The Experts below are selected from a list of 327 Experts worldwide ranked by ideXlab platform
Antonio Fasanella - One of the best experts on this subject based on the ideXlab platform.
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development of a sterne based complement fixation test to monitor the humoral response induced by Anthrax Vaccines
Frontiers in Microbiology, 2016Co-Authors: Rosanna Adone, Massimiliano Francia, Michela Sali, Michela Iatarola, Adelia Donatiello, Antonio FasanellaAbstract:Anthrax is a zoonotic disease caused by Bacillus anthracis spore-forming bacterium. Since it is primarily a disease of animals, the control in animals and humans depends on the prevention in livestock, principally cattle, sheep and goats. Most veterinary Vaccines utilize the toxigenic, uncapsulated (pXO1+/pXO2–) B.anthracis strain 34F2 which affords protection through the production of neutralizing antibodies directed to the toxin components Protective Antigen (PA), Lethal Factor (LF) and Edema Factor (EF). The titration of specific antibodies in sera of vaccinated animals is crucial to evaluate the efficacy of the vaccination and to obtain epidemiological information for an effective Anthrax surveillance. In this study, we developed a Sterne-based Complement Fixation Test to detect specific antibodies induced in animals vaccinated with Sterne 34F2 . We assessed its efficacy in laboratory animals and under field conditions by monitoring the humoral response induced by vaccination in cattle. The results indicated that the Sterne-based CFT is able to identify vaccinated animals with a good sensitivity and specificity offering many benefits especially with regard to costs, standardization and reproducibility of the assay procedure.
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development of a sterne based complement fixation test to monitor the humoral response induced by Anthrax Vaccines
Frontiers in Microbiology, 2016Co-Authors: Rosanna Adone, Massimiliano Francia, Michela Sali, Michela Iatarola, Adelia Donatiello, Antonio FasanellaAbstract:Anthrax is a zoonotic disease caused by Bacillus anthracis spore-forming bacterium. Since it is primarily a disease of animals, the control in animals, and humans depend on the prevention in livestock, principally cattle, sheep, and goats. Most veterinary Vaccines utilize the toxigenic, uncapsulated (pXO1+/pXO2-) B. anthracis strain 34F2 which affords protection through the production of neutralizing antibodies directed to the toxin components Protective Antigen (PA), Lethal Factor (LF), and Edema Factor (EF). The titration of specific antibodies in sera of vaccinated animals is crucial to evaluate the efficacy of the vaccination and to obtain epidemiological information for an effective Anthrax surveillance. In this study, we developed a Sterne-based Complement Fixation Test (CFT) to detect specific antibodies induced in animals vaccinated with Sterne 34F2. We assessed its efficacy in laboratory animals and under field conditions by monitoring the humoral response induced by vaccination in cattle. The results indicated that the Sterne-based CFT is able to correctly identify vaccinated animals. It proved to be a very sensitive and specific test. Moreover, the Sterne-based CFT offers many benefits with regard to costs, standardization and reproducibility of the assay procedure.
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protective activity and immunogenicity of two recombinant Anthrax Vaccines for veterinary use
Vaccine, 2008Co-Authors: Antonio Fasanella, Rosanna Adone, Fiorella Tonello, Giuliano Garofolo, Lucia Muraro, Alessandra Carattoli, C MontecuccoAbstract:In this study, the efficacy of two experimental Vaccines against Bacillus anthracis toxinaemia was evaluated in the rabbit model. A recombinant Protective Antigen (rPA) mutant and a trivalent vaccine (TV) composed by the rPA, a inactive mutant of Lethal Factor (mLF-Y728A; E735A) and a inactive mutant of Edema Factor (mEF-K346R), both emulsified with mineral oils, were evaluated for their immunogenicity and protective activity in New Zealand white rabbits. Rabbits vaccinated subcutaneously with rPA and TV rapidly produced high level of anti-PA, anti-LF and anti-EF antibodies, which were still present 6 months later. In the efficacy test, these Vaccines protected 100% of rabbits challenged with B. anthracis virulent strain 0843 one week after the vaccination. Moreover, all animals vaccinated twice with rPA and TV, resisted B. anthracis infection 6 months later. Our data indicate that rPA and TV could be good vaccine candidates for inducing protection against B. anthracis infection in target animal host. They could successfully be used in an emergency with simultaneous long-acting antibiotics to halt incubating infections or during an Anthrax epidemic.
