Lethal Toxin Neutralization Research Articles

Immunological Evidence of Variation in Exposure and Immune Response to Bacillus anthracis in Herbivores of Kruger and Etosha National Parks.

Exposure and immunity to generalist pathogens differ among host species and vary across spatial scales. Anthrax, caused by a multi-host bacterial pathogen, Bacillus anthracis, is enzootic in Kruger National Park (KNP), South Africa and Etosha National Park (ENP), Namibia. These parks share many of the same potential host species, yet the main anthrax host in one (greater kudu (Tragelaphus strepsiceros) in KNP and plains zebra (Equus quagga) in ENP) is only a minor host in the other. We investigated species and spatial patterns in anthrax mortalities, B. anthracis exposure, and the ability to neutralize the anthrax lethal toxin to determine if observed host mortality differences between locations could be attributed to population-level variation in pathogen exposure and/or immune response. Using serum collected from zebra and kudu in high and low incidence areas of each park (18- 20 samples/species/area), we estimated pathogen exposure from anti-protective antigen (PA) antibody response using enzyme-linked immunosorbent assay (ELISA) and lethal toxin neutralization with a toxin neutralization assay (TNA). Serological evidence of pathogen exposure followed mortality patterns within each system (kudus: 95% positive in KNP versus 40% in ENP; zebras: 83% positive in ENP versus 63% in KNP). Animals in the high-incidence area of KNP had higher anti-PA responses than those in the low-incidence area, but there were no significant differences in exposure by area within ENP. Toxin neutralizing ability was higher for host populations with lower exposure prevalence, i.e., higher in ENP kudus and KNP zebras than their conspecifics in the other park. These results indicate that host species differ in their exposure to and adaptive immunity against B. anthracis in the two parks. These patterns may be due to environmental differences such as vegetation, rainfall patterns, landscape or forage availability between these systems and their interplay with host behavior (foraging or other risky behaviors), resulting in differences in exposure frequency and dose, and hence immune response.

Aerosolized Intratracheal Inoculation of Recombinant Protective Antigen (rPA) Vaccine Provides Protection Against Inhalational Anthrax in B10.D2-Hc0 Mice.

Anthrax caused by Bacillus anthracis is a fatal zoonotic disease with a high lethality and poor prognosis. Inhalational anthrax is the most severe of the three forms of anthrax. The currently licensed commercial human anthrax vaccines require a complex immunization procedure for efficacy and have side effects that limit its use in emergent situations. Thus, development of a better anthrax vaccine is necessary. In this study, we evaluate the potency and efficacy of aerosolized intratracheal (i.t.) inoculation with recombinant protective antigen (rPA) subunit vaccines against aerosolized B. anthracis Pasteur II spores (an attenuated strain) challenge in a B10.D2-Hc0 mouse (deficient in complement component C5) model. Immunization of rPA in liquid, powder or powder reconstituted formulations via i.t. route conferred 100% protection against a 20× LD50 aerosolized Pasteur II spore challenge in mice, compared with only 50% of subcutaneous (s.c.) injection with liquid rPA. Consistently, i.t. inoculation of rPA vaccines induced a higher lethal toxin (LeTx) neutralizing antibody titer, a stronger lung mucosal immune response and a greater cellular immune response than s.c. injection. Our results demonstrate that immunization with rPA dry powder vaccine via i.t. route may provide a stable and effective strategy to improve currently available anthrax vaccines and B10.D2-Hc0 mice challenged with B. anthracis attenuated strains might be an alternative model for anthrax vaccine candidate screening.

Insufficient Anthrax Lethal Toxin Neutralization Is Associated with Antibody Subclass and Domain Specificity in the Plasma of Anthrax-Vaccinated Individuals.

Anthrax vaccine adsorbed (AVA) is a significant line of defense against bioterrorist attack from Bacillus anthracis spores. However, in a subset of individuals, this vaccine may produce a suboptimal quantity of anti-protective antigen (PA), antibodies that are poorly neutralizing, and/or antibody titers that wane over time, necessitating annual boosters. To study individuals with such poor responses, we examine the properties of anti-PA in a subset of vaccinated individuals that make significant quantities of antibody but are still unable to neutralize toxin. In this cohort, characterized by poorly neutralizing antibody, we find that increased IgG4 to IgG1 subclass ratios, low antibody avidity, and insufficient antibody targeting domain 4 associate with improper neutralization. Thus, future vaccines and vaccination schedules should be formulated to improve these deficiencies.

