Analysis of Antibody Responses to Protective Antigen-Based Anthrax Vaccines through Use of Competitive Assays

ABSTRACT 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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Characterization of the Native Form of Anthrax Lethal Factor for Use in the Toxin Neutralization Assay

Clinical and Vaccine Immunology ◽

10.1128/cvi.00046-13 ◽

2013 ◽

Vol 20 (7) ◽

pp. 986-997 ◽

Cited By ~ 2

Author(s):

Hang Lu ◽

Jason Catania ◽

Katalin Baranji ◽

Jie Feng ◽

Mili Gu ◽

...

Keyword(s):

Protective Antigen ◽

Lethal Factor ◽

Neutralization Assay ◽

Dominant Factor ◽

Anthrax Lethal Toxin ◽

Serum Samples ◽

Native Form ◽

Anthrax Lethal Factor ◽

Methionine Residues ◽

Anthrax Vaccines

ABSTRACTThe cell-based anthrax toxin neutralization assay (TNA) is used to determine functional antibody titers of sera from animals and humans immunized with anthrax vaccines. The anthrax lethal toxin is a critical reagent of the TNA composed of protective antigen (PA) and lethal factor (LF), which are neutralization targets of serum antibodies. Cytotoxic potency of recombinant LF (rLF) lots can vary substantially, causing a challenge in producing a renewable supply of this reagent for validated TNAs. To address this issue, we characterized a more potent rLF variant (rLF-A) with the exact native LF amino acid sequence that lacks the additional N-terminal histidine and methionine residues present on the commonly used form of rLF (rLF-HMA) as a consequence of the expression vector. rLF-A can be used at 4 to 6 ng/ml (in contrast to 40 ng/ml rLF-HMA) with 50 ng/ml recombinant PA (rPA) to achieve 95 to 99% cytotoxicity. In the presence of 50 ng/ml rPA, both rLF-A and rLF-HMA allowed for similar potencies (50% effective dilution) among immune sera in the TNA. rPA, but not rLF, was the dominant factor in determining potency of serum samples containing anti-PA antibodies only or an excess of anti-PA relative to anti-rLF antibodies. Such anti-PA content is reflected in immune sera derived from most anthrax vaccines in development. These results support that 7- to 10-fold less rLF-A can be used in place of rLF-HMA without changing TNA serum dilution curve parameters, thus extending the use of a single rLF lot and a consistent, renewable supply.

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Antibody Responses to MAP 1B and Other Cowdria ruminantium Antigens Are Down Regulated in Cattle Challenged with Tick-Transmitted Heartwater

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.8.2.388-396.2001 ◽

2001 ◽

Vol 8 (2) ◽

pp. 388-396 ◽

Cited By ~ 17

Author(s):

S. M. Semu ◽

T. F. Peter ◽

D. Mukwedeya ◽

A. F. Barbet ◽

F. Jongejan ◽

...

Keyword(s):