Stephen F. Little - One of the best experts on this subject based on the ideXlab platform.
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a comparison of the adaptive immune response between recovered Anthrax patients and individuals receiving three different Anthrax Vaccines
PLOS ONE, 2016Co-Authors: Thomas R Laws, Stephen F. Little, Tinatin Kuchuloria, Nazibriola Chitadze, Wendy M Webster, Amanda K Debes, Salome Saginadze, Nikoloz Tsertsvadze, Mariam Chubinidze, Robert G RivardAbstract:Several different human Vaccines are available to protect against Anthrax. We compared the human adaptive immune responses generated by three different Anthrax Vaccines or by previous exposure to cutaneous Anthrax. Adaptive immunity was measured by ELISPOT to count cells that produce interferon (IFN)-γ in response to restimulation ex vivo with the Anthrax toxin components PA, LF and EF and by measuring circulating IgG specific to these antigens. Neutralising activity of antisera against Anthrax toxin was also assayed. We found that the different exposures to Anthrax antigens promoted varying immune responses. Cutaneous Anthrax promoted strong IFN-γ responses to all three antigens and antibody responses to PA and LF. The American AVA and Russian LAAV Vaccines induced antibody responses to PA only. The British AVP vaccine produced IFN-γ responses to EF and antibody responses to all three antigens. Anti-PA (in AVA and LAAV vaccinees) or anti-LF (in AVP vaccinees) antibody titres correlated with toxin neutralisation activities. Our study is the first to compare all three Vaccines in humans and show the diversity of responses against Anthrax antigens.
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structural and immunological analysis of Anthrax recombinant protective antigen adsorbed to aluminum hydroxide adjuvant
Clinical and Vaccine Immunology, 2012Co-Authors: Leslie Wagner, Anita Verma, Karine Reiter, Bruce D Meade, Stephen F. Little, Rebecca A. Brady, David L Narum, Drusilla L BurnsAbstract:New Anthrax Vaccines currently under development are based on recombinant protective antigen (rPA) and formulated with aluminum adjuvant. Because long-term stability is a desired characteristic of these Vaccines, an understanding of the effects of adsorption to aluminum adjuvants on the structure of rPA is important. Using both biophysical and immunological techniques, we compared the structure and immunogenicity of freshly prepared rPA-Alhydrogel formulations to that of formulations stored for 3 weeks at either room temperature or 37°C in order to assess the changes in rPA structure that might occur upon long-term storage on aluminum adjuvant. Intrinsic fluorescence emission spectra of tryptophan residues indicated that some tertiary structure alterations of rPA occurred during storage on Alhydrogel. Using anti-PA monoclonal antibodies to probe specific regions of the adsorbed rPA molecule, we found that two monoclonal antibodies that recognize epitopes located in domain 1 of PA exhibited greater reactivity to the stored formulations than to freshly prepared formulations. Immunogenicity of rPA-Alhydrogel formulations in mice was assessed by measuring the induction of toxin-neutralizing antibodies, as well as antibodies reactive to 12-mer peptides spanning the length of PA. Mice immunized with freshly prepared formulations developed significantly higher toxin-neutralizing antibody titers than mice immunized with the stored preparations. In contrast, sera from mice immunized with stored preparations exhibited increased reactivity to nine 12-mer peptides corresponding to sequences located throughout the rPA molecule. These results demonstrate that storage of rPA-Alhydrogel formulations can lead to structural alteration of the protein and loss of the ability to elicit toxin-neutralizing antibodies.
