Toxin Neutralization Assay Research Articles

Shiga toxin-induced apoptosis is more efficiently inhibited by dimeric recombinant hybrid-IgG/IgA immunoglobulins than by the parental IgG monoclonal antibodies

Shiga toxin 1 (Stx1) is a virulence factor of enterohaemorrhagic Escherichia coli strains such as O157:H7 and Shigella dysenteriae. To prevent entry of Stx1 from the mucosal surface, an immunoglobulin A (IgA) specific for Stx1 would be useful. Due to the difficulty of producing IgA monoclonal antibodies (mAb) against the binding subunit of Stx1 (Stx1B) in mice, we took advantage of recombinant technology that combines the heavy chain variable region from Stx1B-specific IgG1 mAb and the Fc region from IgA. The resulting hybrid IgG/IgA was stably expressed in Chinese hamster ovary cells as a dimeric hybrid IgG/IgA. We separated the dimeric hybrid IgG/IgA from the monomeric one by size-exclusion chromatography. The dimer fraction, confirmed by immunoblot analyses, was used for toxin neutralization assays. The dimeric IgG/IgA was shown to neutralize Stx1 toxicity toward Vero cells by assaying their viability. To compare the relative effectiveness of the dimeric hybrid IgG/IgA and parental IgG1 mAb, Stx1-induced apoptosis was examined using 2 different cell lines, Ramos and Vero cells. The hybrid IgG/IgA inhibited apoptosis more efficiently than the parental IgG1 mAb in both cases. The results indicated that the use of high affinity binding sites as variable regions of IgA would increase the utility of IgA specific for virulence factors.

In vivo mapping of a protective linear neutralizing epitope at the N-terminus of alpha hemolysin from Staphylococcus aureus

Staphylococcus aureus is responsible for a large and diverse burden of human disease associated with significant morbidity and mortality. The dynamic challenge of this pathogen is exemplified by the emergence of highly virulent community-associated methicillin-resistant S. aureus strain USA300, which threatens both healthy and vulnerable individuals and constitutes a public health imperative in the United States. Though S. aureus employs many virulence factors that enable infectivity and evasion of host defenses, evidence suggests that the increased production of alpha hemolysin may be a critical contributor to the increased virulence of USA300. To enable and inform immunological targeting of alpha hemolysin, we sought to precisely map a neutralizing epitope that we hypothesized existed in the N-terminal domain. Using an in vivo mapping strategy employing peptide immunogens and an optimized in vitro toxin neutralization assay, we identified a linear neutralizing determinant in the N-terminal 19 amino acids of alpha hemolysin. Affinity purified rabbit antibody against this neutralizing epitope was shown to be highly effective at mitigating dermonecrosis in inbred and outbred mice challenged with USA300. To our knowledge, this is the first description of a linear neutralizing epitope in alpha hemolysin, and the delineation of this determinant should inform and facilitate the rational design and development of an efficacious, epitope-focused or multivalent vaccine against S. aureus.

Safety and immunogenicity of the pneumococcal pneumolysin derivative PlyD1 in a single-antigen protein vaccine candidate in adults

BackgroundPneumococcal vaccines based on conserved protein antigens have the potential to offer expanded protection against Streptococcus pneumoniae. ObjectiveThis study examined the safety and immunogenicity in adults of three doses of a pneumococcal single-antigen protein vaccine candidate formulated with aluminum hydroxide adjuvant and recombinantly derived, highly detoxified, genetically mutated pneumolysin protein (PlyD1). MethodsThis phase I, randomized, placebo-controlled, observer-blinded, dose-escalating study enrolled adults (18–50 years). In a pilot safety study, participants received a single injection of 10μg PlyD1 and were observed for 24h. Following review of the pilot safety data, participants were randomized (2:1) to receive two injections of PlyD1 at one of three doses or placebo 30 days apart. Assignment of second injection and successive dose cohorts was made after blinded safety reviews after each injection at each dose level. Safety endpoints included rates of solicited injection site reactions, solicited systemic reactions, unsolicited adverse events (AEs), serious AEs (SAEs), and safety laboratory tests. Immunogenicity endpoints included geometric mean concentrations of anti-PlyD1 IgG as determined by ELISA and functional assessment in an in vitro toxin neutralization assay. ResultsThe study included a total of 100 participants, including 10 in the pilot study and 90 in the randomized study. None of the participants in the pilot study had SAEs, allergic reactions, or other safety concerns. Ninety participants received two doses of or placebo (n=30) or active vaccine candidate at 10 (n=20), 25 (n=20), or 50μg (n=20). No vaccine-related SAE or discontinuation due to an AE occurred. Most solicited reactions were mild and transient. The most frequently reported solicited reactions were pain at the injection site and myalgia. Antigen-specific IgG levels and functional activity showed dose-related increases. When comparing the three dose levels, a plateau effect was observed at the 25μg dose. ConclusionsAll dose levels were safe and immunogenic. Repeat vaccination significantly increased the level of anti-PlyD1 antibodies. Functional antibody activity was demonstrated in sera from vaccinated individuals (ClinicalTrails.gov no. NCT01444352).

