and control protein (ovalbumin; 2?g) were
electrophoresed in non-denaturing PAGE and then transblotted
onto PVDF membranes . Membranes were blocked by addi-
tion of 1% bovine serum albumin and 1% human serum for 30min.
Subsequently, 0.033–33?g/ml of TSST-1, TSST-1 (Q136A), or TSST-
1 (G31S/S32P) was incubated with the membranes for 24h at
room temperature. The membranes were then washed and incu-
bated successively with rabbit antibodies against TSST-1, alkaline
phosphatase-conjugated antibodies against rabbit IgG, and finally
substrate, with washing between steps.
2.15. Kd determination for TSST-1 binding to CD40
Various concentrations of TSST-1, ranging from 0.033?g/ml to
33?g/ml, were incubated individually with 2?g CD40 on PVDF
membranes overnight to ensure equilibrium in binding. Subse-
quently, the membranes were washed and incubated successively
with rabbit antibodies against TSST-1, alkaline phosphatase-
conjugated antibodies against rabbit IgG, and finally substrate. The
density of protein bands was compared to standard amounts of
purified TSST-1 treated similarly, with concentrations compared
by NIH program ImageJ (http://rsbweb.nih.gov/ij/). For Kd deter-
mination, Scatchard analysis was performed.
2.16. CD40 pull-down assay
Magnetic beads (Dynabeads, Invitrogen Life Sciences, Grand
Island, NY) coated with protein A were treated with goat IgG anti-
bodies against TSST-1, then TSST-1, and finally CD40 (2?g), with
washing between steps and after incubation with CD40. The resul-
tant preparations were treated with sodium dodecyl sulfate (SDS)
PAGE sample buffer, electrophoresed by SDS-PAGE , and then
tested by Western immunoblotting for CD40.
2.17. Monoclonal antibodies that neutralize CD40 ligand binding
to CD40 competition with TSST-1 for CD40 binding on HVECs
Monoclonal antibodies against CD40 alone (×CD40; 20?l undi-
luted), TSST-1 alone (100?g/ml), isotype-matched monoclonal
antibodies against streptococcal pyrogenic exotoxin A (×SPEA)
and monoclonal antibodies against CD40+TSST-1, and monoclonal
antibodies against streptococcal pyrogenic exotoxin+TSST-1 were
incubated with HVECs for 6h. Subsequently IL-8 production was
measured by ELISA. As an important control, we showed the same
of cytokine production from HVECs.
Unpaired Student’s t test was used to compare fever responses
between groups. Log-rank test was used to compare differences in
to adjust for multiple pairwise comparisons between groups. In
by Student’s t test analysis.
3.1. Rabbit antibody responsiveness
We determined the ability of rabbits to develop protective anti-
body responses to native TSST-1 as an in vivo model to understand
the mechanism for the lack of protective antibody responses in
humans. Rabbits, as opposed to mice, are highly susceptible to
superantigens and make an excellent model for studying factors
important for the development of TSS [32,33]. Immunization of
20 Dutch-belted rabbits with 25?g/dose of native TSST-1 emulsi-
fied in incomplete adjuvant every-other-week for three injections
resulted in only 10/20 rabbits developing antibody titers against
TSST-1 and those were >10,000 as tested by ELISA, where titer
refers to the reciprocal of the last well dilution to give a positive
color change above background. For comparison, humans who are
susceptible to TSS have antibody titers of ≤40 against TSST-1, and
A.R. Spaulding et al. / Vaccine 30 (2012) 5099–5109
Fig. 1. (A) Pyrogenicity±standard deviation over a 4h period and enhancement of lethal LPS shock (Alive/Total) by 500?g/kg TSST-1 (?), G31S/S32P (?), H135A (?), and
Q136A (?). TSST-1, G31S/S32P, H135A, and Q136A were administered intravenously at 0h; LPS (100?g/kg) was administered intravenously at 4h, just after taking the 4h
temperatures. Alive/Total refers to the number of animals that survived as measured 48h post LPS injection. (B) Pyrogenicity on day 2±standard deviation and lethality over
a 7 day period due to TSST-1, G31S/S32P, H135A, and Q136A administered subcutaneously as 1000?g/miniosmotic pump.
humans who do not develop TSS have titers of ≥80 [32,33]. Thus,
the 10 rabbits that developed antibodies may be considered hyper-
immune to TSST-1.
the lower limit of our detection. These 10 non-responsive animals
were next continuously immunized monthly for up to 6 months or
for as long as they survived. The rabbits were also monitored for
development of antibodies to TSST-1 by ELISA monthly. All 10 ani-
mals succumbed to the vaccination attempts, with 7 dying after 6
months. At all tested time-points, all of these 10 rabbits had anti-
body titers of ≤10. Thus, the rabbit model appears to duplicate the
human situation in that a significant percentage of both humans
and rabbits appear unable to develop antibody responses to TSST-
1. We have observed the same phenomenon for rabbit antibody
responses to the superantigens SEB and SEC (data not shown).