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advances in the development of next generation Anthrax Vaccines
Vaccine, 2009Co-Authors: Arthur M Friedlander, Stephen F. LittleAbstract:Anthrax, a disease of herbivores, only rarely infects humans. However, the threat of using Bacillus anthracis, the causative agent, to intentionally produce disease has been the impetus for development of next-generation Vaccines. Two licensed Vaccines have been available for human use for several decades. These are composed of acellular culture supernatants containing the protective antigen (PA) component of the Anthrax toxins. In this review we summarize the various approaches used to develop improved Vaccines. These efforts have included the use of PA with newer adjuvants and delivery systems, including bacterial and viral vectors and DNA Vaccines. Attempts to broaden the protection afforded by PA-based Vaccines have focused on adding other B. anthracis components, including spore and capsule antigens.
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an in vivo passive protection assay for the evaluation of immunity in ava vaccinated individuals
Vaccine, 2008Co-Authors: John F Hewetson, B E Ivins, Phillip R. Pittman, Stephen F. Little, Sarah L Norris, Wendy M Johnson, Edward J Brown, Carl J NielsenAbstract:Samples of human plasma from Anthrax vaccine adsorbed (AVA, BioThrax™)-vaccinated individuals were used to demonstrate passive protection of A/J mice from a lethal challenge with the Sterne strain of Anthrax bacteria. The maximum concentration of human anti-protective antigen IgG in mouse sera 24 h after injection of 260 μg of anti-PA IgG was 134 μg/ml, declining to 91 μg/ml at 72 h (half-life = 101.7 h). Mice showed significant survival (p ≤ 0.001) after injection of serial dilutions up to 1:4 of the standard plasma and challenged with 100 LD50. Similarly, mice injected with the standard anti-AVA plasma and challenged up to 5 days post-treatment also survived (p ≤ 0.001). Using a cohort of human plasma to measure passive protection, the best correlation between passive protection and an in vitro assay was found to be with the quantitative toxin neutralization assay (minimum fold increase in odds of survival: 2.71, p = 0.0062). These results demonstrate a reliable in vivo neutralization method that correlates with standard in vitro measures of neutralizing antibody levels in plasma from individuals vaccinated with the standard Anthrax vaccine. This analytical method may provide additional opportunities to compare the efficacy of improved Anthrax Vaccines with the licensed vaccine.
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An in vivo passive protection assay for the evaluation of immunity in AVA-vaccinated individuals ☆
Vaccine, 2008Co-Authors: John F Hewetson, B E Ivins, Phillip R. Pittman, Stephen F. Little, Sarah L Norris, Wendy M Johnson, J. Edward Brown, Carl J NielsenAbstract:Samples of human plasma from Anthrax vaccine adsorbed (AVA, BioThrax™)-vaccinated individuals were used to demonstrate passive protection of A/J mice from a lethal challenge with the Sterne strain of Anthrax bacteria. The maximum concentration of human anti-protective antigen IgG in mouse sera 24 h after injection of 260 μg of anti-PA IgG was 134 μg/ml, declining to 91 μg/ml at 72 h (half-life = 101.7 h). Mice showed significant survival (p ≤ 0.001) after injection of serial dilutions up to 1:4 of the standard plasma and challenged with 100 LD50. Similarly, mice injected with the standard anti-AVA plasma and challenged up to 5 days post-treatment also survived (p ≤ 0.001). Using a cohort of human plasma to measure passive protection, the best correlation between passive protection and an in vitro assay was found to be with the quantitative toxin neutralization assay (minimum fold increase in odds of survival: 2.71, p = 0.0062). These results demonstrate a reliable in vivo neutralization method that correlates with standard in vitro measures of neutralizing antibody levels in plasma from individuals vaccinated with the standard Anthrax vaccine. This analytical method may provide additional opportunities to compare the efficacy of improved Anthrax Vaccines with the licensed vaccine.
Conrad P Quinn - One of the best experts on this subject based on the ideXlab platform.