Ability of ELISA and a toxin neutralization assay to detect changes in immunogenicity of a recombinant Bacillus anthracis protective antigen vaccine upon storage

We examined the capability of a mouse immunogenicity assay to detect improper storage of a recombinant protective antigen (rPA)-based anthrax vaccine formulated with an aluminum adjuvant, using ELISA and a toxin neutralization assay (TNA) to measure the antibody response to rPA. The vaccine was stored at 4 °C, room temperature (RT) or 37 °C for one, four and eight weeks and used for immunization, along with freshly prepared vaccine. Results showed that, contrary to ELISA, TNA is suitable to detect a loss of immunogenicity of the rPA vaccine following its exposure to RT for a period of eight weeks and to 37 °C for a period as short as 1 week.

Analysis of Defined Combinations of Monoclonal Antibodies in Anthrax Toxin Neutralization Assays and Their Synergistic Action

Antibodies against the protective antigen (PA) component of anthrax toxin play an important role in protection against disease caused by Bacillus anthracis. In this study, we examined defined combinations of PA-specific monoclonal antibodies for their ability to neutralize anthrax toxin in cell culture assays. We observed additive, synergistic, and antagonistic effects of the antibodies depending on the specific antibody combination examined and the specific assay used. Synergistic toxin-neutralizing antibody interactions were examined in more detail. We found that one mechanism that can lead to antibody synergy is the bridging of PA monomers by one antibody, with resultant bivalent binding of the second antibody. These results may aid in optimal design of new vaccines and antibody therapies against anthrax.

Phase 1 Study of a Recombinant Mutant Protective Antigen of Bacillus anthracis

A phase 1 study of a recombinant mutant protective antigen (rPA) vaccine was conducted in 186 healthy adults aged 18 to 45 years. Volunteers were randomized to receive one of three formulations of rPA (formalin treated, alum adsorbed, or both), in 10- or 20-μg dosages each, or the licensed vaccine, AVA. Three injections were given at 2-month intervals and a 4th 1 year after the 3rd. Vaccinees were examined at the clinic once following each injection, at 48 to 72 h postinjection. Adverse reactions were recorded in diaries for 7 days. Sera were collected before each injection and 1 week after the 1st, 2 weeks after the 3rd and 4th, and 1 year after the 4th. Serum anti-PA IgG was assayed by enzyme-linked immunosorbent assay (ELISA) and toxin neutralization assay (TNA). All formulations at both dosages were safe and immunogenic, inducing booster responses, with the highest antibody levels following the 4th injection (354 to 732 μg/ml). The lowest levels were induced by the formalin-only-treated rPA; there was no statistical difference between levels induced by alum-adsorbed and formalin-treated/alum-adsorbed rPA or by the two dosages. The antibody levels declined in all groups during the 1-year intervals after the 3rd and 4th injections but less so during the 2nd year, after the 4th injection (fold decreases were 10 to 25 versus 3.4 to 7.0, P < 0.001). There were too few AVA recipients for statistical comparisons, but their antibody levels followed those of rPA. Anti-rPA measured by ELISA correlated with TNA titers (r = 0.97). These data support studying alum-adsorbed rPA in children.

Humoral immunity and injection-site reactions in cattle vaccinated with a multivalent clostridial vaccine administered via subcutaneous injection or via transdermal needle-free injection

