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. 2013 Oct;20(10):1549-58.
doi: 10.1128/CVI.00371-13. Epub 2013 Aug 7.

Modified opsonization, phagocytosis, and killing assays to measure potentially protective antibodies against pneumococcal surface protein A

Calvin C Daniels  1 , Kyung-Hyo Kim, Robert L Burton, Shaper Mirza, Melissa Walker, Janice King, Yvette Hale, Patricia Coan, Dong-Kwon Rhee, Moon H Nahm, David E Briles
Affiliations

Modified opsonization, phagocytosis, and killing assays to measure potentially protective antibodies against pneumococcal surface protein A (V体育ios版)

Calvin C Daniels et al. Clin Vaccine Immunol. 2013 Oct.

V体育2025版 - Abstract

The standard opsonophagocytosis killing assay (OPKA) for antibodies to pneumococcal capsular polysaccharide was modified to permit an evaluation of the protection-mediating antibodies to pneumococcal surface protein A (PspA). We found that by increasing the incubation time with the complement and phagocytes from 45 min to 75 min, the protective activity was readily detected. In another modification, we used a capsule type 2 target strain that expressed PspA but not pneumococcal surface protein C (PspC). With these modifications separately or in combination, rabbit antisera to the recombinant α-helical or proline-rich domains of PspA mediated >50% killing of the target strain. The ability of normal human sera to mediate the killing of pneumococci in this modified OPKA correlated with their levels of antibodies to PspA and their ability to protect mice against fatal infection with a type 3 strain. Passive protection of mice against pneumococci and killing in the modified OPKA were lost when normal human sera were adsorbed with recombinant PspA (rPspA) on Sepharose, thus supporting the potential utility of the modified OPKA to detect protective antibodies to PspA. In the standard OPKA, monoclonal antibodies to PspA were strongly protective in the presence of subprotective amounts of anti-capsule. Thus, the currently established high-throughput OPKA for antibodies to capsule could be modified in one of two ways to permit an evaluation of the opsonic efficacy of antibodies to PspA VSports手机版. .