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Anthrax vaccine induced antibodies provide cross species prediction of survival to aerosol challenge
Science Translational Medicine, 2012Co-Authors: Dean Follmann, Jarad Schiffer, Conrad P Quinn, Gregory V Stark, Robert Kohberger, Freyja Lynn, Edwin O NuzumAbstract:Because clinical trials to assess the efficacy of Vaccines against Anthrax are not ethical or feasible, licensure for new Anthrax Vaccines will likely involve the Food and Drug Administration’s “Animal Rule,” a set of regulations that allow approval of products based on efficacy data only in animals combined with immunogenicity and safety data in animals and humans. U.S. government–sponsored animal studies have shown Anthrax vaccine efficacy in a variety of settings. We examined data from 21 of those studies to determine whether an immunological bridge based on lethal toxin neutralization activity assay (TNA) can predict survival against an inhalation Anthrax challenge within and across species and genera. The 21 studies were classified into 11 different settings, each of which had the same animal species, vaccine type and formulation, vaccination schedule, time of TNA measurement, and challenge time. Logistic regression models determined the contribution of vaccine dilution dose and TNA on prediction of survival. For most settings, logistic models using only TNA explained more than 75% of the survival effect of the models with dose additionally included. Cross-species survival predictions using TNA were compared to the actual survival and shown to have good agreement (Cohen’s k ranged from 0.55 to 0.78). In one study design, cynomolgus macaque data predicted 78.6% survival in rhesus macaques (actual survival, 83.0%) and 72.6% in rabbits (actual survival, 64.6%). These data add support for the use of TNA as an immunological bridge between species to extrapolate data in animals to predict Anthrax vaccine effectiveness in humans.
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Vaccination of Rhesus Macaques with the Anthrax Vaccine Adsorbed Vaccine Produces a Serum Antibody Response That Effectively Neutralizes Receptor-Bound Protective Antigen In Vitro
Clinical and vaccine immunology : CVI, 2010Co-Authors: Kristin H Clement, Thomas L. Rudge, Carol L K Sabourin, Nancy A Niemuth, April M Brys, Heather J. Mayfield, Lena A. Carlton, Arelis Hester, Conrad P QuinnAbstract:Anthrax toxin (ATx) is composed of the binary exotoxins lethal toxin (LTx) and edema toxin (ETx). They have separate effector proteins (edema factor and lethal factor) but have the same binding protein, protective antigen (PA). PA is the primary immunogen in the current licensed vaccine Anthrax vaccine adsorbed (AVA [BioThrax]). AVA confers protective immunity by stimulating production of ATx-neutralizing antibodies, which could block the intoxication process at several steps (binding of PA to the target cell surface, furin cleavage, toxin complex formation, and binding/translocation of ATx into the cell). To evaluate ATx neutralization by anti-AVA antibodies, we developed two low-temperature LTx neutralization activity (TNA) assays that distinguish antibody blocking before and after binding of PA to target cells (noncomplexed [NC] and receptor-bound [RB] TNA assays). These assays were used to investigate anti-PA antibody responses in AVA-vaccinated rhesus macaques (Macaca mulatta) that survived an aerosol challenge with Bacillus anthracis Ames spores. Results showed that macaque anti-AVA sera neutralized LTx in vitro, even when PA was prebound to cells. Neutralization titers in surviving versus nonsurviving animals and between prechallenge and postchallenge activities were highly correlated. These data demonstrate that AVA stimulates a myriad of antibodies that recognize multiple neutralizing epitopes and confirm that change, loss, or occlusion of epitopes after PA is processed from PA83 to PA63 at the cell surface does not significantly affect in vitro neutralizing efficacy. Furthermore, these data support the idea that the full-length PA83 monomer is an appropriate immunogen for inclusion in next-generation Anthrax Vaccines.
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Use of Anthrax vaccine in the United States: recommendations of the Advisory Committee on Immunization Practices (ACIP), 2009.
