The rBV A/B and rF1V vaccine candidates were initially developed at the US Army Medical Research Institute for Infectious Diseases (USAMRIID). They were transferred to JVAP (and subsequently to DVC) in an early stage of development. The target indication of these vaccines is protection of adults 18 to 55 years of age from disease caused by inhalational exposure to BoNT/A1, BoNT/B1, or Y. pestis.
The rBV A/B vaccine candidate [12
] comprises the recombinant 50
kDa carboxy-terminal of the heavy chains of Antigen A and Antigen B expressed individually from Pichia pastoris
using a methanol induction system. Antigen A is derived from the BoNT/A1 expressed by C. botulinum
strain NCTC 2916 (group I, proteolytic), and Antigen B is derived from BoNT/B1 expressed by C. botulinum
strain Danish (group I, proteolytic). Antigen A was modified to prevent proteolytic cleavage at the N-terminus during gene expression by removing the codons coding for proteolytically susceptible amino acids. Each 0.5
mL dose of rBV A/B-40 consists of a 1
1 mixture of 20μ
g Antigen A and 20μ
g Antigen B adsorbed to Alhydrogel.
The rF1V vaccine candidate for plague [13
] comprises the F1 capsular protein and the V virulence protein of Y. pestis
Colorado 92 (CO92) fused into a single protein, which is produced in Escherichia coli
) and formulated with Alhydrogel.
For any product, successful progress towards licensure involves the integration of different functional groups to develop a scalable manufacturing process from the research and development (R&D) proof-of-concept, coordinate the logistics between the release of manufactured material suitable for testing, and stage the appropriate nonclinical, and clinical studies. The progress of the rBV A/B and rF1V vaccines is described below for manufacturing, nonclinical and clinical efforts.
5.1. Manufacturing Processes
The rBV A/B and rF1V vaccines were initially developed at USAMRIID and were transferred to advanced development at the stage indicated by the gray arrows in . The black arrows indicate the stage of manufacturing development for each product. The products successfully overcame the following challenges to advance to this stage of manufacturing: (1) technology transfer to a contract manufacturing organization and process redesign due to equipment changes, (2) development of manufacturing methods that support the production of clinical trial material and can be validated, (3) scale-up issues associated with either an increase in the number of required troop equivalent doses or nonscalable technological steps, (4) developing needed reagents and analytical methods for product quantitation, purity, and process impurities, and (5) determining conditions that support product stability throughout the manufacturing process.
Stages of the manufacturing process, product transition to DVC (grey arrows), and current status (black arrows) of the development efforts for rF1V and rBV A/B vaccines. Some activities may be conducted concurrently.
The Pichia pastoris
master and working cell banks for the Antigen A and Antigen B expression strains used to produce the rBV A/B vaccine were generated by expanding accession cell banks produced for each antigen and characterized for purity, identity, and suitability according to the FDA and International Conference on Harmonisation (ICH) guidance. Antigen A and Antigen B are manufactured and adsorbed to Alhydrogel separately and then combined to form the final formulated bulk vaccine. The manufacturing process is at commercial scale (100
L for Antigen A and 600
L for Antigen B). Current Good Manufacturing Practices (CGMP) fill/finish activities were conducted at a 5,000
vial level to support the Phase 1B and Phase 2 clinical trials. Currently the formulated FDP manufacturing process is being scaled to the full commercial scale (approximately 300,000
The fused rF1V protein comprising the F1 capsular protein and V virulence protein of Y. pestis
is produced in E. coli
and formulated with the adjuvant Alhydrogel. The sequence encoding the rF1V antigen was derived from plasmid pPW731 produced at USAMRIID [13
] and was initially expressed from pT5.F1V.1 cell banks but later transitioned to expression from pPW731 cell banks due to intellectual property constraints. Both expression systems use the same regulatory elements for gene expression and the same antibiotic resistance gene for plasmid maintenance. The cell banks were characterized for purity, identity, and suitability according to the FDA and ICH guidance. The manufacturing process was scaled to a final commercial process, which is 1,500
L (working volume for fermentation) and 500
L purification scale.
Clinical lots for each vaccine were manufactured and released for use in the completed Phase 1 and the ongoing Phase 2 trials, described below. A stability program is ongoing and was designed to establish, maintain, and execute a testing strategy that is compliant with the FDA and ICH guidance.
5.2. Nonclinical Studies to Support the Animal Rule Requirements
Animal models are critically important for FDA's Animal Rule licensure, in that they are used to assess vaccine efficacy, and the vaccine-induced animal immune responses are compared to human immune responses to predict clinical benefit. The “Draft Guidance for Industry-Animal Models-Essential Elements to Address Efficacy Under the Animal Rule” released in 2009 was followed to guide the design and execution of nonclinical studies. The nonclinical plans are integrated with the clinical development plans for each vaccine to support the comparison of immune responses across species and to enable the selection of the appropriate human dosage.
The animal studies described below were conducted at accredited facilities under the oversight of an assigned Study Director and attending veterinarian and performed according to the Institute- and the DoD-approved animal protocols. Every effort was made to minimize the suffering and distress of animals exposed to challenge agents or subjected to procedures, using approved anesthetics (1 to 6
mg/kg Telazol for macaques and isoflurane for mice). Biostatisticians were consulted in the study design phase to ensure the study used the proper number of animals needed to achieve interpretable data. Animals were observed multiple times per day for signs of clinical illness during the in-life phase. Macaques were anesthetized and humanely euthanized with an overdose of a euthanasia agent containing pentobarbital when meeting preapproved euthanasia criteria such as decreased body temperature to <93.0°F, >20% loss of body weight from prechallenge weight, respiratory distress/failure, significant reduction in activity (e.g., unable to right itself, complete lack of activity, persistent prostration, or total paralysis), or signs of pneumonia. Mice were euthanized using CO2
gas, and guinea pigs were euthanized by a barbiturate overdose injected intraperitoneally or directly into the heart after the animals were anesthetized.
