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Clinical and Vaccine Immunology, July 2008, p. 1134-1137, Vol. 15, No. 7
1071-412X/08/$08.00+0     doi:10.1128/CVI.00091-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Modified Caspase-3 Assay Indicates Correlation of Caspase-3 Activity with Immunity of Nonhuman Primates to Yersinia pestis Infection

Susan Welkos,^1* Sarah Norris,² and Jeffrey Adamovicz³

Bacteriology Division, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), 1425 Porter Street, Fort Detrick, Frederick, Maryland 21702,¹ Biostatistical Services Division, USAMRIID, Frederick, Maryland 21702,² Midwest Research Institute, Frederick, MD 21702³

Received 14 March 2008/ Returned for modification 17 April 2008/ Accepted 8 May 2008

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Defined candidate human vaccines for treating infection by Yersinia pestis, the agent of plague, have been developed. To facilitate evaluation of the vaccines' efficacy, the in vitro caspase-3 assay for cytotoxicity-neutralizing activity was modified and reevaluated. Immune serum-mediated caspase-3 neutralizing activity correlated with protection against infection in a nonhuman primate vaccine model of plague immunity.

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Yersinia pestis, the etiologic agent of plague, has a significant potential to be utilized as a biothreat or a bioweapon (6, 7). Countermeasures, especially those that are effective against pneumonic plague, are critically needed. New candidate vaccines consisting of the F1 capsule antigen and the LcrV, or V, virulence antigen have been developed (5, 10). Both the F1 and the V antigens are highly immunogenic, and they stimulate antibodies that confer passive protection. As the efficacy of these new vaccines cannot be tested in humans, it is essential to develop in vitro surrogate assays that are valid predictors of immunity. We recently described the development of macrophage (M)-based correlate assays of immunity to infection by Y. pestis (1). These assays included a microtiter fluorometric assay of the apoptosis-specific enzyme caspase-3 in J774.A1 Ms, and a fluorescence-activated cell sorter (FACS)-based live-dead staining assay of terminal necrosis of human-derived HL60 cells (1). Sera from mice and nonhuman primates (NHP) vaccinated with the F1-V fusion protein vaccine (5) were evaluated for the levels at which they neutralized the cytotoxicity induced by infection of Ms with Y. pestis or the Y. pseudotuberculosis strain Yptb (pTrcV) (1). The results of both the FACS live-dead assay and the caspase-3 assay for serum cytotoxicity neutralization activity (CNA) correlated well with immunity to plague in mice. However, the caspase-3 assay results did not correlate as well as those of the FACS assay with survival in vaccinated NHP, the species that appears to best model human responses to plague, and the FACS assay was proposed as a candidate in vitro correlate assay. Nonetheless, compared to the FACS assay, the caspase-3 assay is easier to perform and analyze, it is faster and requires less expensive equipment, and it is more amenable to large-scale testing of sera from vaccinees. In addition, NIAID- and DOD-sponsored research of new biodefense countermeasures stipulates the development of a validated in vitro correlate assay(s) of immunity (4). Such validated assays are also required, as part of the advanced development, to secure FDA licensure of the F1-V human plague vaccine; and phase 1 and 2 clinical trials of the F1-V fusion vaccine have been initiated (2). Results of prevalidation studies using the FACS and caspase-3 assays (data not shown) indicated that the latter would more readily fulfill the validation requirements that include demonstration of assay reproducibility, repeatability, and robustness (3, 8). Thus, the purposes of this study were to modify and optimize the performance of the caspase-3 procedure and to test this modified assay as a potential in vitro predictor of immunity in vaccinated NHP. Sera from a large, recently completed study done with cynomolgus macaques to test F1-V vaccine efficacy were available for use, as described below.

Numerous changes were made to the caspase-3 microtiter assay, and a complete protocol is shown in Table 1. Changes were made in the following: the cell and bacterial culture media, the final concentration of bacteria in the assay, the amounts of antibody used in pretreatment and titration steps, and the assay incubation periods. Also, greater emphasis was given to important aspects of bacterial propagation, and more details were provided for statistical analyses of data (Table 1). The modified procedure was evaluated initially by titrating the CNA of a rabbit anti-V immunoglobulin G (IgG) preparation, which was used as the positive control in the original M assay (1). A good dose response was observed in twofold titration assays as determined by regression analysis. Whereas rabbit anti-V IgG diluted 1/100 protected 85% of the cells, the highest dilution of 1/1,600 protected only 3% of the Ms against Yersinia infection-induced apoptosis (Fig. 1), and a 50% reciprocal neutralization titer value of 457 was determined, as described in the legend to Fig. 1.

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TABLE 1. Modified caspase-3 assay for serum neutralization of macrophage cytotoxicity

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FIG. 1. Titration of the CNA of the rabbit anti-V IgG positive control. Twofold dilutions of the IgG were assayed in the caspase-3 microtiter fluorescence assay. Caspase-3 levels represent a marker for M cytotoxicity (apoptotic cell death) associated with the Yersinia infection. The data shown (dashed line with symbols) are the mean percentages of decrease in caspase-3 values for each dilution tested (in quadruplicate). These values were analyzed by four-parameter nonlinear regression (solid line) using GraphPad Prism5 software. The dilution interpolated to represent 50% neutralizing activity in this analysis was 1/457 (log 2.66); r2 value, 0.9998.