Repeat Dose Toxicity Study of the AV7909 Anthrax Vaccine Candidate in Juvenile Rats.

AV7909 is a next-generation anthrax vaccine candidate indicated for post-exposure prophylaxis of exposure to Bacillus anthracis. AV7909 consists of the Anthrax Vaccine Adsorbed (AVA) bulk drug substance and the immunostimulatory Toll-like receptor 9 agonist oligodeoxynucleotide adjuvant, CPG 7909. Safety testing for pediatric population is warranted to support the potential emergency use of AV7909 in children. This study was conducted to investigate the local tolerance and potential systemic toxicity and their reversibility in juvenile rats by repeat intramuscular injections of the AV7909 vaccine candidate. Animals were dosed on postnatal day (PND) 21 (at weaning), PND 28, and PND 35, with the test article (AV7909), the adjuvant alone (Alhydrogel + CPG 7909), or sterile water for injection. Core group animals were necropsied on PND 37 and recovery group on PND 49. Study end points included survival, clinical observations, injection site observations, body weights, clinical pathology (hematology, coagulation, and clinical chemistry), pro-inflammatory biomarker analysis (alpha-2 macroglobulin [A2M] and alpha-1 acid glycoprotein [AGP]), and anatomic pathology. Immune response to vaccination was measured using the high-throughput anthrax lethal toxin neutralization assay (htpTNA). The AV7909 vaccine candidate produced no apparent systemic or local toxicity. The AGP and A2M levels were elevated in both the adjuvant-alone and AV7909 groups at the end of treatment but were comparable to control levels by the end of the recovery period. All animals in the AV7909 group demonstrated a robust neutralizing antibody response. The results indicate that AV7909 has a favorable safety profile in juvenile rats.

Process intensification in peptide manufacturing: Recombinant lethal toxin neutralizing factor (rLTNF) as a case study

Process intensification is necessary to create economical processes. Cleavage reaction is one of the critical unit operations in peptide manufacturing processes as it involves cutting of concatemer expressed to obtain monomer. In this paper, solubilization and cleavage reaction have been merged into a single unit operation so as to allow for simultaneous solubilization and cleavage. Critical variables such as urea concentration, calcium chloride concentration, pH, and enzyme loading were optimized using quality by design (QbD) principles. The subsequent RP-HPLC unit operation was also intensified with respect to elution gradient and product stability in elution buffer so as to facilitate direct freeze-drying and storage. The proposed three-step process was analysed for its economics and compared with the previous generation process, showing significant improvements including a 21% reduction in batch time, 27% increase in productivity, and 30% reduction in manufacturing cost. The work illustrates the effectiveness of applying QbD principles and process intensification for creation of a more efficient manufacturing bioprocess.

Immunogenicity and safety of a novel recombinant protective antigen anthrax vaccine (GC1109), a randomized, single-blind, placebo controlled phase II clinical study

BackgroundThe demand on effective and safe anthrax vaccine is increasing as a part of the preparedness for possible bioterrorism in the future. We performed a randomized, single-blind, placebo controlled phase II clinical study to evaluate the immunogenicity and safety of a novel recombinant protective antigen (rPA) anthrax vaccine, GC1109, in healthy adult volunteers. MethodsParticipants were randomized to experiment groups (0.3 mL, 0.5 mL, and 1.0 mL of GC1109) or placebo group (normal saline 0.5 mL) in 2:2:2:1 ratio. They received respective vaccines intramuscularly at 0, 4 and 8 weeks. Immunogenicity was evaluated by seroconversion rate and geometric mean titer (GMT) of lethal toxin neutralizing assay (TNA) and anti-PA IgG by ELISA. Safety was assessed by laboratory tests, and solicited and unsolicited adverse events on diary cards. Results30, 29, 30 participants were randomized to 0.3, 0.5, and 1.0 mL of GC1109 groups, respectively, while 15 to placebo group. 92 participants received all three doses. In per-protocol analysis, TNA GMTs at week 12 were 296.5, 285.2, and 433.2 in the three groups, respectively. Seroconversion rates measured by ELISA were 100% at week 12 in the three groups. Local and systemic vaccine-related adverse events were frequent; however, most of them were mild, and no serious events were observed. ConclusionsA new rPA anthrax vaccine GC1109 was immunogenic after three doses of intramuscular administration, and was well-tolerated.