MMWR. Recommendations and reports : Morbidity and mortality weekly report. Recommendations and reports, 2010Co-Authors: Jennifer Gordon Wright, Conrad P Quinn, S Shadomy, Nancy MessonnierAbstract:These recommendations from the Advisory Committee on Immunization Practices (ACIP) update the previous recommendations for Anthrax vaccine adsorbed (AVA) (CDC. Use of Anthrax vaccine in the United States: Recommendations of the Advisory Committee on Immunization Practices [ACIP]. MMWR 2000;49:1-20; CDC. Use of Anthrax vaccine in response to terrorism: supplemental recommendations of the Advisory Committee on Immunization Practices [ACIP]. MMWR 2002;51:1024-6) and reflect the status of Anthrax vaccine supplies in the United States. This statement 1) provides updated information on Anthrax epidemiology; 2) summarizes the evidence regarding the effectiveness and efficacy, immunogenicity, and safety of AVA; 3) provides recommendations for pre-event and preexposure use of AVA; and 4) provides recommendations for postexposure use of AVA. In certain instances, recommendations that did not change were clarified. No new licensed Anthrax Vaccines are presented. Substantial changes to these recommendations include the following: 1) reducing the number of doses required to complete the pre-event and preexposure primary series from 6 doses to 5 doses, 2) recommending intramuscular rather than subcutaneous AVA administration for preexposure use, 3) recommending AVA as a component of postexposure prophylaxis in pregnant women exposed to aerosolized Bacillus anthracis spores, 4) providing guidance regarding preexposure vaccination of emergency and other responder organizations under the direction of an occupational health program, and 5) recommending 60 days of antimicrobial prophylaxis in conjunction with 3 doses of AVA for optimal protection of previously unvaccinated persons after exposure to aerosolized B. anthracis spores.
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Use of Anthrax Vaccine in the United States: Recommendations of the Advisory Committee on Immunization Practices (ACIP), 2009 (Morbidity and Mortality Weekly Report. Volume 59, Number RR-6, July 23, 2010)
2010Co-Authors: Jennifer Gordon Wright, Conrad P Quinn, S Shadomy, Nancy MessonnierAbstract:Abstract : These recommendations from the Advisory Committee on Immunization Practices (ACIP) update the previous recommendations for Anthrax vaccine adsorbed (AVA) (CDC. Use of Anthrax vaccine in the United States: Recommendations of the Advisory Committee on Immunization Practices [ACIP]. MMWR 2000;49:1-20; CDC. Use of Anthrax vaccine in response to terrorism: supplemental recommendations of the Advisory Committee on Immunization Practices [ACIP]. MMWR 2002;51:1024-6) and reflect the status of Anthrax vaccine supplies in the United States. This statement 1) provides updated information on Anthrax epidemiology; 2) summarizes the evidence regarding the effectiveness and efficacy, immunogenicity, and safety of AVA; 3) provides recommendations for pre-event and preexposure use of AVA; and 4) provides recommendations for postexposure use of AVA. In certain instances, recommendations that did not change were clarified. No new licensed Anthrax Vaccines are presented. Substantial changes to these recommendations include the following: 1) reducing the number of doses required to complete the pre-event and preexposure primary series from 6 doses to 5 doses, 2) recommending intramuscular rather than subcutaneous AVA administration for preexposure use, 3) recommending AVA as a component of postexposure prophylaxis in pregnant women exposed to aerosolized Bacillus anthracis spores, 4) providing guidance regarding preexposure vaccination of emergency and other responder organizations under the direction of an occupational health program, and 5) recommending 60 days of antimicrobial prophylaxis in conjunction with 3 doses of AVA for optimal protection of previously unvaccinated persons after exposure to aerosolized B. anthracis spores.
Rosanna Adone - One of the best experts on this subject based on the ideXlab platform.