3.2. Immunization against mutant TSST-1 proteins
We next performed studies to evaluate whether or not rabbits
could develop antibodies to TSST-1 mutants that were inactivated
in ability to bind to MHC II or V?-TCR. For these studies, three site-
specific mutants were constructed in tstH, leading to production of
TSST-1 proteins G31S/S32P that fails to interact with MHC II and
H135A and Q136A that fail to bind V?-TCR . These three pro-
teins were used separately to immunize 10 Dutch-belted rabbits
each, with 25?g/dose for three every-other-week injections. Upon
drawing blood one week after the third injection, all 10 animals in
each group (30 total) had antibody titers of >10,000 against native
TSST-1 as tested by ELISA. These data indicate the prior failure of
50% of rabbits to develop antibody responses resulted from TSST-1
induced dysregulation of immune responses, rather than genetic
inability to recognize TSST-1 as a foreign protein.
3.3. Residual toxicity of TSST-1 mutants
and Q136A) as proof of principle for ability to produce effective
superantigen toxoids. None of the 30 immunized rabbits above
exhibited signs of TSS as a result of vaccination, suggesting the
three mutant proteins were biologically inactivated. We tested the
extent of inactivation of the mutant proteins through three assays,
maximizing the chances to observe residual toxicity.
of 5 Dutch-belted rabbits per group intravenously, followed by
100?g/kg of LPS at the 4h time-point. Native TSST-1 as expected
caused high fevers, whereas all 3 mutants were non-pyrogenic
(p<0.001 for comparison of TSST-1 to any mutant) (Fig. 1A). Addi-
tionally, all 5 rabbits receiving native TSST-1 followed by LPS,
succumbed within 1h, but none of the 5 rabbits receiving mutant
TSST-1 proteins followed by LPS succumbed by 48h (p<0.008 for
TSST-1 compared to any mutant) (Fig. 1A). These data suggest that
all 3 mutant proteins were ≥500,000-fold inactivated, and thus
could be considered as toxoids.
Administration of the 3 TSST-1 mutants (1000?g/animal;
143?g/day or 10× LD50) in miniosmotic pumps to 5 rabbits/group
did not induce fevers, as measured on day 2 post-implantation
(p<0.001 for TSST-1 compared to any mutant), did not cause any
TSS symptoms, and did not cause deaths in any animals (Fig. 1B).
In contrast, native TSST-1 was pyrogenic, induced TSS symptoms,
and caused the deaths of all 5 animals by 48h (p<0.008 for TSST-1
compared to any mutant).
Native TSST-1 was superantigenic across the toxin range from
10?g/well down to 10−6?g/well (Fig. 2). None of the 3 TSST-1
mutants exhibited superantigenic activity, even at the 10?g/well
dose. These studies indicate the superantigencity of the mutants
was reduced by >107-fold.
Immunization against TSST-1 mutants protects rabbits from
native TSST-1 lethality. With evidence that the three mutant TSST-
1 proteins (G31S/S32P, H135A, and Q136A) were converted into
biologically inactive proteins that stimulate antibodies reactive
against TSST-1 as tested by ELISA, we examined the ability of these
proteins to elicit protective antibodies against the native toxin.
Ten rabbits per group were immunized three times with the indi-
vidual TSST-1 mutants, their antibody titers were determined to
be >10,000, and then these rabbits and control, non-immunized
animals (5/group), were challenged one week after the last immu-
nization with otherwise lethal doses of native TSST-1, either
the 5 rabbits per group developed fevers when challenged with
A.R. Spaulding et al. / Vaccine 30 (2012) 5099–5109
Fig. 2. Superantigenicity±standard deviation of TSST-1 (?), G31S/S32P (?), H135A
(?), and Q136A (?) for rabbit splenocytes in a 4-day assay. Rabbit splenocytes
(2×105/well) were incubated with TSST-1 and mutants for 3 days, and then 1?Ci
3H-thymidine per well added for 24h. DNA was harvested, and counts per minute
determined as a measure of T cell proliferation.
TSST-1 in the LPS enhancement model, and none of the 5 ani-
mals/group succumbed after being given LPS at the 4h time-point.
In contrast, all 5 control, non-immunized animals developed TSST-
1 induced fevers, and all succumbed in <6h post-administration of
LPS. In the miniosmotic pump model, none of the 5 animals/group
developed fevers, as measured on day 2 post-implantation, none
trol, non-immunized animals showed fevers, and all succumbed by
2 days post-implantation.
3.4. TSST-1 neutralization by antibodies
week with the mutant proteins was pooled. These pooled sera and
pooled sera from pre-vaccinated animals were tested in vitro for
ability to neutralize TSST-1 superantigenicity, as tested with rab-
bit splenocytes and 1?g/well of native TSST-1 (Fig. 3). In these
assays, undiluted and 1/10 and 1/100 diluted sera from immune
Fig. 3. Comparison of pooled rabbit sera from non-immune animals versus
animals hyperimmune to TSST-1 mutants G31S/S32P, H135A, and Q136A to
inhibit superantigenicity of TSST-1 (1?g/well), as tested in a 4 day assay with
rabbit splenocytes. Splenocytes were incubated with designated dilutions of
sera+TSST-1 for 3 days, and then 1?Ci3H-thymidine added for 24h. DNA was
harvested and counts/min determined as a measure of lymphocyte prolifera-
tion. Counts/min splenocytes+TSST-1=110,801±8647. Counts/min splenocytes
cal ?-toxin alone and ?-toxin mixed with TSST-1 mutants G31S/S32P and Q136A.
Titers represent the reciprocal of the serum dilution to give a positive color change
by ELISA when tested against ?-toxin.
animals completely neutralized TSST-1 superantigenicity; even
pre-immune pooled serum failed to neutralize superantigenicity.