Anthrax lethal toxin (LeTx) neutralization by PA domain specific antisera

Anthrax associated causalities in humans and animals are implicated mainly due to the action of two exotoxins that are secreted by the bacterium Bacillus antharcis during the infection. These exotoxins comprise of three protein components namely protective antigen (PA), lethal factor (LF) and edema factor (EF). The protective antigen is the common toxin component required to form both lethal toxin (LeTx) and edema toxin (EdTx). The LeTx is formed, when PA combines with LF and EdTx is formed when PA combines with EF. Therapeutic interventions aiming to neutralize these key effectors of anthrax pathology would therefore, provide an effective means to counter the toxicity imposed by the anthrax toxins on the host. The present work describes the lethal toxin neutralization potential of polyclonal antisera developed against the individual domains of the protective antigen component of the anthrax toxin. The individual domains were produced as recombinant proteins in E. coli and validated with peptide mass fingerprinting by MALDI-TOF analysis and corresponding mice polyclonal antisera by western blotting. Each domain specific antibody titre and isotype was ascertained by ELISA. The isotyping revealed the predominance of IgG1 isotype. The toxin neutralizing potential of these domain specific antisera were evaluated by in-vitro cell viability MTT assay, employing J774.1 mouse macrophage cell line against LeTx (0.25 μg ml−1 PA and 0.125 μg ml−1 LF concentrations). Among the four domain specific antisera, the antiserum against PA domain IV could neutralize LeTx with high efficiency. No significant neutralization of LeTx was observed with other domain specific antibodies. Results indicate that antibodies to r-PA domain IV could be explored further as therapeutic anti toxin molecule along with appropriate antibiotic regimens against anthrax.

Correlation between anthrax lethal toxin neutralizing antibody levels and survival in guinea pigs and nonhuman primates vaccinated with the AV7909 anthrax vaccine candidate.

The anthrax vaccine candidate AV7909 is being developed as a next generation vaccine for a post-exposure prophylaxis (PEP) indication against anthrax. AV7909 consists of the Anthrax Vaccine Adsorbed (AVA, BioThrax®) bulk drug substance adjuvanted with the immunostimulatory oligodeoxynucleotide (ODN) compound, CPG 7909. The addition of CPG 7909 to AVA enhances both the magnitude and the kinetics of antibody responses in animals and human subjects, making AV7909 a suitable next-generation vaccine for use in a PEP setting. The studies described here provide initial information on AV7909-induced toxin-neutralizing antibody (TNA) levels associated with the protection of animals from lethal Bacillus anthracis challenge. Guinea pigs or nonhuman primates (NHPs) were immunized on Days 0 and 28 with various dilutions of AV7909, AVA or a saline or Alhydrogel+CPG 7909 control. Animals were challenged via the inhalational route with a lethal dose of aerosolized B. anthracis (Ames strain) spores and observed for clinical signs of disease and mortality. The relationship between pre-challenge serum TNA levels and survival following challenge was determined in order to calculate a threshold TNA level associated with protection. Immunisation with AV7909 induced a rapid, highly protective TNA response in guinea pigs and NHPs. Surprisingly, the TNA threshold associated with a 70% probability of survival for AV7909 immunized animals was substantially lower than the threshold which has been established for the licensed AVA vaccine. The results of this study suggest that the TNA threshold of protection against anthrax could be modified by the addition of an immune stimulant such as CPG 7909 and that the TNA levels associated with protection may be vaccine-specific.