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development of a sterne based complement fixation test to monitor the humoral response induced by Anthrax Vaccines
Frontiers in Microbiology, 2016Co-Authors: Rosanna Adone, Massimiliano Francia, Michela Sali, Michela Iatarola, Adelia Donatiello, Antonio FasanellaAbstract:Anthrax is a zoonotic disease caused by Bacillus anthracis spore-forming bacterium. Since it is primarily a disease of animals, the control in animals and humans depends on the prevention in livestock, principally cattle, sheep and goats. Most veterinary Vaccines utilize the toxigenic, uncapsulated (pXO1+/pXO2–) B.anthracis strain 34F2 which affords protection through the production of neutralizing antibodies directed to the toxin components Protective Antigen (PA), Lethal Factor (LF) and Edema Factor (EF). The titration of specific antibodies in sera of vaccinated animals is crucial to evaluate the efficacy of the vaccination and to obtain epidemiological information for an effective Anthrax surveillance. In this study, we developed a Sterne-based Complement Fixation Test to detect specific antibodies induced in animals vaccinated with Sterne 34F2 . We assessed its efficacy in laboratory animals and under field conditions by monitoring the humoral response induced by vaccination in cattle. The results indicated that the Sterne-based CFT is able to identify vaccinated animals with a good sensitivity and specificity offering many benefits especially with regard to costs, standardization and reproducibility of the assay procedure.
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development of a sterne based complement fixation test to monitor the humoral response induced by Anthrax Vaccines
Frontiers in Microbiology, 2016Co-Authors: Rosanna Adone, Massimiliano Francia, Michela Sali, Michela Iatarola, Adelia Donatiello, Antonio FasanellaAbstract:Anthrax is a zoonotic disease caused by Bacillus anthracis spore-forming bacterium. Since it is primarily a disease of animals, the control in animals, and humans depend on the prevention in livestock, principally cattle, sheep, and goats. Most veterinary Vaccines utilize the toxigenic, uncapsulated (pXO1+/pXO2-) B. anthracis strain 34F2 which affords protection through the production of neutralizing antibodies directed to the toxin components Protective Antigen (PA), Lethal Factor (LF), and Edema Factor (EF). The titration of specific antibodies in sera of vaccinated animals is crucial to evaluate the efficacy of the vaccination and to obtain epidemiological information for an effective Anthrax surveillance. In this study, we developed a Sterne-based Complement Fixation Test (CFT) to detect specific antibodies induced in animals vaccinated with Sterne 34F2. We assessed its efficacy in laboratory animals and under field conditions by monitoring the humoral response induced by vaccination in cattle. The results indicated that the Sterne-based CFT is able to correctly identify vaccinated animals. It proved to be a very sensitive and specific test. Moreover, the Sterne-based CFT offers many benefits with regard to costs, standardization and reproducibility of the assay procedure.
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protective activity and immunogenicity of two recombinant Anthrax Vaccines for veterinary use
Vaccine, 2008Co-Authors: Antonio Fasanella, Rosanna Adone, Fiorella Tonello, Giuliano Garofolo, Lucia Muraro, Alessandra Carattoli, C MontecuccoAbstract:In this study, the efficacy of two experimental Vaccines against Bacillus anthracis toxinaemia was evaluated in the rabbit model. A recombinant Protective Antigen (rPA) mutant and a trivalent vaccine (TV) composed by the rPA, a inactive mutant of Lethal Factor (mLF-Y728A; E735A) and a inactive mutant of Edema Factor (mEF-K346R), both emulsified with mineral oils, were evaluated for their immunogenicity and protective activity in New Zealand white rabbits. Rabbits vaccinated subcutaneously with rPA and TV rapidly produced high level of anti-PA, anti-LF and anti-EF antibodies, which were still present 6 months later. In the efficacy test, these Vaccines protected 100% of rabbits challenged with B. anthracis virulent strain 0843 one week after the vaccination. Moreover, all animals vaccinated twice with rPA and TV, resisted B. anthracis infection 6 months later. Our data indicate that rPA and TV could be good vaccine candidates for inducing protection against B. anthracis infection in target animal host. They could successfully be used in an emergency with simultaneous long-acting antibiotics to halt incubating infections or during an Anthrax epidemic.
Drusilla L Burns - One of the best experts on this subject based on the ideXlab platform.