The data suggest the mechanism of immunizing against TSST-1
lethality is neutralization of superantigenicity.
3.5. TSST-1 (Q136A) and TSST-1 (G31S/S32P) function as
adjuvants to stimulate antibody responses to other antigens
In a previous study with streptococcal superantigens, it was
suggested that non-toxic mutant superantigens may have intrin-
sic adjuvant activity . This observation was formally tested
in immunization studies using a second staphylococcal antigenic
toxin (?-toxin) with and without two TSST-1 mutants (G31S/S32P
one week after immunization. Rabbits immunized with ?-toxin
alone developed immune response antibody titers that increased
from 100 after the first immunization to 600 after the third immu-
nization (Fig. 4). In contrast, co-immunization with ?-toxin and
either TSST-1 (G31S/S32P) or TSST-1 (Q136A) resulted in antibody
titers to ?-toxins increasing from 200 to 300 after the first immu-
nization to nearly 106after the third immunization. These data
indicate that the MHC II mutant TSST-1 (G31S/S32P) and the TCR
mutant TSST-1 (Q136A) function as effective adjuvants.
3.6. Possible mechanism of adjuvanticity through the immune
co-stimulatory molecule CD40
Our vaccination studies were performed in rabbits because
these animals, like humans, are highly susceptible to the toxic
effects of superantigens. However, studies in rabbits restrict our
ability to determine the mechanism of adjuvanticity. Because of
nal epithelial cells (HVECs) to determine possible mechanisms of
intrinsic adjuvanticity, based on our hypothesis that adjuvanticity
must occur through TSST-1 and immune cell receptor interactions
independent of superantigen interaction with MHC II and V?-TCR
that could explain amplified antibody responses.
Our prior studies examined changes in HVEC gene expres-
sion following exposures to TSST-1 by microarray analysis
. In addition to increasing the expression of cytokines and
chemokines, these studies indicated that CD40 RNA transcription
was up-regulated when ATCC HVECs were incubated with TSST-1
(unpublished data). CD40 is an important immune co-stimulatory
A.R. Spaulding et al. / Vaccine 30 (2012) 5099–5109
Fig. 5. Wild-type TSST-1, TSST-1 (Q136A), and TSST-1 (G31S/S32P) bind to non-denatured CD40 in Western immunoblots. CD40 (2?g/lane) was subject to non-denaturing
PAGE and either stained with Coomassie blue (lane 1) or transblotted to PVDF membranes. Blots were incubated consecutively with TSST-1 proteins, rabbit antibodies against
TSST-1, alkaline phosphatase-conjugated antibodies against rabbit IgG, and substrate.
molecule required for optimal production of antibodies by B cells
. Additionally, our studies determined that HVECs lack MHC
II molecules on their surfaces (data not shown). Thus, these cells
provided an important cell line that we could use to determine if
TSST-1 interacts with CD40 as the potential receptor needed for
Through use of non-denaturing PAGE, we demonstrated that
TSST-1, TSST-1 (Q136A), and TSST-1 (G31S/S32P) bound to CD40 in
Western immunoblots (Fig. 5), but did not bind to electrophoresed
ovalbumin as a negative control (not shown). The binding of all
three TSST-1 proteins appeared comparable. The comparable bind-
with V?-TCR (Q136) and ?-chain MHC II (G31/S32) do not interact
Through use of a fixed concentration of CD40 in Western
immunoblots (2?g), combined with incubation with dilutions of
TSST-1 ranging from 0.033?g/ml to 33?g/ml, and comparison to
standard amounts of TSST-1, the Kd of the interaction of CD40 with
TSST-1, as determined by NIH program ImageJ and Skatchard anal-
ysis, was approximately 2.7×10−6M.
In order to have an independent method to assess CD40 inter-
action with TSST-1, we used pull-down assays to confirm binding.
In this assay, magnetic beads coated with protein A were treated
with goat IgG antibodies against TSST-1, then TSST-1, and finally
CD40 (2?g), with washing between steps and after incubation
with CD40. The resultant preparations were treated with sodium
dodecyl sulfate (SDS) PAGE sample buffer, electrophoresed by
SDS-PAGE, and then tested by Western immunoblotting for CD40
with CD40. In the presence of TSST-1 on the beads, more CD40 was
pulled down than in the absence of TSST-1, confirming that TSST-1
bound to CD40.
We hypothesized that co-incubation of TSST-1 and monoclonal
antibodies that neutralize CD40 interaction with CD40 ligand on T
cells with HVECs would result in interference with IL-8 chemokine
production. Unexpectedly, we observed a nearly 3-fold synergy
in IL-8 chemokine production when both TSST-1 and monoclonal
antibodies against CD40 were incubated with the HVECs compared
to TSST-1 alone (Fig. 7); the monoclonal antibodies to CD40 did not
antibody (monoclonal antibodies against streptococcal pyrogenic
exotoxin A (SPEA) did not synergize with TSST-1 to cause amplified
IL-8 production. Finally, the same monoclonal antibodies against
HVECs (data not shown).
co-stimulatory molecule CD40, which is required for optimal stim-
ulation of B cell to produce neutralizing antibodies. This may
Fig. 6. Wild-type TSST-1 immobilized on beads pulls-down CD40. TSST-1 was
immobilized on magnetic Dynabeads and then used to bind purified CD40. Control
beads lacked TSST-1 and were treated comparably with CD40. After washing, beads
were treated with SDS-PAGE buffer and samples electrophoresed in SDS-PAGE gels.
oclonal antibodies to CD40, alkaline phosphatase-conjugated antibodies to mouse
immunoglobulin, and then substrate.