Tick Infestation ◽

Repeated Exposure ◽

Antibody Responses ◽

Enzyme Linked Immunosorbent Assay ◽

Infected Cattle ◽

Igg Antibodies ◽

Igg Antibody ◽

Cowdria Ruminantium ◽

Infection Pressure ◽

Specific Igg

ABSTRACT Serological diagnosis of heartwater or Cowdria ruminantium infection has been hampered by severe cross-reactions with antibody responses to related ehrlichial agents. A MAP 1B indirect enzyme-linked immunosorbent assay that has an improved specificity and sensitivity for detection of immunoglobulin G (IgG) antibodies has been developed to overcome this constraint (A. H. M. van Vliet, B. A. M. Van der Zeijst, E. Camus, S. M. Mahan, D. Martinez, and F. Jongejan, J. Clin. Microbiol. 33:2405–2410, 1995). When sera were tested from cattle in areas of endemic heartwater infection in Zimbabwe, only 33% of the samples tested positive in this assay despite a high infection pressure (S. M. Mahan, S. M. Samu, T. F. Peter, and F. Jongejan, Ann. N.Y. Acad. Sci 849:85–87, 1998). To determine underlying causes for this observation, the kinetics of MAP 1B-specific IgG antibodies in cattle after tick-transmitted C. ruminantium infection and following recovery were investigated. Sera collected weekly over a period of 52 weeks from 37 cattle, which were naturally or experimentally infected with C. ruminantium via Amblyomma hebraeum ticks, were analyzed. MAP 1B-specific IgG antibody responses developed with similar kinetics in both field- and laboratory-infected cattle. IgG levels peaked at 4 to 9 weeks after tick infestation and declined to baseline levels between 14 and 33 weeks, despite repeated exposure to infected ticks and the establishment of a carrier state as demonstrated by PCR and xenodiagnosis. Some of the serum samples from laboratory, and field-infected cattle were also analyzed by immunoblotting and an indirect fluorescent-antibody test (IFAT) to determine whether this observed seroreversion was specific to the MAP 1B antigen. Reciprocal IFAT and immunoblot MAP 1-specific antibody titres peaked at 5 to 9 weeks after tick infestation but also declined between 30 and 45 weeks. This suggests that MAP 1B-specific IgG antibody responses and antibody responses to other C. ruminantium antigens are down regulated in cattle despite repeated exposure to C. ruminantium via ticks. Significantly, serological responses to the MAP 1B antigen may not be a reliable indicator of C. ruminantium exposure in cattle in areas of endemic heartwater infection.

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Genital Antibody Responses in Mice after Intranasal Infection with an Attenuated Candidate Vector Strain ofBordetella pertussis

Infection and Immunity ◽

10.1128/iai.68.2.485-491.2000 ◽

2000 ◽

Vol 68 (2) ◽

pp. 485-491 ◽

Cited By ~ 9

Author(s):

Nathalie Mielcarek ◽

Inger Nordström ◽

Franco D. Menozzi ◽

Camille Locht ◽

Jan Holmgren

Keyword(s):

Pertussis Toxin ◽

Genital Tract ◽

Intranasal Administration ◽

Protective Antigen ◽

Antibody Responses ◽

Toxin Gene ◽

Igg Antibodies ◽

Intranasal Infection ◽

Sexually Transmitted ◽

Ofbordetella Pertussis

ABSTRACT Intranasal administration of live attenuated Bordetella pertussis, from which the pertussis toxin gene has been deleted, has previously been shown to give rise to high levels of serum immunoglobulin G (IgG) antibodies against both the protective antigen filamentous hemagglutinin (FHA) and heterologous antigens genetically fused to FHA. Here, we extend these results by demonstrating that anti-FHA IgA and IgG antibodies are also produced in the genital tract of mice, both in the vagina and in the uterus, after a single intranasal administration of B. pertussis. By comparing the immune responses induced after infection with wild-type virulentB. pertussis with that induced by infection with an attenuated pertussis toxin-deficient strain, we conclude that pertussis toxin produced by the virulent bacteria does not modify antibody production to FHA in the genital tract of B. pertussis-infected mice. The intranasal infection with either the attenuated or the virulent B. pertussis strain also led to the development of immunologic memory that could be efficiently boosted with purified FHA administered either intranasally or intravaginally to give rise to a significant increase in the levels of specific IgA and IgG produced locally in the genital tract, as well as of specific antibodies in the serum. These observations suggest that attenuatedB. pertussis could be a promising vector for intranasal administration to induce antibody responses against antigens from sexually transmitted pathogens fused to FHA.

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Protective Antigen and Toxin Neutralization Antibody Patterns in Anthrax Vaccinees Undergoing Serial Plasmapheresis

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.12.6.713-721.2005 ◽

2005 ◽

Vol 12 (6) ◽

pp. 713-721 ◽

Cited By ~ 19

Author(s):

Phillip R. Pittman ◽

Susan F. Leitman ◽

Julio G. Barrera Oro ◽

Sarah L. Norris ◽

Nina M. Marano ◽

...