Lethal factor antibodies contribute to lethal toxin neutralization in recipients of anthrax vaccine precipitated

A major difference between two currently licensed anthrax vaccines is presence (United Kingdom Anthrax Vaccine Precipitated, AVP) or absence (United States Anthrax Vaccine Adsorbed, AVA) of quantifiable amounts of the Lethal Toxin (LT) component Lethal Factor (LF). The primary immunogen in both vaccine formulations is Protective Antigen (PA), and LT-neutralizing antibodies directed to PA are an accepted correlate of vaccine efficacy; however, vaccination studies in animal models have demonstrated that LF antibodies can be protective. In this report we compared humoral immune responses in cohorts of AVP (n=39) and AVA recipients (n=78) matched 1:2 for number of vaccinations and time post-vaccination, and evaluated whether the LF response contributes to LT neutralization in human recipients of AVP. PA response rates (≥95%) and PA IgG concentrations were similar in both groups; however, AVP recipients exhibited higher LT neutralization ED50 values (AVP: 1464.0±214.7, AVA: 544.9±83.2, p<0.0001) and had higher rates of LF IgG positivity (95%) compared to matched AVA vaccinees (1%). Multiple regression analysis revealed that LF IgG makes an independent and additive contribution to the LT neutralization response in the AVP group. Affinity purified LF antibodies from two independent AVP recipients neutralized LT and bound to LF Domain 1, confirming contribution of LF antibodies to LT neutralization. This study documents the benefit of including an LF component to PA-based anthrax vaccines.

Cross-species prediction of human survival probabilities for accelerated anthrax vaccine absorbed (AVA) regimens and the potential for vaccine and antibiotic dose sparing

Anthrax vaccine adsorbed (AVA, BioThrax) was recently approved by the Food and Drug Administration (FDA) for a post-exposure prophylaxis (PEP) indication in adults 18–65years of age. The schedule is three doses administered subcutaneous (SC) at 2-week intervals (0, 2, and 4weeks), in conjunction with a 60-day course of antimicrobials. The Public Health Emergency Medical Countermeasures Enterprise (PHEMCE) developed an animal model to support assessment of a shortened antimicrobial PEP duration following Bacillus anthracis exposure. A nonhuman primate (NHP) study was completed to evaluate the efficacy of a two dose anthrax vaccine absorbed (AVA) schedule (0, 2weeks) aerosol challenged with high levels of B. anthracis spores at week4– the time point at which humans would receive the third vaccination of the approved PEP schedule. Here we use logistic regression models to combine the survival data from the NHP study along with serum anthrax lethal toxin neutralizing activity (TNA) and anti-PA IgG measured by enzyme linked immunosorbent assay (ELISA) data to perform a cross-species analysis to estimate survival probabilities in vaccinated human populations at this time interval (week4 of the PEP schedule). The bridging analysis demonstrated that high levels of NHP protection also yield high predicted probability of human survival just 2weeks after the second dose of vaccine with the full or half antigen dose regimen. The absolute difference in probability of human survival between the full and half antigen dose was estimated to be at most approximately 20%, indicating that more investigation of the half-antigen dose for vaccine dose sparing strategies may be warranted.

Comparative analysis of the immunologic response induced by the Sterne 34F2 live spore Bacillus anthracis vaccine in a ruminant model

The Sterne 34F2 live spore vaccine (SLSV) developed in 1937 is the most widely used veterinary vaccine against anthrax. However, literature on the immunogenicity of this vaccine in a target ruminant host is scarce. In this study, we evaluated the humoral response to the Bacillus anthracis protective antigen (rPA), a recombinant bacillus collagen-like protein of anthracis (rBclA), formaldehyde inactivated spores (FIS) prepared from strain 34F2 and a vegetative antigen formulation prepared from a capsule and toxin deficient strain (CDC 1014) in Boer goats. The toxin neutralizing ability of induced antibodies was evaluated using an in vitro toxin neutralization assay. The protection afforded by the vaccine was also assessed in vaccinates. Anti-rPA, anti-FIS and lethal toxin neutralizing titres were superior after booster vaccinations, compared to single vaccinations. Qualitative analysis of humoral responses to rPA, rBclA and FIS antigens revealed a preponderance of anti-FIS IgG titres following either single or double vaccinations with the SLSV. Antibodies against FIS and rPA both increased by 350 and 300-fold following revaccinations respectively. There was no response to rBclA following vaccinations with the SLSV. Toxin neutralizing titres increased by 80-fold after single vaccination and 700-fold following a double vaccination. Lethal challenge studies in naïve goats indicated a minimum infective dose of 36 B. anthracis spores. Single and double vaccination with the SLSV protected 4/5 and 3/3 of goats challenged with>800 spores respectively. An early booster vaccination following the first immunization is suggested in order to achieve a robust immunity. Results from this study indicate that this crucial second vaccination can be administered as early as 3 months after the initial vaccination.

