A Case Study for Critical Reagent Qualification for Ligand Binding Assays Using Equivalence Test Methodology.

Qualifying critical reagents in ligand binding assays by parallel testing of current and candidate reagent lots is recommended by regulatory agencies and industry groups, but specific guidance on the format of reagent qualification experiments is limited. Equivalence testing is a statistically sound approach that is consistent with the objective of critical reagent qualification. We present power analysis for equivalence regions ranging from 1.25- to 1.5-fold multiples of the GM ratio (centered on 1) of current and candidate lots, over a range of assay variability from 5 to 30% coefficient of variation (CV). A 1.25-fold equivalence region can be tested using 6 to 12 plates per lot for assays with up to 15% CV but is not practical for more variable assays. For these assays, wider equivalence regions are justified so long as care is taken to avoid assay drift and the assay remains suitable for the intended use. The equivalence test method is illustrated using historical data from passing and failing reagent qualification experiments. Simulation analysis was performed to support the design of qualification experiments using 6, 12, or 18 plates per lot over a broad range of assay variability. A challenge in implementing the equivalence test approach is selecting an appropriate equivalence region. Equivalence regions providing 90% power using 12 plates/lot were consistent with 1.5σ bounds, which are recommended for equivalence testing of critical quality attributes of biosimilars.

Comparing antibody assays as correlates of protection against COVID-19 in the COVE mRNA-1273 vaccine efficacy trial.

The best assay or marker to define mRNA-1273 vaccine-induced antibodies as a correlate of protection (CoP) is unclear. In the COVE trial, participants received two doses of the mRNA-1273 COVID-19 vaccine or placebo. We previously assessed IgG binding antibodies to the spike protein (spike IgG) or receptor binding domain (RBD IgG) and pseudovirus neutralizing antibody 50 or 80% inhibitory dilution titer measured on day 29 or day 57, as correlates of risk (CoRs) and CoPs against symptomatic COVID-19 over 4 months after dose. Here, we assessed a new marker, live virus 50% microneutralization titer (LV-MN50), and compared and combined markers in multivariable analyses. LV-MN50 was an inverse CoR, with a hazard ratio of 0.39 (95% confidence interval, 0.19 to 0.83) at day 29 and 0.51 (95% confidence interval, 0.25 to 1.04) at day 57 per 10-fold increase. In multivariable analyses, pseudovirus neutralization titers and anti-spike binding antibodies performed best as CoRs; combining antibody markers did not improve correlates. Pseudovirus neutralization titer was the strongest independent correlate in a multivariable model. Overall, these results supported pseudovirus neutralizing and binding antibody assays as CoRs and CoPs, with the live virus assay as a weaker correlate in this sample set. Day 29 markers performed as well as day 57 markers as CoPs, which could accelerate immunogenicity and immunobridging studies.

Single-Shot ChAd3-MARV Vaccine in Modified Formulation Buffer Shows 100% Protection of NHPs.

Marburg virus (MARV) is a virus of high human consequence with a case fatality rate of 24-88%. The global health and national security risks posed by Marburg virus disease (MVD) underscore the compelling need for a prophylactic vaccine, but no candidate has yet reached regulatory approval. Here, we evaluate a replication-defective chimpanzee adenovirus type 3 (ChAd3)-vectored MARV Angola glycoprotein (GP)-expressing vaccine against lethal MARV challenge in macaques. The ChAd3 platform has previously been reported to protect against the MARV-related viruses, Ebola virus (EBOV) and Sudan virus (SUDV), and MARV itself in macaques, with immunogenicity demonstrated in macaques and humans. In this study, we present data showing 100% protection against MARV Angola challenge (versus 0% control survival) and associated production of GP-specific IgGs generated by the ChAd3-MARV vaccine following a single dose of 1 × 1011 virus particles prepared in a new clinical formulation buffer designed to enhance product stability. These results are consistent with previously described data using the same vaccine in a different formulation and laboratory, demonstrating the reproducible and robust protective efficacy elicited by this promising vaccine for the prevention of MVD. Additionally, a qualified anti-GP MARV IgG ELISA was developed as a critical pre-requisite for clinical advancement and regulatory approval.