A.R. Spaulding et al. / Vaccine 30 (2012) 5099–5109
Fig. 7. HVECs treated with TSST-1 and monoclonal antibodies against CD40 that
tion. Monoclonal antibodies against CD40 alone (×CD40; 20?l undiluted), TSST-1
alone (100?g/ml), isotype-matched monoclonal antibodies against streptococcal
pyrogenic exotoxin A (×SPEA) and monoclonal antibodies against CD40+TSST-1,
and monoclonal antibodies against streptococcal pyrogenic exotoxin+TSST-1 were
sequently IL-8 production was measured by ELISA. Bars represent standard error of
account for the TSST-1 mutant toxoid adjuvanticity. It is likely that
native TSST-1 interacts more prominently with MHC II and V?-TCR
to mask the adjuvant effect.
Tri-valent vaccine prevents S. aureus pneumonia. We have
shown previously that immunity to TSST-1 and SEC protects rab-
bits from intra-pulmonary challenge with TSST-1, SEC or USA200
S. aureus producing TSST-1 and SEC . Other investigators have
lethal pneumonia . We thus evaluated the ability of a triva-
lent vaccine, composed of TSST-1 (G31S/S32P), a low dose of native
SEC, and combined with a non-toxic dose of ?-toxin (H35L) (5
rabbits) or wild-type ?-toxin (6 rabbits) to protect from lethal
pneumonia with a high dose challenge with USA200 S. aureus
MNPE (2×109CFUs) that produces high levels of TSST-1, SEC, and
?-toxin. We also evaluated immunization against the non-toxic
mutant of ?-toxin (H35L) (5 animals) or native ?-toxin (5 ani-
mals) alone to protect rabbits from similar challenge with S. aureus
MNPE. Since we observed no differences in antibody responses
or protection from pneumonia in rabbits that had been immu-
nized with ?-toxin (H35L) versus wild-type ?-toxin (low dose
immunization), we combined the groups in data presented. All
animals were immunized every-other-week for 3 injections in
incomplete adjuvant, shown to have high antibody titers (>10,000)
against all three native toxins by ELISA, and were challenged
intra-pulmonary one week after the last immunization, along with
non-immune controls, with 2×109MNPE. There were significant
differences in survivals among the groups (p<0.001). For rab-
bits immunized against the trivalent vaccine containing TSST-1
lethal pneumonia (Fig. 8A). In contrast, all 11 non-immunized
animals succumbed to the lethal challenge (p<0.001). Rabbits
immunized with the ?-toxin H35L alone or native ?-toxin alone
showed delayed deaths due to challenge with MNPE, but ulti-
mately, 9/11 succumbed (p=0.001, compared to non-immunized
controls). Rabbits immunized against the trivalent vaccine had
better survival than rabbits immunized against ?-toxin (H35L or
native) alone (p<0.001).
Pentavalent vaccine prevents infective endocarditis and sepsis.
We have previously shown that TSST-1, SEC, and staphylococcal ?-
toxin contribute to infective endocarditis [9,12]. These toxins are
produced by USA200 S. aureus strains, the strains most commonly
associated with persistent bacteremia and infective endocarditis
. We hypothesized that prior vaccination against the three
combined with vaccination against TSST-1 (G31S/S32P) and SEC,
may protect rabbits from infective endocarditis and sepsis due to
Rabbits (4–5/group) were immunized against these 5 proteins
or ?-toxin H35L alone every-other-week for 3 injections. Con-
trol animals remained non-immunized. After immunization, all
animals were highly immune to each toxin by ELISA, and then
all immune plus non-immunized animals were challenged one
week after the last immunization with MNPE in our previously
established model of infective endocarditis and sepsis. Rabbits pre-
Fig. 8. (A) Protection of rabbits from lethal S. aureus USA200 pneumonia by vaccination against TSST-1 (G31S/S32P)+SEC+?-toxin (H35L or wild-type) and partial protection
by vaccination against ?-toxin (H35L). Rabbits (11/group) were immunized three times with antigens, TSST-1 (G31S/S32P)+SEC+?-toxin (H35L or wild-type) as a cocktail
(?) or ?-toxin (H35L) alone (?), or remained non-immunized (?). Antigens were emulsified in incomplete adjuvant and immune animals plus non-immune control animals
challenged intrapulmonary with 2×109S. aureus MNPE. Rabbits immunized against TSST-1 (G31S/S32P)+SEC+?-toxin (H35L or wild-type) were significantly protected
from lethality compared to non-vaccinated animals or animals vaccinated against ?-toxin (H35L or wild-type) alone (p<0.001). Animals vaccinated against ?-toxin (H35L or
wild-type) were significantly delayed in lethality compared to non-vaccinated controls (p=0.001). (B) Protection of rabbits from lethal sepsis by immunization against five
S. aureus exotoxins. Rabbits were immunized three times with TSST-1 (G31S/S32P), SEC, ?-toxin (H35L), ?-toxin, and ?-toxin (?) or ?-toxin (H35L) alone (?), or remained
non-vaccinated (?). Challenge organism was intravenous USA200 S. aureus MNPE (2×108/2ml volume in PBS).