Anthrax Toxin-Expressing Bacillus cereus Isolated from an Anthrax-Like Eschar

Bacillus cereus isolates have been described harboring Bacillus anthracis toxin genes, most notably B. cereus G9241, and capable of causing severe and fatal pneumonias. This report describes the characterization of a B. cereus isolate, BcFL2013, associated with a naturally occurring cutaneous lesion resembling an anthrax eschar. Similar to G9241, BcFL2013 is positive for the B. anthracis pXO1 toxin genes, has a multi-locus sequence type of 78, and a pagA sequence type of 9. Whole genome sequencing confirms the similarity to G9241. In addition to the chromosome having an average nucleotide identity of 99.98% when compared to G9241, BcFL2013 harbors three plasmids with varying homology to the G9241 plasmids (pBCXO1, pBC210 and pBFH_1). This is also the first report to include serologic testing of patient specimens associated with this type of B. cereus infection which resulted in the detection of anthrax lethal factor toxemia, a quantifiable serum antibody response to protective antigen (PA), and lethal toxin neutralization activity.

Humoral and Cell-Mediated Immune Responses to Alternate Booster Schedules of Anthrax Vaccine Adsorbed in Humans.

Protective antigen (PA)-specific antibody and cell-mediated immune (CMI) responses to annual and alternate booster schedules of anthrax vaccine adsorbed (AVA; BioThrax) were characterized in humans over 43 months. Study participants received 1 of 6 vaccination schedules: a 3-dose intramuscular (IM) priming series (0, 1, and 6 months) with a single booster at 42 months (4-IM); 3-dose IM priming with boosters at 18 and 42 months (5-IM); 3-dose IM priming with boosters at 12, 18, 30, and 42 months (7-IM); the 1970 licensed priming series of 6 doses (0, 0.5, 1, 6, 12, and 18 months) and two annual boosters (30 and 42 months) administered either subcutaneously (SQ) (8-SQ) or IM (8-IM); or saline placebo control at all eight time points. Antibody response profiles included serum anti-PA IgG levels, subclass distributions, avidity, and lethal toxin neutralization activity (TNA). CMI profiles included frequencies of gamma interferon (IFN-γ)- and interleukin 4 (IL-4)-secreting cells and memory B cells (MBCs), lymphocyte stimulation indices (SI), and induction of IFN-γ, IL-2, IL-4, IL-6, IL-1β, and tumor necrosis factor alpha (TNF-α) mRNA. All active schedules elicited high-avidity PA-specific IgG, TNA, MBCs, and T cell responses with a mixed Th1-Th2 profile and Th2 dominance. Anti-PA IgG and TNA were highly correlated (e.g., month 7,r(2)= 0.86,P< 0.0001, log10 transformed) and declined in the absence of boosters. Boosters administered IM generated the highest antibody responses. Increasing time intervals between boosters generated antibody responses that were faster than and superior to those obtained with the final month 42 vaccination. CMI responses to the 3-dose IM priming remained elevated up to 43 months. (This study has been registered at ClinicalTrials.gov under registration no. NCT00119067.).

Comprehensive analysis and selection of anthrax vaccine adsorbed immune correlates of protection in rhesus macaques.