To evaluate injection-site reactions and serum antibody titers in cattle vaccinated with a clostridial vaccine administered SC or via needle-free transdermal injection. Sixteen 11-to 12-month-old Herefords. Cattle in 2 groups were vaccinated on days 0 and 28 with a commercially available multivalent clostridial vaccine administered SC or transdermally Injection sites and serum antibody titers were evaluated at several time points after vaccination. Serum antibody titers against Clostridium perfringens beta toxin, Clostridium novyi alpha toxin, and Clostridium septicum alpha toxin were determined with an ELISA; Clostridium sordellii lethal toxin titers were determined with a toxin neutralization assay. Firm injection site swellings developed in cattle vaccinated via either route; however, at several observation times, swellings were significantly smaller in cattle vaccinated transdermally. Serum titers against C perfringens beta toxin and C septicum alpha toxin did not differ significantly between groups after vaccination; serum titers against C novyi alpha toxin were not significantly different between groups, except on days 10 and 56, when they were significantly higher in cattle vaccinated SC. Titers against C sordellii lethal toxin were significantly higher in cattle vaccinated SC on several days after vaccination, but titers were not significantly different after day 49. Transdermal vaccination of cattle resulted in serum antibody titers that were similar to those induced via SC vaccination and caused injection-site reactions that were significantly smaller. Transdermal vaccination may be an effective technique for vaccinating cattle against clostridial diseases while minimizing local reactions that often develop after clostridial vaccination.

Qualitative and quantitative parameters predict functional humoral response to alum-adjuvanted immunization. (52.4)

Abstract Adjuvants are crucial vaccine components for enhancing immune response to immunogens. Many potential adjuvant materials have been studied for their ability to augment the response to antigens while maintaining low toxicity. A US FDA approved adjuvant routinely used in humans is alum. The aim of this study was to comprehensively measure the ways in which innate immune signals induced by alum manifest in terms of the effect on the adaptive immune response to recombinant protective antigen (PA) from Bacillus anthracis. C57Bl/6 mice were given a single subcutaneous immunization with PA +/- alum at the base of each limb. Serum and draining lymph nodes were collected after immunization (days 1-70). On day 71, saline or PA only boost was administered, followed by additional serum and tissue collection (days 78-85). Using multi-color immunohistochemistry it was shown beginning day 10 that alum induces a rapid and strong germinal center response in the draining lymph node. This observation correlates with quantitative and qualitative changes in PA-specific antibody assessed by ELISA and Biacore (SPR). In addition, augmented antibody response to PA+alum resulted in increased antibody functionality, as determined by anthrax toxin neutralization assays. This study demonstrates a novel multi-parameter approach to evaluation of antigen-specific responses to immunization and the impact of adjuvants. This approach will be useful in the development and testing of novel adjuvants.

Development and validation of serological methods for human vaccine potency testing: Case study of an anthrax vaccine

Only one anthrax vaccine (Anthrax Vaccine Adsorbed, AVA) is licensed in the US to date, although new vaccines are under development. Logistic difficulties with the potency testing of AVA, plus humane considerations, have prompted the development of an alternative test method that can be used as a potency test not only for AVA, but also for newer vaccines. A potency test is not limited to measuring the concentration and quality of the antigen in the final formulation at the time of vaccine release, but should also detect changes in these characteristics during the dating period of the product, to ensure that the vaccine has retained its potency. We have developed a mouse immunogenicity test with potential for use as a potency test for anthrax vaccines. This model is based on the measurement of antibodies induced by a fixed dose of antigen. The test consists of two stages: a) the induction of antibodies in mice with one pre-selected test dose of vaccine; and b) the measurement of the response. We have established the test dose for AVA and experimental vaccines based on the use of anthrax Protective Antigen (PA). Two types of assays, an ELISA and a toxin neutralization assay (TNA) have been employed to measure antibodies to PA. TNA may be more useful in predicting vaccine efficacy, since it measures the neutralizing activity of sera against the cytotoxic effect of the toxin formed by PA when associated with Lethal Factor. However, anti-PA ELISA is less demanding from a technical point of view. Therefore, if ELISA were capable of detecting accurately changes in antigen quantity and quality, then it could be selected as the antibody-measuring test. Studies in our laboratory suggest that even though ELISA and TNA results are correlated, they may not be strictly interchangeable for quantification of anti-PA antibodies after a single immunization of mice with a test dose of anthrax vaccine. We have also found that TNA is better suited than ELISA to detect changes in immunogenicity caused by vaccine exposure to high temperature for very short periods (two minutes). These results open the possibility that an immunogenicity test in which TNA is used to quantify the anti-PA antibody responses can be used to measure vaccine potency of anthrax vaccines at the end of the manufacturing process and periodically after the finished product has been placed in storage, instead of an active protection test that requires lethal challenge.

