Interlaboratory Comparison of Results of an Anthrax Lethal Toxin Neutralization Assay for Assessment of Functional Antibodies in Multiple Species

Omland, Kristian Shawn; Brys, April M.; Lansky, David M.; Clement, Kristin H.; Lynn, Freyja

doi:10.1128/cvi.00003-08

Cited by 32 publications

(34 citation statements)

References 23 publications

(34 reference statements)

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“…Fc␥RII/III-dependent neutralization contributed to a greater extent when PA was employed at a concentration of 400 ng/ml than when it was employed at 50 ng/ml. For most of the experiments presented in this report, we chose to use PA at a concentration of 50 ng/ml since this concentration has been used in assays to evaluate clinical samples (9,24,26). Interestingly, we did not observe this PA concentration dependence for the RAW 264.7 cell-based assay.…”

Section: Discussionmentioning

confidence: 95%

Analysis of the Fc Gamma Receptor-Dependent Component of Neutralization Measured by Anthrax Toxin Neutralization Assays

Verma

Ngundi

Meade

et al. 2009

Clin Vaccine Immunol

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Anthrax toxin neutralization assays are used to measure functional antibody levels elicited by anthrax vaccines in both preclinical and clinical studies. In this study, we investigated the magnitude and molecular nature of Fc gamma (Fc␥) receptor-dependent toxin neutralization observed in commonly used forms of the anthrax toxin neutralization assay. Significantly more Fc␥ receptor-dependent neutralization was observed in the J774A.1 cell-based assay than in the RAW 264.7 cell-based assay, a finding that could be due to the larger numbers of Fc␥ receptors that we found on J774A.1 cells by using flow cytometry. Thus, the extent to which Fc␥ receptor-dependent neutralization contributes to the total neutralization measured by the assay depends on the specific cell type utilized in the assay. Using Fc␥ receptor blocking monoclonal antibodies, we found that at least three murine Fc␥ receptor classes, IIB, III, and IV, can contribute to Fc␥ receptor-dependent neutralization. When antibodies elicited by immunization of rabbits with protective-antigen-based anthrax vaccines were analyzed, we found that the magnitude of Fc␥ receptor-dependent neutralization observed in the J774A.1 cell-based assay was dependent on the concentration of protective antigen utilized in the assay. Our results suggest that the characteristics of the antibodies analyzed in the assay (e.g., species of origin, isotype, and subclass), as well as the assay design (e.g., cell type and protective antigen concentration), could significantly influence the extent to which Fc␥ receptor-dependent neutralization contributes to the total neutralization measured by anthrax toxin neutralization assays. These findings should be considered when interpreting anthrax toxin neutralization assay output.

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Section: Discussionmentioning

confidence: 95%

Analysis of the Fc Gamma Receptor-Dependent Component of Neutralization Measured by Anthrax Toxin Neutralization Assays

Verma

Ngundi

Meade

et al. 2009

Clin Vaccine Immunol

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“…Licensure based on immune correlates is being considered for other vaccines. In the case of anthrax, a lethal toxin neutralization assay likely will be used to correlate the protection offered by new anthrax vaccines in animal models to its immunogenicity in humans (24).…”

Section: Discussionmentioning

confidence: 99%

A Murine Genital-Challenge Model Is a Sensitive Measure of Protective Antibodies against Human Papillomavirus Infection

