Serum dilution

Serum dilution Serum dilution

OD

405

nm

OD

405

nm

OD

405

nm

PNAG CP5

CP8

A B

C

Supplemental Figure 1: ELISA analysis of IgG antibody binding in the pre-immune sera of the 9 groups of mice immunized with S. aureus antigens: (A) Binding to PNAG (0.06 ug/well), (B) Binding to CP5 (3 ug/well), (C) Binding to CP8 (3 ug/well). Positive controls used for (A), (B) and (C) are presented in Figure 2d.

OD

405

nm

OD

405

nm

OD

405

nm

Serum dilution

PNAG

CP5

CP8

Serum dilutionSerum dilution

Serum dilution

OD

405

nm

OD

405

nm

OD

405

nm

A B

C D

PNAG CP5

CP8

A. Binding to PNAG (0.06 ug/well). B. Binding to CP5 (3 ug/well), (C) Binding to CP8 (3 ug/well). Mice immunized with the indicated antigens. Dashed lines: no immune response to PNAG, CP5 or CP8 detected in sera from mice injected with vaccines not containing these antigens. D: Top, center and bottom: positive controls used for (A): Rabbit polyclonal antibodies to PNAG, (B): Mouse monoclonal antibodies to CP5 and (C): Rabbit polyclonal antibodies to CP8.

Supplemental Figure 2: IgG responses to S. aureus antigens following immunization

Combinations of antigens given together Combinations of antigens given separately

Perc

ent S

. aur

eus

New

man

kill

ed

(dPNAG-TT + CP5-TT)

(dPNAG-TT + CP8-TT)

(dPNAG-TT + CP5-TT + CP8-TT)

(dPNAG-TT) + (CP5-TT)

(dPNAG-TT) + (CP8-TT) (dPNAG-TT) + (CP5-TT + CP8-TT)

10 40 160

A

10 40 160 10 40 160 10 40 160 10 40 160 10 40 160

Supplemental Figure 3A:Killing of S. aureus Newman (CP5) at the indicated concentration in antisera from mice immunized with combinations of dPNAG-TT, CP5-TT and CP8-TT administrated together or separately into different flanks but at the same time. No killing >30% was detected in any antiserum. Controls: Pre-immune sera.

Serum dilutions

Pre-immune sera

Combinations of antigens given together Combinations of antigens given separately

Perc

ent S

. aur

eus

New

man

kill

ed

(dPNAG-TT + CP5-TT)

(dPNAG-TT + CP8-TT)

(dPNAG-TT + CP5-TT + CP8-TT)

(dPNAG-TT) + (CP5-TT) (dPNAG-TT) +

(CP8-TT) (dPNAG-TT) + (CP5-TT + CP8-TT)

10 40 160

B

10 40 160 10 40 160 10 40 160 10 40 160 10 40 160

Supplemental Figure 3B:Killing of S. aureus PS80 (CP8) at the indicated concentration in antisera from mice immunized with combinations of dPNAG-TT, CP5-TT and CP8-TT administrated together or separately into different flanks but at the same time. No killing >30% was detected in any antiserum. Controls: Pre-immune sera.

Serum dilutions

Pre-immune sera

Supplemental Figure 3C: Killing of S. aureus LAC (Non-typable) by sera from mice immunized with the indicated antigens. Only antibody to dPNAG-TT mediated killing >30%. Control: Pre-immune serum.

Antigens administrated alone Combinations of antigens given together

Combinations of antigens given separately

Perc

ent S

. aur

eus

LAC

kille

d

CP5-TTCP8-TT

(dPNAG-TT+CP5-TT)

(dPNAG-TT+CP8-TT)

(dPNAG-TT+CP-TT5 + CP8-TT)

(dPNAG-TT)+ (CP5-TT) (dPNAG) +

(CP8-TT)(dPNAG-TT) + (CP5-TT+CP8-TT)

Control

Immune sera

Pre-immune sera

dPNAG-TT

ica+capicacap

S. aureus MN8 mutants used for the absorption

ica+capicacap

ica+capcap

S. aureus Newman mutants used for the absorption

ica+capcap

Serum dilutionSerum dilution

Serum dilution

A B

C D Serum dilution

S. aureus Newman mutants used for absorption

S. aureus MN8 mutants used for the absorption

Supplemental Figure 4: OPKA after specific absorptions of the sera from the mice immunized with combinations of antigen injected together. Killing of S. aureus Newman (CP5) (A and C) or PS80 (CP8) (B and D) in antisera of mice immunized with dPNAG-TT+ CP5-TT (A) , dPNAG-TT+CP8-TT (B), or dPNAG-TT+CP5-TT+CP8-TT (C-D), after absorption with S. aureus double mutants (cap+ica) to leave behind antibodies to both PNAG and CP or with individual ica or cap mutants to leave behind only antibodies to PNAG or to CP, respectively. Bars indicate average of 4 replicates per assay. Control: No complement in assay tube.

ica+capicacap

S. aureus MN8 mutants used for the absorption

ica+capicacap

ica+capcap

S. aureus Newman mutants used for the absorption

ica+capcap

Serum dilutionSerum dilution

Serum dilution

A B

C DSerum dilution

Supplemental Figure 5: OPKA after absorption of antisera from the mice immunized with combinations of antigens injected into separate flanks. Killing of S. aureus Newman (CP5) (A and C) or PS80 (CP8) (B and D) in antisera from mice immunized with dPNAG-TT & CP5-TT (A); , dPNAG-TT & CP8-TT (B); or dPNAG-TT & CP5-TT+CP8-TT (C-D); injected separately after absorption with S. aureus double mutant (cap+ica) to leave behind antibodies to both PNAG and CP or with individual ica or cap mutants to leave behind only antibodies to PNAG or CP, respectively. Bars indicate average of 4 replicates per assay. Controls: No complement.