Enhanced sensitivity to Botulinum type A neurotoxin of human neuroblastoma SH-SY5Y cells after differentiation into mature neuronal cells

Research on more sensitive cell based assays is essential to assess neutralising activities of Botulinum neurotoxins antibodies. A reproducible and robust differentiation protocol was applied on human neuroblastoma SH-SY5Y cells generating neuronal cultures stable for 2?3 weeks. The presence of specific neuronal markers was assessed with labelled antibodies. Accurate and reproducible noradrenaline release levels were obtained which were significantly higher in differentiated cells and correlated with vesicle turnover assessments. The suitability of differentiated SH-SY5Y cells for detecting low levels of Botulinum type A and for toxin neutralisation assay was evidenced using functional end points relevant to Botulinum neurotoxicity.

Reduction of Immunogenicity of Anthrax Vaccines Subjected to Thermal Stress, as Measured by a Toxin Neutralization Assay

We report that a toxin neutralization assay (TNA) can detect a decrease in the immunogenicity of anthrax vaccines as a consequence of brief exposure to elevated temperature. This attribute of TNA may help in adopting immunogenicity as a replacement of the current potency test, which involves protection from lethal challenge.

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

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.

Induction of Neutralizing Antibody Responses to Anthrax Protective Antigen by Using Influenza Virus Vectors: Implications for Disparate Immune System Priming Pathways

Viral vectors based on influenza virus, rabies virus (RV), and vaccinia virus (VV) were used to express large polypeptide segments derived from the Bacillus anthracis protective antigen (PA). For the infectious influenza virus vector and recombinant VV constructs, the receptor binding domain (RBD or domain 4) or the lethal and edema factor binding domain (LEF or domain 1') were engineered into functional chimeric hemagglutinin (HA) glycoproteins. In the case of the RV vector, the viral glycoprotein (G) was used as a carrier for RBD in an inactivated form of the vector. These constructs were examined by using multiple homologous and heterologous prime/boost immunization regimens in order to optimize the induction of alpha-PA antibody responses. Several immunization combinations were shown to induce high titers of antibody recognizing the anthrax RBD and LEF domains, as well as the full-length PA protein in mice. The heterologous prime/boost immunization regimens that involved an initial intranasal administration of a live influenza virus vector, followed by an intramuscular boost with either the killed RV vector or the VV vector, were particularly effective, inducing antigen-specific antibodies at levels severalfold higher than homologous or alternative heterologous protocols. Furthermore, sera from several groups of the immunized mice demonstrated neutralization activity in an in vitro anthrax toxin neutralization assay. In some cases, such toxin-neutralizing activity was notably high, indicating that the mechanisms by which immunity is primed by live influenza virus vectors may have beneficial properties.

Vaccination of Rabbits with an Alkylated Toxoid Rapidly Elicits Potent Neutralizing Antibodies against Botulinum Neurotoxin Serotype B

New Zealand White (NZW) rabbits were immunized with several different nontoxic botulinum neurotoxin serotype B (BoNT/B) preparations in an effort to optimize the production of a rapid and highly potent, effective neutralizing antibody response. The immunogens included a recombinant heavy chain (rHc) protein produced in Escherichia coli, a commercially available formaldehyde-inactivated toxoid, and an alkylated toxoid produced by urea-iodoacetamide inactivation of the purified active toxin. All three immunogens elicited an antibody response to BoNT/B, detected by enzyme-linked immunosorbent assay (ELISA) and by toxin neutralization assay, by the use of two distinct mouse toxin challenge models. The induction period and the ultimate potency of the observed immune response varied for each immunogen, and the ELISA titer was not reliably predictive of the potency of toxin neutralization. The kinetics of the BoNT/B-specific binding immune response were nearly identical for the formaldehyde toxoid and alkylated toxoid immunogens, but immunization with the alkylated toxoid generated an approximately 10-fold higher neutralization potency that endured throughout the study, and after just 49 days, each milliliter of serum was capable of neutralizing 10(7) 50% lethal doses of the toxin. Overall, the immunization of rabbits with alkylated BoNT/B toxoid appears to have induced a neutralizing immune response more rapid and more potent than the responses generated by vaccination with formaldehyde toxoid or rHc preparations.