Assays for the Evaluation of the Immune Response to Marburg and Ebola Sudan Vaccination-Filovirus Animal Nonclinical Group Anti-Marburg Virus Glycoprotein Immunoglobulin G Enzyme-Linked Immunosorbent Assay and a Pseudovirion Neutralization Assay.

Since the discovery of the Marburg virus (MARV) in 1967 and Ebola virus (EBOV) in 1976, there have been over 40 reported outbreaks of filovirus disease with case fatality rates greater than 50%. This underscores the need for efficacious vaccines against these highly pathogenic filoviruses. Due to the sporadic and unpredictable nature of filovirus outbreaks, such a vaccine would likely need to be vetted through the U.S. Food and Drug Administration (FDA), following the Animal Rule or similar European Medicines Agency (EMA) regulatory pathway. Under the FDA Animal Rule, vaccine-induced immune responses correlating with survival of non-human primates (NHPs), or another well-characterized animal model, following lethal challenge, will need to be bridged for human immune response distributions in clinical trials. A correlate of protection has not yet been identified for the filovirus disease, but antibodies, specifically anti-glycoprotein (GP) antibodies, are believed to be critical in providing protection against the filovirus disease following vaccination and are thus a strong candidate for a correlate of protection. Thus, species-neutral methods capable of the detection and bridging of these antibody immune responses, such as methods to quantify anti-GP immunoglobulin G (IgG)-binding antibodies and neutralizing antibodies, are needed. Reported here is the development and qualification of two Filovirus Animal Nonclinical Group (FANG) anti-GP IgG Enzyme-Linked Immunosorbent Assays (ELISAs) to quantify anti-MARV and anti-Sudan virus (SUDV) IgG antibodies in human and NHP serum samples, as well as the development of pseudovirion neutralization assays (PsVNAs) to quantify MARV- and SUDV-neutralizing antibodies in human and NHP serum samples.

Mucosal Challenge Ferret Models of Ebola Virus Disease.

Recent studies have shown the domestic ferret (Mustela putorius furo) to be a promising small animal model for the study of Ebola virus (EBOV) disease and medical countermeasure evaluation. To date, most studies have focused on traditional challenge routes, predominantly intramuscular and intranasal administration. Here, we present results from a non-clinical pathogenicity study examining oronasal, oral, and ocular mucosal challenge routes in ferrets. Animals were challenged with 1, 10, or 100 plaque forming units EBOV followed by monitoring of disease progression and biosampling. Ferrets administered virus via oronasal and oral routes met euthanasia criteria due to advanced disease 5-10 days post-challenge. Conversely, all ferrets dosed via the ocular route survived until the scheduled study termination 28-day post-challenge. In animals that succumbed to disease, a dose/route response was not observed; increases in disease severity, febrile responses, serum and tissue viral load, alterations in clinical pathology, and gross/histopathology findings were similar between subjects. Disease progression in ferrets challenged via ocular administration was unremarkable throughout the study period. Results from this study further support the ferret as a model for EBOV disease following oral and nasal mucosa exposure.

Safety and Immunogenicity of a 4-Component Toxoid-Based Staphylococcus aureus Vaccine in Rhesus Macaques.

Staphylococcus aureus is a leading cause of significant morbidity and mortality and an enormous economic burden to public health worldwide. Infections caused by methicillin-resistant S. aureus (MRSA) pose a major threat as MRSA strains are becoming increasingly prevalent and multi-drug resistant. To this date, vaccines targeting surface-bound antigens demonstrated promising results in preclinical testing but have failed in clinical trials. S. aureus pathogenesis is in large part driven by immune destructive and immune modulating toxins and thus represent promising vaccine targets. Hence, the objective of this study was to evaluate the safety and immunogenicity of a staphylococcal 4-component vaccine targeting secreted bi-component pore-forming toxins (BCPFTs) and superantigens (SAgs) in non-human primates (NHPs). The 4-component vaccine proved to be safe, even when repeated vaccinations were given at a dose that is 5 to 10- fold higher than the proposed human dose. Vaccinated rhesus macaques did not exhibit clinical signs, weight loss, or changes in hematology or serum chemistry parameters related to the administration of the vaccine. No acute, vaccine-related elevation of serum cytokine levels was observed after vaccine administration, confirming the toxoid components lacked superantigenicity. Immunized animals demonstrated high level of toxin-specific total and neutralizing antibodies toward target antigens of the 4-component vaccine as well as cross-neutralizing activity toward staphylococcal BCPFTs and SAgs that are not direct targets of the vaccine. Cross-neutralization was also observed toward the heterologous streptococcal pyogenic exotoxin B. Ex vivo stimulation of PBMCs with individual vaccine components demonstrated an overall increase in several T cell cytokines measured in supernatants. Immunophenotyping of CD4 T cells ex vivo showed an increase in Ag-specific polyfunctional CD4 T cells in response to antigen stimulation. Taken together, we demonstrate that the 4-component vaccine is well-tolerated and immunogenic in NHPs generating both humoral and cellular immune responses. Targeting secreted toxin antigens could be the next-generation vaccine approach for staphylococcal vaccines if also proven to provide efficacy in humans.

