Capsular polysaccharides are an important immune evasion mechanism for Staphylococcus aureus

Figures & data

Table 1. Capsule expression reduces non-specific kill of S aureus strains in OPA

Strain	Growth time (hr)^a	Control IgG (MFI)	CP5 (MFI)^b	CP8 (MFI)^c	Protein A (MFI)	%kill^d
PFESA0158 (CP5)	4	163	283		972	-61
	6	372	472		953	-54
	22	649	8,858		966	-30
PFESA0186 (CP8)	4	85		574	32,389	-30
	6	101		621	34,015	-28
	22	160		28,378	28,999	-7

^a PFESA0158 and PFESA0186 were grown in TSB to different growth phase and their CP expression levels were determined using flow cytometry. ^bStrain PFESA0158 was stained with CP5 mAb and compared with isotype matched mIgG as control. ^cStrain PFESA0186 was stained with CP8 mAb and compared with isotype matched mIgG as control. ^dNegative number denotes non-specific kill. Data are average of four independent experiments.

Figure 1. Enhanced non-specific killing with increasing complement concentration in OPA. (A) PFESA0158 and (B) PFESA0186 strains were incubated with either 2%, 5% or 10% complement plus HL60 cells at 37°C for 60 min. Negative and positive percentage in the bar graphs denotes kill and growth, respectively The surviving bacteria were counted using a CTL immunospot® reader. Percent kill/growth was calculated by comparing the bacterial counts at T0 (input) and at 60 min post-phagocytosis incubation step. Each bar is average of four independent experiments ± standard deviation.

Table 2. MRSA and MSSA strains expressing high levels of capsule are less susceptible to non-specific killing in OPA

Strain	Capsular type	Methicillin susceptibility	Control IgG (MFI)	CP5 (MFI)^b	CP8 (MFI)^c	Protein A (MFI)	% growth^d
PFESA0158	5	MSSA	54	20,000		64	48
PFESA0046	5	MRSA	37	9,887		633	61
PFESA0048	5	MRSA	42	16,400		1,205	55
PFESA0054	5	MRSA	161	23,500		61	61
PFESA0186	8	MSSA	36		33,300	38	24
PFESA0012	8	MRSA	70		21,300	41	6
PFESA0110	8	MRSA	62		18,700	5,017	-4
PFESA0120	8	MRSA	101		20,600	3,062	28

^a Strains were grown overnight in modified Frantz media and their CP expression levels were determined using flow cytometry. ^bCP5 strains were stained with CP5 mAb and compared with isotype matched mIgG control. ^cCP8 strains were stained with CP8 mAb and compared with isotype matched mIgG control. ^dPositive number denotes growth.

Figure 2. CP5 conjugate vaccine induced immune sera that effectively kill MRSA strain PFESA0046. Sera samples (7 and 8) from rhesus macaques vaccinated with the CP5-CRM₁₉₇ conjugate was tested for OP activity against PFESA0046 strain. Non-vaccinated serum samples (2 and 3) were used as negative controls for the assay. The titer is defined as the reciprocal of the highest serum dilution that kills 50% of the test bacteria. The CFU associated with 50% bacterial killing is indicated by the horizontal line.

Figure 3. CP8 conjugate vaccine induced immune sera that effectively kill MSSA strain PFESA0186. Sera samples (5 and 6) from rhesus macaques vaccinated with the CP8-CRM₁₉₇ conjugate was tested for OP activity against PFESA0186 strain. Non-vaccinated serum samples (1 and 2) were used as negative controls for the assay. The titer is defined as the reciprocal of the highest serum dilution that kills 50% of the test bacteria. The CFU associated with 50% bacterial killing is indicated by the horizontal line.

Table 3. OPA titers against MRSA strains expressing CP5 and SpA antigen

	PFESA0046^d			PFESA0048^e
Sample #^a	GMT^b	95% CI Lower	95% CI Upper	GMT	95% CI Lower	95% CI Upper
1	50^c	50	50	50	50	50
2	50	50	50	50	50	50
3	50	50	50	50	50	50
4	50	50	50	50	50	50
5	1,651	958	2,344	2,086	1,685	2,487
6	2,964	1,998	3,931	3,265	2,100	4,429
7	3,711	3,589	3,833	4,646	4,079	5,213
8	4,559	4,163	4,955	4,856	2,857	6,854
9	12,149	8,205	16,092	10,853	9,478	12,228
10	9,836	5,353	14,319	21,244	14,190	28,299
11	22,087	14,534	29,640	21,244	14,190	28,299

^a Samples 1–4 are non-immune and 5–11 are immune sera from rhesus macaques vaccinated with either 10 or 20 μg of CP5-CRM₁₉₇ conjugate vaccine. ^bOPA titers are defined as the reciprocal of the highest serum dilution that kills 50% of the bacteria. Each sample was tested in replicates and geometric mean titers with 95% confidence interval were calculated using data from three independent experiments. ^cNegative samples were arbitrarily assigned a titer of 50, which is half of the lowest serum dilution tested in the OPA. ^dStrain PFESA0046 antigen expression levels (MFI): CP5- 9,887; SpA- 633. ^eStrain PFESA0048 antigen expression levels (MFI): CP5–16,400; SpA- 1,205.

Table 4. OPA titers against MSSA strain expressing CP8 antigen

	PFESA0186^d
Sample #^a	GMT^b	95% CI Lower	95% CI Upper
1	50^c	50	50
2	50	50	50
3	50	50	50
4	50	50	50
5	13,353	12,311	14,396
6	4,845	4,784	4,906
7	6,973	5,317	8,629
8	11,484	8,970	13,998
9	6,969	5,354	8,584

^a Samples 1–4 are non-immune and 5–9 are immune sera from rhesus macaques vaccinated with either 20 or 100 μg of CP8-CRM₁₉₇ conjugate vaccine. ^bOPA titers are defined as the reciprocal of the highest serum dilution that kills 50% of the bacteria. Each sample was tested in replicates and geometric mean titers with 95% confidence interval were calculated using data from two independent experiments. ^cNegative samples were arbitrarily assigned a titer of 50, which is half of the lowest serum dilution tested in the OPA. ^dStrain PFESA0186 antigen expression levels (MFI): CP8- 33,300; SpA- 38

Figure 4. Anti-capsular antibodies show antigen specific killing for MRSA strain PFESA0046 and PFESA0048. CP5 immune rhesus macaque serum OP activity against (A) PFESA0046 and (B) PFESA0048 strains was competed with 1μg of either purified CP5-PS (homologous polysaccharide) or CP8-PS (heterologous polysaccharide). Abrogation of kill activity in the presence of homologous polysaccharide demonstrates that the assay is specific.