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mechanistic analysis of the effect of deamidation on the immunogenicity of Anthrax protective antigen
Clinical and Vaccine Immunology, 2016Co-Authors: Anita Verma, Miriam M Ngundi, Drusilla L BurnsAbstract:ABSTRACT The spontaneous modification of proteins, such as deamidation of asparagine residues, can significantly affect the immunogenicity of protein-based Vaccines. Using a “genetically deamidated” form of recombinant protective antigen (rPA), we have previously shown that deamidation can decrease the immunogenicity of rPA, the primary component of new-generation Anthrax Vaccines. In this study, we investigated the biochemical and immunological mechanisms by which deamidation of rPA might decrease the immunogenicity of the protein. We found that loss of the immunogenicity of rPA vaccine was independent of the presence of adjuvant. We assessed the effect of deamidation on the immunodominant neutralizing B-cell epitopes of rPA and found that these epitopes were not significantly affected by deamidation. In order to assess the effect of deamidation on T-cell help for antibody production elicited by rPA vaccine, we examined the ability of the wild-type and genetically deamidated forms of rPA to serve as hapten carriers. We found that when wild-type and genetically deamidated rPA were modified to similar extents with 2,4-dinitrophenyl hapten (DNP) and then used to immunize mice, higher levels of anti-DNP antibodies were elicited by wild-type DNP-rPA than those elicited by the genetically deamidated DNP-rPA, indicating that wild-type rPA elicits more T-cell help than the genetically deamidated form of the protein. These results suggest that a decrease in the ability of deamidated rPA to elicit T-cell help for antibody production is a possible contributor to its lower immunogenicity.
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use of site directed mutagenesis to model the effects of spontaneous deamidation on the immunogenicity of bacillus anthracis protective antigen
Infection and Immunity, 2013Co-Authors: Anita Verma, Juan L. Arciniega, Miriam M Ngundi, Beth Mcnichol, Rocio I Dominguezcastillo, Juan C Amadormolina, Karine Reiter, Bruce D Meade, Scott Stibitz, Drusilla L BurnsAbstract:Long-term stability is a desired characteristic of Vaccines, especially Anthrax Vaccines, which must be stockpiled for large-scale use in an emergency situation; however, spontaneous deamidation of purified vaccine antigens has the potential to adversely affect vaccine immunogenicity over time. In order to explore whether spontaneous deamidation of recombinant protective antigen (rPA)—the major component of new-generation Anthrax Vaccines—affects vaccine immunogenicity, we created a “genetically deamidated” form of rPA using site-directed mutagenesis to replace six deamidation-prone asparagine residues, at positions 408, 466, 537, 601, 713, and 719, with either aspartate, glutamine, or alanine residues. We found that the structure of the six-Asp mutant rPA was not significantly altered relative to that of the wild-type protein as assessed by circular dichroism (CD) spectroscopy and biological activity. In contrast, immunogenicity of aluminum-adjuvanted six-Asp mutant rPA, as measured by induction of toxin-neutralizing antibodies, was significantly lower than that of the corresponding wild-type rPA vaccine formulation. The six-Gln and six-Ala mutants also exhibited lower immunogenicity than the wild type. While the wild-type rPA vaccine formulation exhibited a high level of immunogenicity initially, its immunogenicity declined significantly upon storage at 25°C for 4 weeks. In contrast, the immunogenicity of the six-Asp mutant rPA vaccine formulation was low initially but did not change significantly upon storage. Taken together, results from this study suggest that spontaneous deamidation of asparagine residues predicted to occur during storage of rPA Vaccines would adversely affect vaccine immunogenicity and therefore the storage life of Vaccines.