Method feasibility for cross-species testing, qualification, and validation of the Filovirus Animal Nonclinical Group anti-Ebola virus glycoprotein immunoglobulin G enzyme-linked immunosorbent assay for non-human primate serum samples.

An anti-Zaire Ebola virus (EBOV) glycoprotein (GP) immunoglobulin G (IgG) enzyme linked immunosorbent assay (ELISA) was developed to quantify the serum levels of anti-EBOV IgG in human and non-human primate (NHP) serum following vaccination and/or exposure to EBOV. This method was validated for testing human serum samples as previously reported. However, for direct immunobridging comparability between humans and NHPs, additional testing was warranted. First, method feasibility experiments were performed to assess cross-species reactivity and parallelism between human and NHP serum samples. During these preliminary assessments, the goat anti-human IgG secondary antibody conjugate used in the previous human validation was found to be favorably cross-reactive with NHP samples when tested at the same concentrations previously used in the validated assay for human sample testing. Further, NHP serum samples diluted in parallel with human serum when tested side-by-side in the ELISA. A subsequent NHP matrix qualification and partial validation in the anti-GP IgG ELISA were performed based on ICH and FDA guidance, to characterize assay performance for NHP test samples and supplement the previous validation for human sample testing. Based on our assessments, the anti-EBOV GP IgG ELISA method is considered suitable for the intended use of testing with both human and NHP serum samples in the same assay for immunobridging purposes.

Interlaboratory comparison for the Filovirus Animal Nonclinical Group (FANG) anti-Ebola virus glycoprotein immunoglobulin G enzyme-linked immunosorbent assay.

The need for an efficacious vaccine against highly pathogenic filoviruses was reinforced by the devastating 2014-2016 outbreak of Ebola virus (EBOV) disease (EVD) in Guinea, Sierra Leone, and Liberia that resulted in over 28,000 cases and over 11,300 deaths. In addition, the 2018-2020 outbreak in the Democratic Republic of the Congo currently has over 3,400 cases and over 2,200 deaths. A fully licensed vaccine and at least one other investigational vaccine are being deployed to combat this EVD outbreak. To support vaccine development and pre-clinical/clinical testing a Filovirus Animal Nonclinical Group (FANG) human anti-EBOV GP IgG ELISA was developed to measure anti-EBOV GP IgG antibodies. This ELISA is currently being used in multiple laboratories. Reported here is a characterization of an interlaboratory statistical analysis of the human anti-EBOV GP IgG ELISA as part of a collaborative study between five participating laboratories. Each laboratory used similar method protocols and reagents to measure anti-EBOV GP IgG levels in human serum samples from a proficiency panel consisting of ten serum samples created by the differential dilution of a serum sample positive for anti-GP IgG antibodies (BMIZAIRE105) with negative serum (BMI529). The total assay variability (inter- and intra-assay variability) %CVs observed at each laboratory ranged from 12.2 to 30.6. Intermediate precision (inter-assay variability) for the laboratory runs ranged from 8.9 to 21.7%CV and repeatability (intra-assay variability) %CVs ranged from 7.2 to 23.7. The estimated slope for the relationship between log10(Target Concentration) and the log10(Observed Concentration) across all five laboratories was 0.95 with a 90% confidence interval of (0.93, 0.97). Equivalence test results showed that the 90% confidence interval for the ratios for the sample-specific mean concentrations at the five individual labs to the overall laboratory consensus value were within the equivalence bounds of 0.80 to 1.25 for each laboratory and test sample, except for six test samples from Lab D, two samples from Lab B1, and one sample from Lab B2. The mean laboratory concentrations for Lab D were less than those from the other laboratories by 20% on average across the serum samples. The evaluation of the proficiency panel at these laboratories provides a limited assessment of assay precision (intermediate precision, repeatability, and total assay variability), dilutional linearity, and accuracy. This evaluation suggests that the within-laboratory performance of the anti-EBOV GP IgG ELISA as implemented at the five laboratories is consistent with the intended use of the assay based on the acceptance criteria used by laboratories that have validated the assay. However, the assessment of between-laboratory performance revealed lower observed concentrations at Lab D and greater variability in assay results at Lab B1 relative to other laboratories.