Keyword(s):

Protective Antigen ◽

Optimal Schedule ◽

Neutralization Assay ◽

Enzyme Linked Immunosorbent Assay ◽

Immune Reactivity ◽

Highly Correlated ◽

Natural Decay ◽

Antibody Levels ◽

Over Time ◽

Donor Plasma

ABSTRACT Recipients of licensed anthrax vaccine (AVA; Biothrax) could serve as a source of hyperimmune plasma and immunoglobulin for therapy and prophylaxis. We measured serum antibodies during serial weekly to biweekly plasmapheresis in 38 individuals previously vaccinated with 4 to 27 doses of AVA. Immunoglobulin G (IgG) to protective antigen (PA) and toxin neutralization assay (TNA) antibody levels were highly correlated (r = 0.86930 and P < 0.0001 for anti-PA concentration versus TNA concentration). Significant decreases in antibody titer and concentration were observed over time when compared for the number of days from the last AVA injection (P < 0.0001 for both anti-PA and TNA concentration) and for the number of days from the first plasmapheresis (P = 0.0007 for anti-PA concentration and P = 0.0025 for TNA concentration). The rate of the decrease in total IgG concentration (half-life [t 1/2] = 198.90 days after first plasmapheresis) was significantly less than the decrease in anti-PA IgG (t 1/2 = 63.53 days) (P < 0.0001), indicating that the reduction in anti-PA IgG was more likely due to natural decay than plasmapheresis. The time since the last injection and the time after initial plasmapheresis are important elements in considering an optimal schedule for collecting anthrax hyperimmune plasma. Good correlation between IgG to PA and TNA antibodies suggests that the anti-PA enzyme-linked immunosorbent assay can be used as a high-throughput screen for functional immune reactivity in donor plasma units.

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Development and Application of a Microneutralization ELISA for the Detection of Antibodies to Bovine Respiratory Syncytial Viruses

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063879500700203 ◽

1995 ◽

Vol 7 (2) ◽

pp. 183-189 ◽

Cited By ~ 1

Author(s):

John A. Ellis ◽

Lori Hassard ◽

Paul S. Morley

Keyword(s):

Indirect Elisa ◽

Neutralization Assay ◽

Antibody Responses ◽

Enzyme Linked Immunosorbent Assay ◽

Inhibition Assay ◽

Virus Neutralization Assay ◽

Fusion Inhibition ◽

Antibody Titers ◽

Respiratory Syncytial Viruses ◽

High Degree

A microneutralization enzyme-linked immunosorbent assay (ELISA) was developed to detect specific antibodies to bovine respiratory syncytial viruses (BRSVs) in cattle sera using a monoclonal antibody to the fusion protein of the virus. Serum from 20 naturally exposed, 24 experimentally infected, and 15 immunized cattle were evaluated using 3 different BRSV isolates. Antibody titers determined with the micro-neutralization ELISA were compared with those derived from a classical virus neutralization assay, an indirect ELISA, and a fusion inhibition assay. These studies demonstrated a high degree of correlation (usually 0.90) among the assays. Furthermore, the results showed that immunization of cattle with one isolate (subgroup) of BRSV induced antibody responses that cross-reacted with at least 2 disparate isolates. These results document the utility of the microneutralization ELISA in assessing functionally important antibody responses to BRSVs in cattle.

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Serum Immunoglobulin G Antibody Responses to Bordetella pertussis Lipooligosaccharide and B.parapertussis Lipopolysaccharide in Children with Pertussis and Parapertussis

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.8.5.1015-1017.2001 ◽

2001 ◽

Vol 8 (5) ◽

pp. 1015-1017 ◽

Cited By ~ 8

Author(s):

Birger Trollfors ◽

Teresa Lagergård ◽

John Taranger ◽

Elisabet Bergfors ◽

Rachel Schneerson ◽

...

Keyword(s):

Immunosorbent Assay ◽

Bordetella Pertussis ◽

Immunoglobulin G ◽

Antibody Responses ◽

Serum Immunoglobulin ◽

Enzyme Linked Immunosorbent Assay ◽

Igg Antibodies ◽

Bordetella Parapertussis ◽

The Individual

ABSTRACT Serum immunoglobulin G (IgG) antibodies against the lipooligosaccharide (LOS) of Bordetella pertussis and the lipopolysaccharide (LPS) of Bordetella parapertussiswere measured by enzyme-linked immunosorbent assay in paired sera from 40 children with pertussis and 14 with parapertussis. Wide differences in the individual responses were noted. Both anti-LOS and -LPS IgG levels increased significantly in the children with pertussis, as did anti-LPS but not anti-LOS in those with parapertussis.