Longet

Schiller

Bobst

et al. 2011

J Virol

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The available virus-like particle (VLP)-based prophylactic vaccines against specific human papillomavirus (HPV) types afford close to 100% protection against the type-associated lesions and disease. Based on papillomavirus animal models, it is likely that protection against genital lesions in humans is mediated by HPV type-restricted neutralizing antibodies that transudate or exudate at the sites of genital infection. However, a correlate of protection was not established in the clinical trials because few disease cases occurred, and true incident infection could not be reliably distinguished from the emergence or reactivation of prevalent infection. In addition, the current assays for measuring vaccine-induced antibodies, even the gold standard HPV pseudovirion (PsV) in vitro neutralization assay, may not be sensitive enough to measure the minimum level of antibodies needed for protection. Here, we characterize the recently developed model of genital challenge with HPV PsV and determine the minimal amounts of VLP-induced neutralizing antibodies that can afford protection from genital infection in vivo after transfer into recipient mice. Our data show that serum antibody levels >100-fold lower than those detectable by in vitro PsV neutralization assays are sufficient to confer protection against an HPV PsV genital infection in this model. The results clearly demonstrate that, remarkably, the in vivo assay is substantially more sensitive than in vitro PsV neutralization and thus may be better suited for studies to establish correlates of protection.Cervical cancer, the second most common cause of cancer death in women worldwide, is associated with high-risk types of human papillomavirus (HPV) infections (27). HPV vaccines based on L1 virus-like particles (VLPs) have been shown to be safe and efficient at preventing infections and precancerous lesions caused by HPV vaccine-related types (26, 33) and now have been commercialized, specifically the HPV6/11/16/18 VLP Gardasil and the HPV16/18 VLP Cervarix vaccines. Neutralizing antibodies (Ab) are thought to be the primary immune mechanism of protection by HPV vaccination, primarily based on preclinical papillomavirus (PV) animal models showing that the passive transfer of immunized sera is protective in naïve rabbits and dogs against skin and oral mucosal challenge, respectively (3, 31). In addition, clinical trials showed that vaccinated individuals developed robust anti-VLP antibody titers in serum (15, 32) and in cervicovaginal secretions (21, 23), and that antibody-mediated cross-type neutralization in in vitro assays largely parallels the cross-type protection in the trials. However, these trials did not allow the establishment of antibody concentrations or thresholds that could be correlated to protection, mainly because too few disease cases occurred (26, 33) and because breakthrough infections could not be unambiguously distinguished from the emergence or reactivation of prevalent infection. In addition, the serological assays that were used in the trials (pr...

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“…(i) TNA assay. Anthrax toxin-neutralizing antibody levels in the serum samples of laboratory animals and human subjects were measured using a validated TNA assay (37,38). The assay colorimetrically determines the viability of cells exposed to LT using a tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), as the reporter or signal system.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of Immunogenicity and Efficacy of Anthrax Vaccine Adsorbed for Postexposure Prophylaxis

Ионин

Hopkins

Pleune

et al. 2013

Clin Vaccine Immunol

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b Antimicrobials administered postexposure can reduce the incidence or progression of anthrax disease, but they do not protect against the disease resulting from the germination of spores that may remain in the body after cessation of the antimicrobial regimen. Such additional protection may be achieved by postexposure vaccination; however, no anthrax vaccine is licensed for postexposure prophylaxis (PEP). In a rabbit PEP study, animals were subjected to lethal challenge with aerosolized Bacillus anthracis spores and then were treated with levofloxacin with or without concomitant intramuscular (i.m.) vaccination with anthrax vaccine adsorbed (AVA) (BioThrax; Emergent BioDefense Operations Lansing LLC, Lansing, MI), administered twice, 1 week apart. A significant increase in survival rates was observed among vaccinated animals compared to those treated with antibiotic alone. In preexposure prophylaxis studies in rabbits and nonhuman primates (NHPs), animals received two i.m. vaccinations 1 month apart and were challenged with aerosolized anthrax spores at day 70. Prechallenge toxin-neutralizing antibody (TNA) titers correlated with animal survival postchallenge and provided the means for deriving an antibody titer associated with a specific probability of survival in animals. In a clinical immunogenicity study, 82% of the subjects met or exceeded the prechallenge TNA value that was associated with a 70% probability of survival in rabbits and 88% probability of survival in NHPs, which was estimated based on the results of animal preexposure prophylaxis studies. The animal data provide initial information on protective antibody levels for anthrax, as well as support previous findings regarding the ability of AVA to provide added protection to B. anthracis-infected animals compared to antimicrobial treatment alone.

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Interlaboratory Comparison of Results of an Anthrax Lethal Toxin Neutralization Assay for Assessment of Functional Antibodies in Multiple Species

Cited by 32 publications

References 23 publications

Analysis of the Fc Gamma Receptor-Dependent Component of Neutralization Measured by Anthrax Toxin Neutralization Assays

Analysis of the Fc Gamma Receptor-Dependent Component of Neutralization Measured by Anthrax Toxin Neutralization Assays

A Murine Genital-Challenge Model Is a Sensitive Measure of Protective Antibodies against Human Papillomavirus Infection

Evaluation of Immunogenicity and Efficacy of Anthrax Vaccine Adsorbed for Postexposure Prophylaxis

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