Development, qualification, and validation of the Filovirus Animal Nonclinical Group anti-Ebola virus glycoprotein immunoglobulin G enzyme-linked immunosorbent assay for human serum samples.

The need for an efficacious vaccine against highly pathogenic filoviruses was reinforced by the recent and devastating 2014-2016 outbreak of Ebola virus (EBOV) disease in Guinea, Sierra Leone, and Liberia that resulted in more than 10,000 casualties. Such a vaccine would need to be vetted through a U.S. Food and Drug Administration (FDA) traditional, accelerated, or Animal Rule or similar European Medicines Agency (EMA) regulatory pathway. Under the FDA Animal Rule, vaccine-induced immune responses correlating with survival of non-human primates (NHPs), or another well-characterized animal model, following lethal EBOV challenge will need to be bridged to human immune response distributions in clinical trials. When possible, species-neutral methods are ideal for detection and bridging of these immune responses, such as methods to quantify anti-EBOV glycoprotein (GP) immunoglobulin G (IgG) antibodies. Further, any method that will be used to support advanced clinical and non-clinical trials will most likely require formal validation to assess suitability prior to use. Reported here is the development, qualification, and validation of a Filovirus Animal Nonclinical Group anti-EBOV GP IgG Enzyme-Linked Immunosorbent Assay (FANG anti-EBOV GP IgG ELISA) for testing human serum samples.

Single-Dose Trivalent VesiculoVax Vaccine Protects Macaques from Lethal Ebolavirus and Marburgvirus Challenge.

Previous studies demonstrated that a single intramuscular (i.m.) dose of an attenuated recombinant vesicular stomatitis virus (rVSV) vector (VesiculoVax vector platform; rVSV-N4CT1) expressing the glycoprotein (GP) from the Mayinga strain of Zaire ebolavirus (EBOV) protected nonhuman primates (NHPs) from lethal challenge with EBOV strains Kikwit and Makona. Here, we studied the immunogenicities of an expanded range of attenuated rVSV vectors expressing filovirus GP in mice. Based on data from those studies, an optimal attenuated trivalent rVSV vector formulation was identified that included rVSV vectors expressing EBOV, Sudan ebolavirus (SUDV), and the Angola strain of Marburg marburgvirus (MARV) GPs. NHPs were vaccinated with a single dose of the trivalent formulation, followed by lethal challenge 28 days later with each of the three corresponding filoviruses. At day 14 postvaccination, a serum IgG response specific for all three GPs was detected in all the vaccinated macaques. A modest and balanced cell-mediated immune response specific for each GP was also detected in a majority of the vaccinated macaques. No matter the level of total GP-specific immune response detected postvaccination, all the vaccinated macaques were protected from disease and death following lethal challenge with each of the three filoviruses. These findings indicate that vaccination with a single dose of attenuated rVSV-N4CT1 vectors each expressing a single filovirus GP may provide protection against the filoviruses most commonly responsible for outbreaks of hemorrhagic fever in sub-Saharan Africa.IMPORTANCE The West African Ebola virus Zaire outbreak in 2013 showed that the disease was not only a regional concern, but a worldwide problem, and highlighted the need for a safe and efficacious vaccine to be administered to the populace. However, other endemic pathogens, like Ebola virus Sudan and Marburg, also pose an important health risk to the public and therefore require development of a vaccine prior to the occurrence of an outbreak. The significance of our research was the development of a blended trivalent filovirus vaccine that elicited a balanced immune response when administered as a single dose and provided complete protection against a lethal challenge with all three filovirus pathogens.