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Detection of Genotype-Specific Antibody Responses to Glycoproteins B and H in Primary and Non-Primary Human Cytomegalovirus Infections by Peptide-Based ELISA

Viruses ◽

10.3390/v13030399 ◽

2021 ◽

Vol 13 (3) ◽

pp. 399

Author(s):

Federica Zavaglio ◽

Loretta Fiorina ◽

Nicolás M. Suárez ◽

Chiara Fornara ◽

Marica De Cicco ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Primary Infection ◽

Antibody Responses ◽

Enzyme Linked Immunosorbent Assay ◽

Igg Antibodies ◽

Igg Antibody ◽

Background Strain ◽

Specific Igg ◽

Cytomegalovirus Infections ◽

Specific Igg Antibodies

Background: Strain-specific antibodies to human cytomegalovirus (HCMV) glycoproteins B and H (gB and gH) have been proposed as a potential diagnostic tool for identifying reinfection. We investigated genotype-specific IgG antibody responses in parallel with defining the gB and gH genotypes of the infecting viral strains. Methods: Subjects with primary (n = 20) or non-primary (n = 25) HCMV infection were studied. The seven gB (gB1-7) and two gH (gH1-2) genotypes were determined by real-time PCR and whole viral genome sequencing, and genotype-specific IgG antibodies were measured by a peptide-based enzyme-linked immunosorbent assay (ELISA). Results: Among subjects with primary infection, 73% (n = 8) infected by gB1-HCMV and 63% (n = 5) infected by gB2/3-HCMV had genotype-specific IgG antibodies to gB (gB2 and gB3 are similar in the region tested). Peptides from the rarer gB4-gB7 genotypes had nonspecific antibody responses. All subjects infected by gH1-HCMV and 86% (n = 6) infected by gH2-HCMV developed genotype-specific responses. Among women with non-primary infection, gB and gH genotype-specific IgG antibodies were detected in 40% (n = 10) and 80% (n = 20) of subjects, respectively. Conclusions: Peptide-based ELISA is capable of detecting primary genotype-specific IgG responses to HCMV gB and gH, and could be adopted for identifying reinfections. However, about half of the subjects did not have genotype-specific IgG antibodies to gB.

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Influence of Exogenous Reproductive Hormones on Specific Antibody Production in Genital Secretions after Vaginal Vaccination with Recombinant Cholera Toxin B Subunit in Humans

Clinical and Vaccine Immunology ◽

10.1128/cvi.13.2.202-207.2006 ◽

2006 ◽

Vol 13 (2) ◽

pp. 202-207 ◽

Cited By ~ 10

Author(s):

Lotta Wassen ◽

Marianne Jertborn

Keyword(s):

Cholera Toxin ◽

Specific Antibody ◽

Large Scale ◽

Reproductive Hormones ◽

Antibody Responses ◽

Enzyme Linked Immunosorbent Assay ◽

Cholera Toxin B Subunit ◽

Igg Antibodies ◽

Igg Antibody ◽

Predictive Values

ABSTRACT The objective of this study was to investigate the influence of exogenous reproductive hormones on the local and systemic production of specific immunoglobulin A (IgA) and IgG antibodies after vaginal vaccination with recombinant cholera toxin subunit B (CTB). Three groups of women using either progesterone-containing intrauterine devices (n = 9), oral contraceptives (n = 8), or no hormonal contraceptive methods (n = 9) were vaginally immunized twice, 2 weeks apart. Cervical secretions, vaginal fluids, and serum were collected before and after vaccination. Total and CTB-specific IgA and IgG antibodies in genital secretions and serum were analyzed by enzyme-linked immunosorbent assay. A majority of the women presented strong CTB-specific IgA and IgG antibody responses in cervicovaginal secretions after vaccination, whereas the antitoxin responses in serum were weaker. Exogenously administered steroid hormones did not seem to have any impact on the production of specific antibodies. Both the frequencies and the magnitudes of IgA and IgG antitoxin responses in genital secretions were comparable among the three immunization groups. An association, in particular for IgA, was found between the magnitudes of the CTB-specific antibody responses in cervical secretions and vaginal fluids after vaccination. The sensitivities and positive predictive values of vaginal antibody analyses to reflect responses in cervical secretions were also high, suggesting that vaginal fluids alone might be used for evaluation of genital immune responses in large-scale vaccination studies in the future.