Sera from a recent study of the efficacy of the F1-V fusion vaccine in cynomolgus macaques were tested with the caspase-3 assay; the results of that study will be reported separately. That study, which involved four cohorts of 16 animals each, assessed the association between vaccine dose and protection against lethal aerosol challenge with the virulent Y. pestis strain C092 (cohorts 2 to 4). It also analyzed the long-term immunity of the vaccine (cohort 1). The NHP in all four groups were vaccinated three times with F1-V by the intramuscular route on days 0, 56, and 182 and were challenged by aerosol exposure to lethal doses of strain C092 at 1 month (cohorts 2 to 4) or 1 year (day 547; cohort 1) after the third vaccine dose. Sera were collected at various times over the course of the prechallenge vaccination period for in vitro evaluation. Further details of the vaccination doses and schedule are described in the figure legends and by Adamovicz et al. (manuscript in preparation). Figure 2 illustrates the caspase-3 results of tests done with sera collected on five separate days from three representative F1-V-vaccinated NHP of cohort 1. These results, as well as those of assays of additional sets of sera and those including sera collected on days 0, 14, 56, 70, 182, 196, and 469 (cohort 4 only) of the vaccination period, confirmed a finding reported previously for the live-dead assays (1). For all four animal groups, the day 70 and day 196 sera, which were collected 2 weeks after the second and third doses of vaccine, respectively, exhibited the greatest extent of M protection compared to that associated with sera collected at other times during vaccination (Fig. 2 and data not shown).

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FIG. 2. Neutralization of Yersinia-mediated J774.A1 macrophage cytotoxicity by immune sera from F1-V-vaccinated cynomolgus macaques, as detected in the caspase-3 fluorescence assay. The caspase-3 data are shown as mean relative fluorescence units (RFU) (± standard deviations) of four replicates per serum sample (1). Tests were done with sera from three representative F1-V-vaccinated NHP of cohort 1 of this vaccine trial, and the results shown are those using sera collected on vaccination days 0, 56, 70, 182, and 196. Data are grouped by the day of serum collection. The three controls were (left to right) uninfected Ms, infected untreated Ms, and Ms infected with bacteria pretreated with the positive control rabbit anti-V IgG. NS, nonsurvivor; S, survivor. *, P 0.0001, compared to the mean value for the day 0 serum-treated samples (analysis of variance and t test).

The relationship between the caspase-3 levels of Ms infected with serum-pretreated Yersinia and the survival of the vaccinated NHP was then evaluated by logistic regression analysis of the pooled cohort data. As illustrated in Fig. 3, the extent to which caspase-3 levels decreased was significantly associated with survival for both the day 70 and the day 196 results (P = 0.0038 and P = 0.0024, respectively). The serum CNA was also highly correlated with the anti-V and anti-F1 enzyme-linked immunosorbent assay titers for both the day 70 and the day 196 sera (P 0.001, data not shown). The significance was observed when the caspase-3 levels measured in Ms infected with the pretreated bacteria were compared to either the experimental negative control level (infected, nontreated Ms; P = 0.0020 to 0.0001; data not shown) or to the caspase-3 levels measured in infected Ms pretreated with day 0 (preimmune) sera from each animal (P < 0.0001) (Fig. 3).

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FIG. 3. The relationship between the level of caspase-3 in infected Ms and the probability of survival after challenge in F1-V-immunized NHP. J774.A1 Ms were infected with bacteria [Yptb (pTrcV)] that had been pretreated with serum collected from F1-V-vaccinated (or nonvaccinated control) cynomolgus macaques on vaccination days 70 (open symbols) and 196 (filled symbols). The effects of the treatment on apoptotic cell death of the Ms were determined with the caspase-3 assay, and the results were expressed as the percent of decrease in caspase-3 levels (%); the latter were calculated based on the day 0 value for each NHP sera set. The significances of the association between the probability of survival after challenge with virulent Y. pestis and the in vitro cytotoxicity-neutralizing activity (percent of decrease in caspase-3) were evaluated by logistic regression. Data were analyzed with SAS/STAT software, version 9.1.3 (SAS system for PC; SAS Institute Inc.).

Thus, the results of the modified caspase-3 microtiter assay for serum-mediated CNA were highly correlated with survival status of F1-V-vaccinated NHP after they were challenged with virulent Y. pestis.

 
The research described herein was supported by JSTO-CBD/DTRA, project no. 5.10047-05-RDB.

The views expressed in this paper are those of the authors and do not reflect the official policy or position of the Department of the Army, Department of Defense, or the United States Government.

 
* Corresponding author. Mailing address: Bacteriology Division, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), 1425 Porter Street, Fort Detrick, Frederick, MD 21702. Phone: (301) 619-4930. Fax: (301) 619-2152. E-mail: susan.welkos{at}amedd.army.mil

Published ahead of print on 14 May 2008.

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Clinical and Vaccine Immunology, July 2008, p. 1134-1137, Vol. 15, No. 7
1071-412X/08/$08.00+0     doi:10.1128/CVI.00091-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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