Humoral and cell-mediated immune correlates of protection (COP) for inhalation anthrax in a rhesus macaque (Macaca mulatta) model were determined. The immunological and survival data were from 114 vaccinated and 23 control animals exposed to Bacillus anthracis spores at 12, 30, or 52 months after the first vaccination. The vaccinated animals received a 3-dose intramuscular priming series (3-i.m.) of anthrax vaccine adsorbed (AVA) (BioThrax) at 0, 1, and 6 months. The immune responses were modulated by administering a range of vaccine dilutions. Together with the vaccine dilution dose and interval between the first vaccination and challenge, each of 80 immune response variables to anthrax toxin protective antigen (PA) at every available study time point was analyzed as a potential COP by logistic regression penalized by least absolute shrinkage and selection operator (LASSO) or elastic net. The anti-PA IgG level at the last available time point before challenge (last) and lymphocyte stimulation index (SI) at months 2 and 6 were identified consistently as a COP. Anti-PA IgG levels and lethal toxin neutralization activity (TNA) at months 6 and 7 (peak) and the frequency of gamma interferon (IFN-γ)-secreting cells at month 6 also had statistically significant positive correlations with survival. The ratio of interleukin 4 (IL-4) mRNA to IFN-γ mRNA at month 6 also had a statistically significant negative correlation with survival. TNA had lower accuracy as a COP than did anti-PA IgG response. Following the 3-i.m. priming with AVA, the anti-PA IgG responses at the time of exposure or at month 7 were practicable and accurate metrics for correlating vaccine-induced immunity with protection against inhalation anthrax.

Anthrax vaccine adsorbed: Further evidence supporting continuing the vaccination series rather than restarting the series when doses are delayed

Whether to restart or continue the series when anthrax vaccine doses are missed is a frequent medical management problem. We applied the noninferiority analysis model to this prospective study comparing the Bacillus anthracis protective antigen (PA) IgG antibody response and lethal toxin neutralization activity at day 28 to the anthrax vaccine adsorbed (AVA) (Biothrax®) administered on schedule or delayed. A total of 600 volunteers were enrolled: 354 in the on-schedule cohort; 246 in the delayed cohort. Differences were noted in immune responses between cohorts (p<0.0001) and among the racial categories (p<0.0001). Controlling for covariates, the delayed cohort was non-inferior to the on-schedule cohort for the rate of 4-fold rise in both anti-PA IgG concentration (p<0.0001) and TNA ED50 titers (p<0.0001); as well as the mean log10-transformed anti-PA IgG concentration (p<0.0001) and the mean log10-transformed TNA ED50 titers (p<0.0001). Providing a missed AVA dose after a delay as long as 5–7 years, elicits anti-PA IgG antibody and TNA ED50 responses that are robust and non-inferior to the responses observed when the 6-month dose is given on-schedule. These important data suggest it is not necessary to restart the series when doses of the anthrax vaccine are delayed as long as 5 or more years.

Protective-antigen (PA) based anthrax vaccines confer protection against inhalation anthrax by precluding the establishment of a systemic infection

An intense effort has been launched to develop improved anthrax vaccines that confer rapid, long lasting protection preferably with an extended stability profile amenable for stockpiling. Protective antigen (PA)-based vaccines are most favored as immune responses directed against PA are singularly protective, although the actual protective mechanism remains to be unraveled. Herein we show that contrary to the prevailing view, an efficacious PA-based vaccine confers protection against inhalation anthrax by preventing the establishment of a toxin-releasing systemic infection. Equally importantly, antibodies measured by the in vitro lethal toxin neutralization activity assay (TNA) that is considered as a reliable correlate of protection, especially for PA protein-based vaccines adjuvanted with aluminum salts appear to be not absolutely essential for this protective immune response.

Effect of delayed anthrax vaccine dose on Bacillus anthracis protective antigen IgG response and lethal toxin neutralization activity

We describe the Bacillus anthracis protective antigen IgG antibody response and the B. anthracis lethal toxin neutralization activity to a delayed dose of anthrax vaccine adsorbed (AVA, BioThrax®) using validated assays. 373 individuals received 1, 2, or 3 priming doses, 18–24 months afterward, they received a delayed dose of AVA. Overall, 23.6% of subjects showed detectable anti-PA IgG before the boost, compared to 99.2% (P<0.0001) 28 days after the boost. Geometric mean anti-PA IgG concentration (GMC) was 1.66μg/mL before and 887.82μg/mL after the boost (P<0.0001). The proportion of individuals with four-fold increase in GMC following the boost ranged from 93.8% to 100%. Robust anti-PA IgG levels and B. anthracis lethal toxin neutralization activity are induced when an AVA dose is delayed as long as two years. These data support continuing with the vaccination schedule when a dose is delayed as long as two years rather than restarting the series.