Assessing the safety and immunogenicity of recombinant vesicular stomatitis virus Ebola vaccine in healthy adults: a randomized clinical trial.

BACKGROUND: The 2013-2016 Ebola virus outbreak in West Africa was the most widespread in history. In response, alive attenuated recombinant vesicular stomatitis virus (rVSV) vaccine expressing Zaire Ebolavirus glycoprotein (rVSVΔG-ZEBOV-GP) was evaluated in humans. METHODS: In a phase 1, randomized, dose-ranging, observer-blind, placebo-controlled trial, healthy adults aged 18-65 years were randomized into 4 groups of 10 to receive one of 3 vaccine doses or placebo. Follow-up visits spanned 180 days postvaccination for safety monitoring, immunogenicity testing and any rVSV virus shedding. RESULTS: Forty participants were injected with rVSVΔG-ZEBOV-GP vaccine (n = 30) or saline placebo (n = 10). No serious adverse events related to the vaccine or participant withdrawals were reported. Solicited adverse events during the 14-day follow-up period were mild to moderate and self-limited, with the exception of injection-site pain and headache. Viremia following vaccination was transient and no longer detectable after study day 3, with no virus shedding in saliva or urine. All vaccinated participants developed serum immunoglobulin G (IgG), as measured by Ebola virus envelope glycoprotein-based enzyme-linked immunosorbent assay (ELISA). Immunogenicity was comparable across all dose groups, and sustained IgG titers were detectable through to the last visit, at study day 180. INTERPRETATION: In this phase 1 study, there were no safety concerns after a single dose of rVSVΔG-ZEBOV-GP vaccine. IgG ELISA showed persistent high titers at 180 days postimmunization. There was a period of reactogenicity, but in general, the vaccine was well tolerated. This study provides evidence of the safety and immunogenicity of rVSVΔG-ZEBOV-GP vaccine and importance of its further investigation. Trial registration: Clinical-Trials.gov no., NCT02374385.

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-1ß, 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.).

Immunogenicity and safety of four different dosing regimens of anthrax vaccine adsorbed for post-exposure prophylaxis for anthrax in adults.

BACKGROUND: Strategies to implement post exposure prophylaxis (PEP) in case of an anthrax bioterror event are needed. To increase the number of doses of vaccine available we evaluated reducing the amount of vaccine administered at each of the vaccinations, and reducing the number of doses administered. METHODS: Healthy male and non-pregnant female subjects between the ages of 18 and 65 were enrolled and randomized 1:1:1:1 to one of four study arms to receive 0.5 mL (standard dose) of vaccine subcutaneously (SQ) at: (A) days 0, 14; (B) days 0 and 28; (C) days 0, 14, and 28; or (D) 0.25 mL at days 0, 14, and 28. A booster was provided on day 180. Safety was assessed after each dose. Blood was obtained on days 0, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 84, 100, 180, and 201 and both Toxin Neutralizing antibody and anti-PA IgG antibody measured. RESULTS: Almost all subjects developed some local reactions with 46-64% reported to be of moderate severity and 3.3% severe during the primary series. Vaccine groups that included a day 14 dose induced a ≥ 4 fold antibody rise in more subjects on days 21, 28, and 35 than the arm without a day 14 dose. However, schedules with a full day 28 dose induced higher peak levels of antibody that persisted longer. The half dose regimen did not induce antibody as well as the full dose study arms. CONCLUSION: Depending on the extent of the outbreak, effectiveness of antibiotics and availability of vaccine, the full dose 0, 28 or 0, 14, 28 schedules may have advantages.

Effect of anthrax immune globulin on response to BioThrax (anthrax vaccine adsorbed) in New Zealand white rabbits.

Development of anthrax countermeasures that may be used concomitantly in a postexposure setting requires an understanding of the interaction between these products. Anthrax immune globulin intravenous (AIGIV) is a candidate immunotherapeutic that contains neutralizing antibodies against protective antigen (PA), a component of anthrax toxins. We evaluated the interaction between AIGIV and BioThrax (anthrax vaccine adsorbed) in rabbits. While pharmacokinetics of AIGIV were not altered by vaccination, the vaccine-induced immune response was abrogated in AIGIV-treated animals.