A.R. Spaulding et al. / Vaccine 30 (2012) 5099–5109
protected from lethal sepsis (Fig. 8B). Vegetation sizes for MNPE
are typically up to 100mg (data not shown). One rabbit from the
pentavalent immunized group died late on day 2 and had a veg-
etation of 6mg with 1×108CFUs. The largest vegetation seen in
the immunized rabbits was 14mg while the smallest was 1mg,
vastly smaller than the typical size associated with MNPE. The data
suggest that prior immunization against these 5 secreted toxins
provided immune protection against otherwise lethal challenge
and significantly reduced vegetation size.
For rabbits previously immunized against ?-toxin H35L alone,
three of four developed small vegetations (2–3mg), and all suc-
cumbed from lethal sepsis (Fig. 8B), though lethality was delayed
compared to the non-immunized control group (day 2 or 3 for
H35L immunized rabbits versus day 1 for control rabbits). There
were significant differences in overall survival among the groups
(p=0.01). The survivals of animals immunized against ?-toxin
alone and pentavalent vaccine were both significantly better than
non-immunized animals (p=0.002 and 0.001, respectively). Fur-
thermore, rabbits immunized against pentavalent vaccine were
also significantly protected from lethal sepsis than rabbits immu-
nized against ?-toxin alone (p=0.004).
All control, non-immunized animals in this study succumbed
to lethal sepsis by 24h post injection of MNPE (Fig. 8B). None of
the animals had significant vegetations, presumably due to the
rapidity with which the animals succumbed, though there was
visual confirmation of small vegetations forming on their aortic
We have shown that immunization with secreted virulence fac-
tors produced by S. aureus protects against pneumonia, infective
endocarditis, and lethal sepsis caused by high-dose challenge with
a highly pathogenic USA200 strain. The challenge organism came
from a patient who succumbed to post-influenza TSS . Immu-
nization against TSST-1, SEC, and ?-toxin protects rabbits against
pneumonia caused by the USA200 strain producing these three
exotoxins, and immunization with a pentavalent vaccine against
TSST-1, SEC, ?-toxin, ?-toxin, and ?-toxin protects rabbits against
non-immune rabbits succumbed to the infections, dying before the
end of the experiments.
Superantigens and cytolysins function critically in a number of
sis, and infective endocarditis [9,13,15,16]. We sought to create
a vaccine based on the most common secreted virulence factors
known to participate in these diseases. The superantigens TSST-1,
SEC, as well as the cytolysins ?-toxin, ?-toxin, and ?-toxin were
prime candidates; however we believe that a more effective vac-
cine will additionally include SEB and SE-like X toxoids. Both of
these exotoxins have been shown recently also to participate in
serious S. aureus illnesses [17,41]. Thus, the studies in the present
research are proof of principle that a polyvalent toxoid vaccine can
be developed to protect against serious staphylococcal infections.
Prior studies reported the inability of people to produce anti-
bodies to superantigens [42–44]. There were two possible reasons
for this: persons have genetic inabilities to recognize and respond
to the superantigens; or these individuals have hyperimmune
responses to the superantigens, resulting in immune dysregulation
and lack of antibody responses. This latter effect has been observed
in patients with streptococcal TSS . We sought to understand
the underlying mechanism, as genetic inability to respond would
hinder using superantigen toxoid vaccines. Typically, only small
antigens with one or two epitopes would be expected to result in
inability to recognize an antigen as foreign. Superantigens range
in size from 19,000 to 30,000 molecular weight, and in com-
parison to other antigens, would be expected to have multiple
epitopes for antibody recognition [46,47]. Although formally pos-
sible, this makes it less likely that genetic inability to respond
accounts for the lack of antibody responses in 20% of adults. We
showed in a rabbit model that 50% of animals appear unable
to develop antibody responses to native TSST-1, suggesting the
same mechanism causes their unresponsiveness as humans. How-
ever, 100% of rabbits are able to make antibody responses when
superantigenicity is removed through mutation. This suggests that
the inability to produce antibodies to superantigens results from
hyper-responsiveness to superantigens, leading to immune dys-
function through an unknown mechanism, rather than genetic
inability to respond.
We also performed extensive studies to show that the TSST-1
mutant proteins used in our studies possess three important prop-
erties of toxoids. First, the mutant proteins contain no detectable
residual toxicities, appearing to be >107-fold inactivated by the
most sensitive test, superantigenicity, as tested in vitro. Addition-
ally, with use of the most sensitive assays in rabbit models of TSS,
we demonstrated that the proteins are >500,000-fold inactivated.
Other studies with use of streptococcal superantigens to develop
toxoid vaccines have also shown that mutation of immune cell
contact residues greatly reduces residual activities [39,48]. From
a safety perspective double mutants such as TSST-1 (G31S/S32P)
would be the most useful in vaccines since reversion to toxicity is
unlikely. Like TSST-1 mutants, our studies and prior studies 
show that the H35L mutation of ?-toxin inactivates the cytolysin
activity. This is particularly important since rabbits are killed by
0.1?g intravenously of native ?-toxin in 1h . Second, prospec-
tive toxoid proteins must be immunogenic, preferably with few
bodies are formed by three injections. Importantly, prior studies
suggested but did not prove that superantigens have intrinsic
adjuvanticities . The present studies demonstrate this activ-
ity clearly, showing that non-toxic superantigen mutants amplify
immune responses to a second antigen, staphylococcal ?-toxin, by
10–100-fold. We have also shown this adjuvanticity with multi-
ple other antigens, including HIV proteins and sheep erythrocytes
(data not shown). Third, toxoids must elicit antibodies that pro-
tect against native toxin. By multiple assays, we demonstrated that
by native TSST-1. The rabbits have antibody titers >10,000. From
prior studies, we and others have shown that healthy humans who
do not develop TSS most often have antibody titers against TSST-1
between 80 and 320 [43,44,49].