Comparison of Three Anthrax Toxin Neutralization Assays

Different types of anthrax toxin neutralization assays have been utilized to measure the antibody levels elicited by anthrax vaccines in both nonclinical and clinical studies. In the present study, we sought to determine whether three commonly used toxin neutralization assays-J774A.1 cell-, RAW 264.7 cell-, and CHO cell-based assays-yield comparable estimates of neutralization activities for sera obtained after vaccination with anthrax vaccines composed of recombinant protective antigen (rPA). In order to compare the assays, sera were assayed alongside a common reference serum sample and the neutralization titers were expressed relative to the titer for the reference sample in each assay. Analysis of sera from rabbits immunized with multiple doses of the rPA vaccine showed that for later bleeds, the quantitative agreement between the assays was good; however, for early bleeds, some heterogeneity in relative neutralization estimates was observed. Analysis of serum samples from rabbits, nonhuman primates, and humans immunized with the rPA vaccine showed that the relative neutralization estimates obtained in the different assays agreed to various extents, depending on the species of origin of the sera examined. We identified differences in the magnitudes of the Fc receptor-mediated neutralization associated with the J774A.1 cell- and RAW 264.7 cell-based assays, which may account for some of the species dependence of the assays. The differences in the relative neutralization estimates among the assays were relatively small and were always less than 2.5-fold. However, because toxin neutralization assays will likely be used to establish the efficacies of new anthrax vaccines, our findings should be considered when assay outputs are interpreted.

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

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

Comparability of ELISA and toxin neutralization to measure immunogenicity of Protective Antigen in mice, as part of a potency test for anthrax vaccines

Complexities of lethal challenge models have prompted the investigation of immunogenicity assays as potency tests of anthrax vaccines. An ELISA and a lethal toxin neutralization assay (TNA) were used to measure antibody response to Protective Antigen (PA) in mice immunized once with either a commercial or a recombinant PA (rPA) vaccine formulated in-house. Even though ELISA and TNA results showed correlation, ELISA results may not be able to accurately predict TNA results in this single immunization model.

Antigen-specific human polyclonal antibodies from hyperimmunized cattle

Antigen-specific human polyclonal antibodies (hpAbs), produced by hyperimmunization, could be useful for treating many human diseases. However, yields from available transgenic mice and transchromosomic (Tc) cattle carrying human immunoglobulin loci are too low for therapeutic applications. We report a Tc bovine system that produces large yields of hpAbs. Tc cattle were generated by transferring a human artificial chromosome vector carrying the entire unrearranged, human immunoglobulin heavy (hIGH) and kappa-light (hIGK) chain loci to bovine fibroblasts in which two endogenous bovine IgH chain loci were inactivated. Plasma from the oldest animal contained >2 g/l of hIgG, paired with either human kappa-light chain (up to approximately 650 microg/ml, fully human) or with bovine kappa- or lambda-light chain (chimeric), with a normal hIgG subclass distribution. Hyperimmunization with anthrax protective antigen triggered a hIgG-mediated humoral immune response comprising a high proportion of antigen-specific hIgG. Purified, fully human and chimeric hIgGs were highly active in an in vitro toxin neutralization assay and protective in an in vivo mouse challenge assay.

An adenoviral vector-based mucosal vaccine is effective in protection against botulism

A replication-incompetent adenoviral vector encoding the heavy chain C-fragment (HC50) of botulinum neurotoxin type C (BoNT/C) was evaluated as a mucosal vaccine against botulism in a mouse model. Single intranasal inoculation of the adenoviral vector elicited a high level of HC50-specific IgG, IgG1 and IgG2a in sera and IgA in mucosal secretions as early as 2 weeks after vaccination. The antigen-specific serum antibodies were maintained at a high level at least until the 27th week. Immune sera showed high potency in neutralizing BoNT/C as indicated by in vitro toxin neutralization assay. The mice receiving single dose of 2 × 107 p.f.u. (plaque-forming unit) of adenoviral vector were completely protected against challenge with up to 104 × MLD50 of BoNT/C. The protective immunity showed vaccine dose dependence from 105 to 2 × 107 p.f.u. of adenoviral vector. In addition, animals receiving single intranasal dose of 2 × 107 p.f.u. adenoviral vector could be protected against 100 × MLD50 27 weeks after vaccination. Animals with preexisting immunity to adenovirus could also be vaccinated intranasally and protected against lethal challenge with BoNT/C. These results suggest that the adenoviral vector is a highly effective gene-based mucosal vaccine against botulism.

Role of the N-Terminal Amino Acid of Bacillus anthracis Lethal Factor in Lethal Toxin Cytotoxicity and Its Effect on the Lethal Toxin Neutralization Assay

The cytotoxic activity of lethal factor (LF), a critical reagent used in the cell-based lethal toxin neutralization assay to assess anthrax vaccines, was shown to depend on the identity of its N-terminal amino acid, which plays a role in the targeting of LF to the proteasome for degradation. These results demonstrate that care must be taken to ensure that LF preparations used in standardized cell-based assays are not altered at their N-terminal ends.