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Neutralizing Activity of Vaccine-Induced Antibodies to Two Bacillus anthracis Toxin Components, Lethal Factor and Edema Factor

Clinical and Vaccine Immunology ◽

10.1128/cvi.00321-07 ◽

2007 ◽

Vol 15 (1) ◽

pp. 71-75 ◽

Cited By ~ 26

Author(s):

Sarah C. Taft ◽

Alison A. Weiss

Keyword(s):

Animal Models ◽

Protective Antigen ◽

Lethal Factor ◽

Antibody Responses ◽

Anthrax Toxin ◽

Enzyme Linked Immunosorbent Assay ◽

Mouse Macrophage ◽

Anthrax Vaccine ◽

Neutralizing Activity ◽

Edema Factor

ABSTRACT Anthrax vaccine adsorbed (AVA; BioThrax), the current FDA-licensed human anthrax vaccine, contains various amounts of the three anthrax toxin components, protective antigen (PA), lethal factor (LF), and edema factor (EF). While antibody to PA is sufficient to mediate protection against anthrax in animal models, it is not known if antibodies to LF or EF contribute to protection in humans. Toxin-neutralizing activity was evaluated in sera from AVA-vaccinated volunteers, all of whom had antibody responses to LF and EF, as well as PA. The contribution of antibodies to LF and EF was assessed using mouse macrophage J774A.1 cells by examining neutralization of LF-induced lysis using alamarBlue reduction and neutralization of EF-induced cyclic AMP increases by enzyme-linked immunosorbent assay. Antibody responses to LF and EF were low compared to those to PA, and the amount of LF or EF in the assay could exceed the amount of antibodies to LF or EF. Higher titers were seen for most individuals when the LF or EF concentration was limiting compared to when LF or EF was in excess, initially suggesting that antibody to LF or EF augmented protection. However, depletion of LF and EF antibodies in sera did not result in a significant decrease in toxin neutralization. Overall, this study suggests that AVA-induced LF and EF antibodies do not significantly contribute to anthrax toxin neutralization in humans and that antibodies to PA are sufficient to neutralize toxin activity.

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

Clinical and Vaccine Immunology ◽

10.1128/cvi.00194-09 ◽

2009 ◽

Vol 16 (10) ◽

pp. 1405-1412 ◽

Cited By ~ 24

Author(s):

Anita Verma ◽

Miriam M. Ngundi ◽

Bruce D. Meade ◽

Roberto De Pascalis ◽

Karen L. Elkins ◽

...

Keyword(s):

Protective Antigen ◽

Neutralization Assay ◽

Anthrax Toxin ◽

Specific Cell ◽

Cell Type ◽

Fcγ Receptor ◽

Gamma Receptor ◽

Preclinical And Clinical Studies ◽

Antibody Levels ◽

Anthrax Vaccines

ABSTRACT 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 (Fcγ) receptor-dependent toxin neutralization observed in commonly used forms of the anthrax toxin neutralization assay. Significantly more Fcγ 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 Fcγ receptors that we found on J774A.1 cells by using flow cytometry. Thus, the extent to which Fcγ receptor-dependent neutralization contributes to the total neutralization measured by the assay depends on the specific cell type utilized in the assay. Using Fcγ receptor blocking monoclonal antibodies, we found that at least three murine Fcγ receptor classes, IIB, III, and IV, can contribute to Fcγ receptor-dependent neutralization. When antibodies elicited by immunization of rabbits with protective-antigen-based anthrax vaccines were analyzed, we found that the magnitude of Fcγ 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 Fcγ 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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