We have explored the possible mechanism of adjuvanticity of
TSST-1 mutants. This effect is not seen with use of wild-type TSST-
1 which is more likely to result in antibody immunosuppression
than in adjuvanticity. Rabbits like humans are highly susceptible to
superantigens, but insufficient reagents are available to determine
the mechanism of adjuvanticity. Thus, we used data obtained from
that function as adjuvants must interact with a host cell recep-
tor other than the two known receptors that cause TSS, namely
MHC II and V?-TCR. Our studies suggest that TSST-1 binds to the
production of antibodies by B cells . Our studies showed that
TSST-1 directly binds to purified CD40 in Western immunoblots
and in CD40 pull-down assays. Additionally, our previous studies
show TSST-1 interaction with HVECs leads to increased chemokine
production despite HVECs lacking T cell receptors and MHC II
molecules . Interestingly, monoclonal antibodies that mimic T
cell CD40 ligand binding to CD40 do not block cytokine production,
A.R. Spaulding et al. / Vaccine 30 (2012) 5099–5109
but in fact lead to synergy in chemokine production. We hypoth-
esize that this effect accounts for the adjuvant effect that leads to
amplified antibody production in the presence of TSST-1 mutant
toxoids. Additional studies are needed to verify this hypothesis.
As proof of principle for protective ability of a cocktail vaccine
against secreted virulence factors, rabbits were immunized with a
trivalent vaccine of TSST-1 (G31S/S32P), native SEC, and ?-toxin
(H35L or wild-type) a protein previously used as to protect mice
from lethal staphylococcal pneumonia. The animals were chal-
producing all three of these secreted virulence factors. All of the
vaccinated animals survived and exhibited immunity to the organ-
isms. These data suggest that these secreted virulence factors are
critical for establishing pulmonary infections, in addition to their
causing lethality. Similarly, a pentavalent vaccine composed of 5
secreted virulence factors significantly protected rabbits against
lethal sepsis and reduced the severity of infective endocarditis due
to challenge with the same organism. In some studies, we immu-
nized rabbits against ?-toxin (H35L) or native ?-toxin only and
challenged with the highly lethal dose of MNPE. Partial protec-
tion was observed. It is possible that greater protection would have
been observed if the challenge dose was not as high. However, the
survival studies with high dose challenge clearly emphasize the
advantage of having multiple secreted factors in the vaccine.
In our studies, we did not have available toxoids for all of the
superantigens and cytolysins we wished to assess. However, our
studies demonstrate that an appropriate vaccine with multiple
toxoids may be protective. In our prior studies, we have already
identified the critical amino acids necessary for activity of SEB and
SEC, just as we have used previous studies to identify the critical
residues in TSST-1 . We have also shown that these toxoids
are highly inactivated . Additionally, others have identified key
residues in ?-toxin , and we have completely inactivated ?-
toxin by mutation of two active site residues . There is no
compelling reason to mutagenize ?-toxin since these heptamer
pore-forming toxins require two different chains for activity .
We suggest that immunization can be accomplished with use of
the shared B chain. Our current efforts are being directed toward
identification of important residues for mutation in SE-like X.
Collectively, these results suggest that a vaccine composed of
common secreted virulence factors produced by S. aureus is capa-
ble of protecting rabbits from lethal staphylococcal disease. Future
studies in our laboratory seek to explore the possibility of adding
in other secreted virulence factors, including SEB and the newly
discovered SE-like X.
Patients with staphylococcal TSS do not develop neutralizing
antibody responses to the superantigen TSST-1, and thus they
remain susceptible to TSS recurrences. This effect results from
immune dysfunction due to TSST-1, rather than genetic inability to
recognize the superantigen as foreign. TSST-1 may be >107inacti-
vated by genetic modification of amino acid residues in the MHC II
and V?-TCR sites; resultant mutants stimulate protective immu-
nity against native TSST-1 and function as adjuvants to amplify
antibody responses to secondary antigens. Immunization against
cocktails of secreted superantigens and cytolysins protects rabbits
from lethal pneumonia, infective endocarditis, and sepsis.
R01 AI074283, R01 AI73366, and U54 AI57153. P.M.S. is a mem-
ber of the Great Lakes Regional Center of Excellence in Biodefense
and Emerging Infectious Diseases. Dr. Juliane Bubeck-Wardenburg
is gratefully acknowledged for providing ?-toxin (H35L) used in
these studies.Contributors: ARS performed direct experimentation
MLP, and PMS performed direct experimentation and data analy-
ses, and edited the manuscript. All have approved the final version
for submission. Conflict of interest statement: None of the authors
have conflict of interests to declare.