Evaluation of immunogenicity and efficacy of anthrax vaccine adsorbed for postexposure prophylaxis.

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

Randomized, double-blind, placebo-controlled, safety and immunogenicity study of 4 formulations of Anthrax Vaccine Adsorbed plus CPG 7909 (AV7909) in healthy adult volunteers.

A new anthrax vaccine that could accelerate the immune response and possibly reduce the number of injections needed for protection would be desirable in a post-exposure setting. This Phase 1 study compared the safety and immunogenicity of 2 IM doses (Days 0 and 14) of 4 formulations of AV7909 (AVA plus CPG 7909) with 2 IM doses of BioThrax(®) (Anthrax Vaccine Adsorbed) and 2 IM doses of saline placebo administered on Days 0 and 14. A total of 105 healthy adults 18-50 years of age were randomized to 1 of 6 study groups: BioThrax (0.5 mL), AV7909 Formulation 1 (0.5 mL AVA+0.5mg CPG 7909), AV7909 Formulation 2 (0.5 mL AVA+0.25mg CPG 7909), AV7909 Formulation 3 (0.25 mL AVA+0.5mg CPG 7909), AV7909 Formulation 4 (0.25 mL AVA+0.25mg CPG 7909), or saline placebo (0.5 mL). All randomized subjects received at least 1 vaccination, and 100 subjects completed the trial. After 2 doses, mean peak normalized toxin neutralizing antibody responses (TNA NF50) in the AV7909 groups were higher than in the BioThrax group. Differences among the 4 AV7909 groups were not statistically significant. Subjects who received AV7909 reached peak titers on Day 28 vs. Day 35 in the BioThrax group. The most common adverse events (AEs) in the BioThrax and AV7909 groups assessed as related to vaccination were injection site reactions. Transient lymphopenia was observed after the first dose in each AV7909 group. Frequencies of injection site and systemic reactions recorded by subjects in diaries for 7 days after each injection were highest with AV7909 Formulation 1. No AEs of special interest (autoimmune events) were observed in the study. Further studies of doses and dosing regimens are planned to assess the immunogenicity and reactogenicity of AV7909.

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.

A new generation of serotype chimeric infectivity-enhanced conditionally replicative adenovirals: the safety profile of ad5/3-Δ24 in advance of a phase I clinical trial in ovarian cancer patients.

Conditionally replicative adenoviral (CRAd) virotherapy represents a promising therapeutic approach for cancer. We have demonstrated that a serotype chimeric adenoviral 5/3 fiber-knob modification achieves enhanced ovarian cancer infectivity, conditional replication, and oncolytic activity. This study evaluated the safety of intraperitoneal (IP) Ad5/3-Δ24 in advance of a phase I clinical trial in gynecologic cancers. Syrian hamster cohorts were treated with IP Ad5/3-Δ24 or control buffer for 3 consecutive days and euthanized on study days 8, 17, 57, and 89. Blood and tissue samples were harvested from each animal. For biodistribution studies, presence and quantitation of viral levels within samples were determined via quantitative polymerase chain reaction. For safety studies, animals were assessed for adverse vector-related tissue or laboratory effects. In the biodistribution study, low levels of Ad5/3-Δ24 DNA were noted outside of the abdominal cavity. Viral DNA levels in tissues obtained from the peritoneal cavity peaked at day 8 and declined thereafter. In the safety study, no specific histopathologic changes were attributable to virus administration. Hematologic findings noted in the 1 × 10(11) viral particles (vp)/dose group on Days 4 and/or 8 were indicative of an Ad5/3-Δ24-specific generalized inflammatory response; these findings resolved by day 56. The no observable adverse effect level was determined to be 1 × 10(10) vp/dose. This study elucidates the safety profile of IP administration of the serotype chimeric infectivity-enhanced CRAd, Ad5/3-Δ24, and provides guidance for a planned phase I trial for patients with recurrent gynecologic cancers.

Vaccination of rhesus macaques with the anthrax vaccine adsorbed vaccine produces a serum antibody response that effectively neutralizes receptor-bound protective antigen in vitro.

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.