Stochastic humoral immunity to Bacillus anthracis protective antigen: Identification of anti-peptide IgG correlating with seroconversion to Lethal Toxin neutralization

A substantial fraction of individuals vaccinated against anthrax have low to immeasurable levels of serum Lethal Toxin (LeTx)-neutralizing activity. The only known correlate of protection against Bacillus anthracis in the currently licensed vaccine is magnitude of the IgG response to Protective Antigen (PA); however, some individuals producing high serum levels of anti-PA IgG fail to neutralize LeTx in vitro. This suggests that non-protective humoral responses to PA may be immunodominant in some individuals. Therefore, to better understand why anthrax vaccination elicits heterogeneous levels of protection, this study was designed to elucidate the relationship between anti-PA fine specificity and LeTx neutralization in response to PA vaccination. Inbred mice immunized with recombinant PA produced high levels of anti-PA IgG and neutralized LeTx in vitro and in vivo. Decapeptide binding studies using pooled sera reproducibly identified the same 9 epitopes. Unexpectedly, sera from individual mice revealed substantial heterogeneity in the anti-PA IgG and LeTx neutralization responses, despite relative genetic homogeneity, shared environment and exposure to the same immunogen. This heterogeneity permitted the identification of specificities that correlate with LeTx-neutralizing activity. IgG binding to six decapeptides comprising two PA epitopes, located in domains I and IV, significantly correlate with seroconversion to LeTx neutralization. These results indicate that stochastic variation in humoral immunity is likely to be a major contributor to the general problem of heterogeneity in vaccine responsiveness and suggest that vaccine effectiveness could be improved by approaches that focus the humoral response toward protective epitopes in a greater fraction of vaccinees.

Anthrax vaccine-induced antibodies provide cross-species prediction of survival to aerosol challenge.

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 κ 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.

A Three-Dose Intramuscular Injection Schedule of Anthrax Vaccine Adsorbed Generates Sustained Humoral and Cellular Immune Responses to Protective Antigen and Provides Long-Term Protection against Inhalation Anthrax in Rhesus Macaques

A 3-dose (0, 1, and 6 months) intramuscular (3-IM) priming series of a human dose (HuAVA) and dilutions of up to 1:10 of anthrax vaccine adsorbed (AVA) provided statistically significant levels of protection (60 to 100%) against inhalation anthrax for up to 4 years in rhesus macaques. Serum anti-protective antigen (anti-PA) IgG and lethal toxin neutralization activity (TNA) were detectable following a single injection of HuAVA or 1:5 AVA or following two injections of diluted vaccine (1:10, 1:20, or 1:40 AVA). Anti-PA and TNA were highly correlated (overall r(2) = 0.89 for log(10)-transformed data). Peak responses were seen at 6.5 months. In general, with the exception of animals receiving 1:40 AVA, serum anti-PA and TNA responses remained significantly above control levels at 28.5 months (the last time point measured for 1:20 AVA), and through 50.5 months for the HuAVA and 1:5 and 1:10 AVA groups (P < 0.05). PA-specific gamma interferon (IFN-γ) and interleukin-4 (IL-4) CD4(+) cell frequencies and T cell stimulation indices were sustained through 50.5 months (the last time point measured). PA-specific memory B cell frequencies were highly variable but, in general, were detectable in peripheral blood mononuclear cells (PBMC) by 2 months, were significantly above control levels by 7 months, and remained detectable in the HuAVA and 1:5 and 1:20 AVA groups through 42 months (the last time point measured). HuAVA and diluted AVA elicited a combined Th1/Th2 response and robust immunological priming, with sustained production of high-avidity PA-specific functional antibody, long-term immune cell competence, and immunological memory (30 months for 1:20 AVA and 52 months for 1:10 AVA). Vaccinated animals surviving inhalation anthrax developed high-magnitude anamnestic anti-PA IgG and TNA responses.