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Figures

Fig 1
Fig 1
Comparison of killing of wild-type S. pneumoniae D39 (A) and its pspC mutant TRE118 (B) in OPKA-1A. The sample dilutions start at 1:4 and extend through 3-fold dilutions to 1:8,748. The solid horizontal line in each panel represents 100% survival of the bacteria. The dashed horizontal line in each panel represents 50% killing. ○, human pool 22 (containing Abs to type 2 PS); ▲, rabbit antisera to family 1 PspA; △, rabbit antisera to family 2 PspA. The antiserum to family 2 was adsorbed with family 1 PspA to make it relatively specific for family 2 (46). The target strains express family 1 PspA.
Fig 2
Fig 2
Comparisons of combined 45-min versus sequential (30 plus 45 min) incubations on killing of the antibody-coated pspC mutant, TRE118, in OPKA-2A and OPKA-2B. In the OPKA-2A, antibody-coated bacteria were incubated with BRC (complement) and HL-60 cells for 45 min. In OPKA-2B, the antibody-coated bacteria had separate sequential incubations with BRC for 30 min and HL-60 cells for 30 min. Rabbit serum specific for the family 1 PspA α-helical domain (A) and rabbit antiserum specific for the proline-rich domain containing a nonproline block (B) were diluted 1:10, 1:30, or 1:90 and evaluated for killing in OPKA-2A and OPKA-2B. A human serum sample J3 from a nonimmunized adult (C) was diluted 1:30, 1:100, and 1:300 and tested in OPKA-2A and OPKA-2B. In each panel, the percentages of the target bacteria killed in OPKA-2A are shown in black; the results for OPKA-2B are shown in red. The greater killing in OPKA-2B than in OPKA-2A was statistically significant at P values of 0.0009, 0.0004, and 0.035 for panels A, B, and C, respectively. The means and standard errors are shown for each data set. To calculate statistical significance between the treatments with each serum, we first ranked all 6 data points (three from the OPKA-2A and three from the OPKA-2B) at each dilution. Next (A and B), the ranked data points all for all three dilutions were grouped into two groups of 9 according to assay type. Next, the 9 data points for OPKA-2A were compared with the 9 for OPKA-2B by the Mann-Whitney two-sample rank test. With the human serum (C), the highest dilution showed no killing with either treatment. For this serum, only data from the 1:30 and 1:100 dilutions were used for statistical analysis.
Fig 3
Fig 3
Analysis of 19 normal human serum samples to look for correlations between levels of antibody to PspA or capsule (y axis) and the ability of the sera to protect mice from fatal pneumococcal infection with capsular serotype 3 strain A66.1 (x axis). The correlation between IgG antibody levels to PspA and passive protection was highly significant by Pearson's linear regression correlation (r = 0.71, P = 0.0007). However, there was no correlation between IgG antibody levels to type 3 capsule and passive protection (r = 0.39, P = 0.11). Seven of these sera (open symbols) were also evaluated in the experiment shown in Fig. 4B.
Fig 4
Fig 4
Correlation of killing activity in the OPKA-2B against the TRE118 target strain with passive protection against type 3 infection. (A) A human serum sample selected from the 19 examined in Fig. 3 that was highly protective in mice and a poorly protective human serum sample were examined in the OPKA-2B (at 6 replicates per data point) for their ability to kill strain TRE118. The poorly protective serum showed less killing of TRE118 at all dilutions than the highly protective serum. At 1:30 and 1:90, the two sera differed at a P value of <0.0001 by Student's t test. (B) Seven serum samples were selected from the 19 sera in Fig. 3 (open symbols) that covered the range of activity in the passive protection and anti-PspA assays. All 7 sera were tested at a 1:90 dilution in the OPKA-2B against strain TRE118, with 6 replicates per data point; the mean percentage of OPKA killing is shown. The means and standard errors for each data set are shown. The mean percentage of TRE118 pneumococci killed at a 1:90 dilution showed a significant correlation with passive protection (r = 0.85, P = 0.024) by Pearson's linear regression correlation.
Fig 5
Fig 5
Adsorption of normal human serum with rPspA Sepharose removes its antipneumococcal activity in OPKA-2B against TRE118 target strains and in passive protection against fatal infection. The normal human serum sample J3, also depicted in Fig. 2C, was adsorbed with rPspA Sepharose 4B. The adsorption also resulted in a dilution that reduced the serum concentration to 61% of its original concentration. Correcting for this dilution, the adsorption removed 97.3% of the antibodies to PspA and none of the IgG antibodies to type 3 PS (data not shown). A mock adsorption with BSA-conjugated Sepharose 4B resulted in the same dilution but did not remove Abs to PspA (data not shown). Shown is the mean percent killing and standard error of TRE118 in the OPKA-2B for the no-serum control (Ringer's diluent), the mock-adsorbed serum, and the PspA-adsorbed serum in the bar graphs. A passive protection study was also conducted with Ringer's lactate, mock-adsorbed serum, and PspA-adsorbed serum using 5 mice per group. Each mouse was given 100 μl of a 1:10 dilution of the indicated serum intraperitoneally (i.p.) 1 h prior to i.v. injection of CBA/N mice with 500 CFU of strain A66.1 type 3 S. pneumoniae. The time to moribund is depicted for each mouse as individual data points. The study was terminated at 510 h postchallenge, and all surviving mice are plotted at 510 h. The postadsorption sera showed significantly (*) less killing in the modified OPKA (P = 0.019) and in passive protection (P = 0.02) than did the serum adsorbed with BSA Sepharose.
Fig 6
Fig 6
Synergy between MAbs to capsule and MAbs to PspA in the standard UAB OPKA. For this study, the Hyp3M6 MAb to type 3 PS was purposefully used at a concentration that only weakly mediated killing of the type 3 target strain (●). The anti-PspA MAb XiR278 was used as an ascites fluid without prior dilution. When XiR278 was used by itself, it mediated no (<5%) measurable killing at any dilution (data not shown). Red line, the result when XiR278 is mixed with the anti-type 3 MAb. Open symbols, the results when the anti-type 3 MAb was mixed with an anti-type 6B MAb, which was anticipated not to react with the type 3 PS target strain.
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