S. aureus Newman mutants used for the absorption

S. aureus MN8 mutants used for the absorption

Serum dilutionSerum dilution

Anti-dPNAG-TT+ absorbed NHS(cap)

Anti-dPNAG-TT+ absorbed NHS(ica)

Anti-dPNAG-TT+ Unabsorbed NHS

Anti-dPNAG-TT+ absorbed NHS(cap)

Anti-dPNAG-TT+ absorbed NHS(ica)

Anti-dPNAG-TT+ Unabsorbed NHS

Supplemental Figure 6: Effect of addition of pooled normal human sera on OPK of rabbit antisera raised to dPNAG-TT: OPK of S. aureus Newman (A) or PS80 (B) mediated by rabbit antibody to dPNAG-TT and effect of the addition of pooled NHS, either unabsorbed or absorbed with S. aureus mutants to leave behind only antibodies to PNAG (S. aureus ica) or antibodies to CP (S. aureus cap). Bars indicate average of 4 replicates per assay. Controls lacking complement had a killing <1%.

A B

10 40 160 10 40 160 10 40 160 10 40 160 10 40 160 10 40 160

Serum dilution

Rabbit anti-CP5-TT

NHS

Rabbit anti-CP5-TT +Unabsorbed NHS

Rabbit anti-CP5-TT + NHS absorbed (cap)

104

105

106

107

CFU

(New

man

)/ab

sces

s

Anti-CP5-TT + NHS

Anti-CP5-TT NHS

P<0.01

NS

P<0.05

A

BSupplemental Figure 7: Characteristics of a pool of NHS prepared from individual NHS. Pool prepared from sera with high levels of interference with the OPK of rabbit antibody to CP5-TT. A. OPKA against S. aureus Newman in rabbit antisera raised to CP5-TT , the selected NHS pool and a 1:10 dilution of the rabbit antisera added to indicated dilutions of the NHS, either unabsorbed or absorbed with S. aureus Dcap to remove the antibodies to PNAG. NRS=Normal rabbit serum, No C’=No complement, No PMN=No Polymorphonuclear leukocytes. B. Effect of the NHS pool on reduction in the cfu S. aureus/abscess mediated by rabbit antibody to CP5-TT. Antibody raised to CP5-TT without added NHS significantly reduced the cfu S. aureus/abscess compared with mice injected with antibody raised to CP5-TT into which NHS had been added or mice injected with NHS alone. Overall ANOVA P =0.006, pair-wise comparisons on figures. Challenge: S. aureus Newman (106 cfu).

Rabbit anti-CP8-TT

Rabbit anti-CP8-TT+ NHS adsorbed with E. coli. Rabbit anti-CP8-TT

+NHS adsorbed with E. coli pga

Rabbit anti-CP5-TT

Rabbit anti-CP5-TT+ NHS adsorbed with E. coli.

Rabbit anti-CP5-TT +NHS adsorbed with E. coli pga

A BPe

rcen

t of S

. aur

eus

New

man

kill

ed

Perc

ent o

f S. a

ureu

s PS

80 k

illed

Supplemental Figure 8: Effect on the OPK activity mediated by anti-capsule antibodies of a pool of NHS with and without absorbtion of the anti-PNAG antibodies by E. coli, a non-S. aureus PNAG-producing bacteria. A. OPKA against S. aureus Newman in rabbit antisera raised to CP5-TT, a 1:10 dilution of the rabbit antisera added to indicated dilutions of the NHS, either absorbed with E. coli J to remove the antibodies to PNAG or E. coli J pga to leave behind antibodies to PNAG. NHS=Normal Human Serum, No C’=No complement. B. OPKA against S. aureus PS80 in rabbit antisera raised to CP8-TT, a 1:10 dilution of the rabbit antisera added to indicated dilutions of the NHS, either absorbed with E. coli J to remove the antibodies to PNAG or E. coli J pga to leave behind antibodies to PNAG. NHS=Normal Human Serum, No C’=No complement. Pool prepared from sera with high levels of interference with the OPK of rabbit antibodies to CP5-TT and CP8-TT.

Anti-CP5-TT Anti-CP5-TT+NHS NHS102

103

104C

FU

(N

ewm

an)/

ml o

f b

loo

d

Anti-CP8-TT Anti-CP8-TT+NHS NHS102

103

104

CF

U(P

S80

)/m

l of

blo

od

A B

NSP<0.05

P<0.05

Supplemental Figure 9 : Effect of pooled NHS on the CFU of S. aureus/ml of blood detected in mice passively administered rabbit antibodies to CP5-TT or CP8-TT.A: CFU S. aureus Newman/ml of blood in mice injected with rabbit antibodies to CP5-TT, CP5-TT plus NHS or NHS alone. Overall ANOVA, P<0.0001, Dunnett’s post-hoc test pair-wise comparisons shown on figure. Challenge: S. aureus Newman (2.0x107cfu). B: CFU S. aureus PS80/ml of blood in mice injected with rabbit antibodies to CP8-TT, CP8-TT plus NHS or NHS alone. Overall ANOVA P<0.0001, Dunnett’s post-hoc test pair-wise comparisons shown on figure. Challenge: S. aureus PS80 (5.3x107 cfu). NS-not significant.

NSP<0.05

P<0.05