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structural and immunological analysis of Anthrax recombinant protective antigen adsorbed to aluminum hydroxide adjuvant
Clinical and Vaccine Immunology, 2012Co-Authors: Leslie Wagner, Anita Verma, Karine Reiter, Bruce D Meade, Stephen F. Little, Rebecca A. Brady, David L Narum, Drusilla L BurnsAbstract:New Anthrax Vaccines currently under development are based on recombinant protective antigen (rPA) and formulated with aluminum adjuvant. Because long-term stability is a desired characteristic of these Vaccines, an understanding of the effects of adsorption to aluminum adjuvants on the structure of rPA is important. Using both biophysical and immunological techniques, we compared the structure and immunogenicity of freshly prepared rPA-Alhydrogel formulations to that of formulations stored for 3 weeks at either room temperature or 37°C in order to assess the changes in rPA structure that might occur upon long-term storage on aluminum adjuvant. Intrinsic fluorescence emission spectra of tryptophan residues indicated that some tertiary structure alterations of rPA occurred during storage on Alhydrogel. Using anti-PA monoclonal antibodies to probe specific regions of the adsorbed rPA molecule, we found that two monoclonal antibodies that recognize epitopes located in domain 1 of PA exhibited greater reactivity to the stored formulations than to freshly prepared formulations. Immunogenicity of rPA-Alhydrogel formulations in mice was assessed by measuring the induction of toxin-neutralizing antibodies, as well as antibodies reactive to 12-mer peptides spanning the length of PA. Mice immunized with freshly prepared formulations developed significantly higher toxin-neutralizing antibody titers than mice immunized with the stored preparations. In contrast, sera from mice immunized with stored preparations exhibited increased reactivity to nine 12-mer peptides corresponding to sequences located throughout the rPA molecule. These results demonstrate that storage of rPA-Alhydrogel formulations can lead to structural alteration of the protein and loss of the ability to elicit toxin-neutralizing antibodies.
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Analysis of antibody responses to protective antigen-based Anthrax Vaccines through use of competitive assays.
Clinical and Vaccine Immunology, 2010Co-Authors: Rebecca A. Brady, Bruce D Meade, Anita Verma, Drusilla L BurnsAbstract:The licensed Anthrax vaccine and many of the new Anthrax Vaccines being developed are based on protective antigen (PA), a nontoxic component of Anthrax toxin. For this reason, an understanding of the immune response to PA vaccination is important. In this study, we examined the antibody response elicited by PA-based Vaccines and identified the domains of PA that contribute to that response in humans as well as nonhuman primates (NHPs) and rabbits, animal species that will be used to generate efficacy data to support approval of new Anthrax Vaccines. To this end, we developed a competitive enzyme-linked immunosorbent assay (ELISA), using purified recombinant forms of intact PA and its individual domains. We found that PA-based Vaccines elicited IgG antibodies to each of the four PA domains in all three species. We also developed a competitive toxin neutralization assay, which showed that rabbits, NHPs, and humans all have functional antibody populations that bind to domains 1, 3, and 4. While the domain specificities of the antibody responses elicited by PA-based Vaccines were similar in humans, NHPs, and rabbits, competitive assays suggested that humans may have a more significant secondary population of IgG antibodies that bind to partially unfolded or incorrectly folded PA. These findings provide information that will be useful when linking animal protection data to humans via an antibody bridge to establish efficacy of new Anthrax Vaccines.
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Analysis of antibody responses to protective antigen-based Anthrax Vaccines through use of competitive assays.
Clinical and vaccine immunology : CVI, 2010Co-Authors: Rebecca A. Brady, Bruce D Meade, Anita Verma, Drusilla L BurnsAbstract:The licensed Anthrax vaccine and many of the new Anthrax Vaccines being developed are based on protective antigen (PA), a nontoxic component of Anthrax toxin. For this reason, an understanding of the immune response to PA vaccination is important. In this study, we examined the antibody response elicited by PA-based Vaccines and identified the domains of PA that contribute to that response in humans as well as nonhuman primates (NHPs) and rabbits, animal species that will be used to generate efficacy data to support approval of new Anthrax Vaccines. To this end, we developed a competitive enzyme-linked immunosorbent assay (ELISA), using purified recombinant forms of intact PA and its individual domains. We found that PA-based Vaccines elicited IgG antibodies to each of the four PA domains in all three species. We also developed a competitive toxin neutralization assay, which showed that rabbits, NHPs, and humans all have functional antibody populations that bind to domains 1, 3, and 4. While the domain specificities of the antibody responses elicited by PA-based Vaccines were similar in humans, NHPs, and rabbits, competitive assays suggested that humans may have a more significant secondary population of IgG antibodies that bind to partially unfolded or incorrectly folded PA. These findings provide information that will be useful when linking animal protection data to humans via an antibody bridge to establish efficacy of new Anthrax Vaccines.