 Lowy FD. Staphylococcus aureus infections. N Engl J Med 1998;339:520–32.
superantigens: an update. Annu Rev Microbiol 2001;55:77–104.
 Mulligan ME, Murray-Leisure KA, Ribner BS, Standiford HC, John JF, Korvick
JA, et al. Methicillin-resistant Staphylococcus aureus: a consensus review of the
microbiology, pathogenesis, and epidemiology with implications for preven-
tion and management. Am J Med 1993;94:313–28.
 Mylonakis E, Calderwood SB. Infective endocarditis in adults. N Engl J Med
 BeynonRP, BahlVK, Prendergast
 Bayer AS, Ramos MD, Menzies BE, Yeaman MR, Shen AJ, Cheung AL. Hyper-
production of alpha-toxin by Staphylococcus aureus results in paradoxically
microbicidal proteins. Infect Immun 1997;65:4652–60.
 Fowler Jr VG, Miro JM, Hoen B, Cabell CH, Abrutyn E, Rubinstein E, et al.
Staphylococcus aureus endocarditis: a consequence of medical progress. JAMA
 Miro JM, Anguera I, Cabell CH, Chen AY, Stafford JA, Corey GR, et al. Staphylo-
coccus aureus native valve infective endocarditis: report of 566 episodes from
the International Collaboration on Endocarditis Merged Database. Clin Infect
factor regulation by SrrAB, a two-component system in Staphylococcus aureus.
J Bacteriol 2004;186:2430–8.
 Xiong YQ, Willard J, Yeaman MR, Cheung AL, Bayer AS. Regulation of Staphy-
lococcus aureus alpha-toxin gene (hla) expression by agr, sarA, and sae in vitro
and in experimental infective endocarditis. J Infect Dis 2006;194:1267–75.
 Nienaber JJ, Sharma Kuinkel BK, Clarke-Pearson M, Lamlertthon S, Park L, Rude
TH, et al. Methicillin-susceptible Staphylococcus aureus endocarditis isolates
are associated with clonal complex 30 genotype and a distinct repertoire of
enterotoxins and adhesins. J Infect Dis 2008;204:704–13.
 Huseby MJ, Kruse AC, Digre J, Kohler PL, Vocke JA, Mann EE, et al. Beta toxin
catalyzes formation of nucleoprotein matrix in staphylococcal biofilms. Proc
Natl Acad Sci U S A 2010;107:14407–12.
 Cheung AL, Eberhardt KJ, Chung E, Yeaman MR, Sullam PM, Ramos M, et al.
Diminished virulence of a sar−/agr− mutant of Staphylococcus aureus in the
rabbit model of endocarditis. J Clin Invest 1994;94:1815–22.
et al. Comparison of Staphylococcus aureus strains for ability to cause infective
 Bubeck Wardenburg J, Bae T, Otto M, Deleo FR, Schneewind O. Poring over
pores: alpha-hemolysin and Panton–Valentine leukocidin in Staphylococcus
aureus pneumonia. Nat Med 2007;13:1405–6.
 Bubeck Wardenburg J, Schneewind O. Vaccine protection against Staphylococ-
cus aureus pneumonia. J Exp Med 2008;205:287–94.
 Strandberg KL, Rotschafer JH, Vetter SM, Buonpane RA, Kranz DM, Schlievert
PM. Staphylococcal superantigens cause lethal pulmonary disease in rabbits. J
Infect Dis 2010;202:1690–7.
 Voyich JM, Otto M, Mathema B, Braughton KR, Whitney AR, Welty D, et al. Is
Panton–Valentine leukocidin the major virulence determinant in community-
associated methicillin-resistant Staphylococcus aureus disease. J Infect Dis
 Leder L, Llera A, Lavoie PM, Lebedeva MI, Li H, Sekaly RP, et al. A mutational
analysis of the binding of staphylococcal enterotoxins B and C3 to the T cell
receptor beta chain and major histocompatibility complex class II. J Exp Med
 Murray DL, Earhart CA, Mitchell DT, Ohlendorf DH, Novick RP, Schlievert PM.
Localization of biologically important regions on toxic shock syndrome toxin
1. Infect Immun 1996;64:371–4.
 Murray DL, Prasad GS, Earhart CA, Leonard BA, Kreiswirth BN, Novick RP, et al.
Immunobiologic and biochemical properties of mutants of toxic shock syn-
drome toxin-1. J Immunol 1994;152:87–95.
 Gaskin DK, Bohach GA, Schlievert PM, Hovde CJ. Purification of Staphylococcus
aureus beta-toxin: comparison of three isoelectric focusing methods. Protein
Expr Purif 1997;9:76–82.
 Lin YC, Anderson MJ, Kohler PL, Strandberg KL, Olson ME, Horswill AR, et al.
Proinflammatory exoprotein characterization of toxic shock syndrome Staphy-
lococcus aureus. Biochemistry 2011;50:7157–67.
 MacDonald KL, Osterholm MT, Hedberg CW, Schrock CG, Peterson GF, Jentzen
JM, et al. Toxic shock syndrome. A newly recognized complication of influenza
and influenzalike illness. JAMA 1987;257:1053–8.
BD. Infective endocarditis.BMJ
A.R. Spaulding et al. / Vaccine 30 (2012) 5099–5109 Download full-text
factor comparison between methicillin-resistant and methicillin-sensitive
Staphylococcus aureus, and its relevance to atopic dermatitis. J Allergy Clin
 Schlievert PM, Blomster DA. Production of staphylococcal pyrogenic exotoxin
type C: influence of physical and chemical factors. J Infect Dis 1983;147:
 Schlievert PM, Gahr PJ, Assimacopoulos AP, Dinges MM, Stoehr JA, Har-
mala JW, et al. Aggregation and binding substances enhance pathogenicity
in rabbit models of Enterococcus faecalis endocarditis. Infect Immun 1998;66:
 Blomster-Hautamaa DA, Schlievert PM. Preparation of toxic shock syndrome
toxin-1. Methods Enzymol 1988;165:37–43.
 Jardetzky TS, Brown JH, Gorga JC, Stern LJ, Urban RG, Chi YI, et al. Three-
dimensional structure of a human class II histocompatibility molecule
complexed with superantigen. Nature 1994;368:711–8.
 McCormick JK, Tripp TJ, Llera AS, Sundberg EJ, Dinges MM, Mariuzza RA, et al.
predicts further diversity in MHC class II/superantigen/TCR ternary complexes.
J Immunol 2003;171:1385–92.
 Schlievert PM. Enhancement of host susceptibility to lethal endotoxin
shock by staphylococcal pyrogenic exotoxin type C. Infect Immun 1982;36:
protection of rabbits challenged subcutaneous with toxic shock syndrome tox-
ins. Infect Immun 1991;59:879–84.
 Parsonnet J, Gillis ZA, Richter AG, Pier GB. A rabbit model of toxic shock syn-
drome that uses a constant, subcutaneous infusion of toxic shock syndrome
toxin 1. Infect Immun 1987;55:1070–6.
 Westphal O, Luderitz O, Bister F. Uber die extraktion von vacterium mit phe-
nol/wasser. Z Naturforsch 1952;7b:148–55.
 Schlievert PM, Shands KN, Dan BB, Schmid GP, Nishimura RD. Identification
toxic-shock syndrome. J Infect Dis 1981;143:509–16.
 Peterson M, Ault K, Kremer MJ, Klingelhutz AJ, Davis CC, Squier CA, et al.
Innate immune system is activated by stimulation of vaginal epithelial cells
with Staphylococcus aureus and toxic shock syndrome toxin-1. Infect Immun
chains of hemoglobin inhibit production of Staphylococcus aureus exotoxins.
 Laemmli UK. Cleavage of structural proteins during the assembly of the head
of bacteriophage T4. Nature 1970;227:680–5.
 Roggiani M, Stoehr JA, Olmsted SB, Matsuka YV, Pillai S, Ohlendorf DH, et al.
Toxoids of streptococcal pyrogenic exotoxin A are protective in rabbit models
of streptococcal toxic shock syndrome. Infect Immun 2000;68:5011–7.
 Elgueta R, Benson MJ, de Vries VC, Wasiuk A, Guo Y, Noelle RJ. Molecular
mechanism and function of CD40/CD40L engagement in the immune system.
Immunol Rev 2009;229:152–72.
 Wilson GJ, Seo KS, Cartwright RA, Connelley T, Chuang-Smith ON, Merri-
man JA, et al. A novel core genome-encoded superantigen contributes to
lethality of community-associated MRSA necrotizing pneumonia. PLoS Pathog
 Osterholm MT, Davis JP, Gibson RW, Mandel JS, Wintermeyer LA, Helms CM,
et al. Tri-state toxic-state syndrome study. I. Epidemiologic findings. J Infect
W, et al. Prevalence of toxic shock syndrome toxin 1-producing Staphylococcus
aureus and the presence of antibodies to this superantigen in menstruating
women. J Clin Microbiol 2005;43:4628–34.
 Vergeront JM, Stolz SJ, Crass BA, Nelson DB, Davis JP, Bergdoll MS. Prevalence of
serum antibody to staphylococcal enterotoxin F among Wisconsin residents:
implications for toxic-shock syndrome. J Infect Dis 1983;148:692–8.
 Kotb M, Norrby-Teglund A, McGeer A, El-Sherbini H, Dorak MT, Khurshid A,
et al. An immunogenetic and molecular basis for differences in outcomes of
invasive group A streptococcal infections. Nat Med 2002;8:1398–404.
tions of toxic shock syndrome toxin-1 by use of monoclonal antibodies and
cyanogen bromide-generated toxin fragments. J Immunol 1986;137:3572–6.
 Murphy BG, Kreiswirth BN, Novick RP, Schlievert PM. Localization of a bio-
logically important epitope on toxic-shock-syndrome toxin-1. J Infect Dis
 McCormick JK, Tripp TJ, Olmsted SB, Matsuka YV, Gahr PJ, Ohlendorf DH,
et al. Development of streptococcal pyrogenic exotoxin C vaccine toxoids
that are protective in the rabbit model of toxic shock syndrome. J Immunol
 Bergdoll MS, Crass BA, Reiser RF, Robbins RN, Davis JP. A new staphylococcal
cus aureus isolates. Lancet 1981;1:1017–21.
 Huseby M, Shi K, Brown CK, Digre J, Mengistu F, Seo KS, et al. Structure
and biological activities of beta toxin from Staphylococcus aureus. J Bacteriol
staphylococcal gamma-hemolysin into heteroheptameric transmembrane
pores with alternate subunit arrangements in ratios of 3:4 and 4:3. J Bacteriol