Proof-of-concept studies conducted by USAMRIID using research material and pilot lots demonstrated the immunogenicity and efficacy of rBV A/B and rF1V vaccines in a variety of animal models including rodents and NHPs. The nonclinical development plans for rBV A/B and rF1V continue the testing in stages: (1) performing initial toxicity studies to support the clinical development, (2) developing and characterizing aerosol challenge models, (3) identifying vaccination regimens that induce immune responses similar to the responses observed in clinical volunteers, (4) demonstrating efficacy in animal models, and (5) conducting final pivotal vaccination/aerosol challenge and reproductive toxicity studies.
Good Laboratory Practice- (GLP-) compliant nonclinical safety studies were conducted to support clinical testing of the rBV A/B and rF1V vaccines. These evaluated general toxicity following repeat-dose administration in mice and local reactogenicity of administration of a full human dose of vaccine in rabbits. An additional study to evaluate neurobehavioral toxicity was performed for the rBV A/B vaccine. The vaccines produced no apparent systemic toxicity and only mild inflammation at the injection site. The rBV A/B vaccine produced no apparent neurobehavioral toxicity. Together these nonclinical studies supported the initiation of Phase 1 clinical trials.
An important aspect of developing animal models is the requirement for well-characterized challenge agent. The challenge agents used in the nonclinical efficacy studies are classified as Category A Select Agents by the CDC, and all US facilities that possess or transfer the challenge agent must be registered with the CDC and/or the US Department of Agriculture. The BoNT/A1 and BoNT/B1 were fully characterized to confirm their identity, purity, and strength (biological activity or potency). Protein concentration and biological activity (in terms of MIPLD50 units) of both BoNTs were verified using a micro-Bradford protein assay and mouse (toxin) potency assay, respectively. Testing protocols were established to monitor the real time and accelerated stability of the vialed BoNTs. The stability program includes annual testing to confirm the maintenance of strength and purity using the micro-Bradford protein assay, mouse (toxin) potency assay, SDS-PAGE, and size exclusion chromatography.
The rF1V challenge studies use the CO92 or C12 strains of Y. pestis. To ensure the quality and integrity of these strains, challenge material is grown, characterized, and stored in a three-tiered banking system. The banks are characterized by (1) purity on selective media, (2) titer, (3) phenotype, (4) Gram stain, (5) polymerase chain reaction (PCR) for presence of plasmids and chromosomal marker (pathogenicity island), (6) nitrate reduction, (7) antibiotic susceptibility, (8) growth curve, and (9) glycerol fermentation.
The first requirement of the Animal Rule relates to understanding the pathophysiologic mechanism of toxicity and demonstrating that the pathology is similar to that in humans. This is a significant challenge, especially when there is little information available for human disease (e.g., inhalational botulism). Usually studies in two animal species are required for this purpose, unless the disease is well characterized in one animal species and is an accepted model for the human disease. For the rBV A/B and rF1V vaccine development programs, rodent and NHP models were developed to evaluate disease pathophysiology following inhalational exposure of BoNTs and Y. pestis. The comparisons of the animal hallmarks of disease to what is known for humans are presented in Tables and .
Symptoms following aerosol exposure to BoNT/A1 or BoNT/B1.
Clinical signs, gross pathology and histopathology associated with plague infection1.
The lethality of BoNT/A1 and BoNT/B1 was determined in CD-1 mice and guinea pigs (unpublished) and recently in rhesus macaques [14
]. A stage-wise approach was used to estimate the inhaled median lethal dose (LD50
) and exposure concentration (LCt50
). The pathophysiologic responses to aerosol exposure were evaluated for each species to identify relevant endpoints for efficacy studies. The most relevant pathophysiological responses in mice and rhesus macaques were mortality and development of clinical signs of botulism. Clinical observations in all species were consistent with the recognized pattern of botulism disease progression in humans (). A significant dose response was observed with regard to lethality and the onset and duration of clinical signs in each species. No significant changes in clinical hematology and chemistry and gross and microscopic pathology were observed in mice or rhesus macaques. Changes in physiologic parameters measured by telemetry in rhesus macaques also did not correlate with mortality.
The pathology induced by Y. pestis CO92 was evaluated in animals through clinical chemistries, hematology, telemetry cynomolgus macaques (CMs) only, and detailed histopathology in Swiss Webster mice and CMs. Exposed animals demonstrated multilobar pneumonia, bacterial infiltration of macrophages and lymphoid tissues, fever, sepsis, and death. Data collected from the mouse model development studies estimated the LD50 of CO92 to be approximately 2,000 colony-forming units (cfu).
In the CMs, the inhaled dosage was calculated using the total accumulative tidal volume as measured by plethysmography [15
]. The LD50
was estimated to be 24
cfu by Probit analysis. Telemetry provided useful information on the clinical course of disease not captured by clinical observations. A rise in temperature routinely coincided with the loss of diurnal rhythm, while increased heart and respiration rate followed by inactivity strongly correlated with a lethal outcome. All CMs with Y. pestis
positive blood cultures died from pneumonic plague. The pathology in the lungs of all CMs was consistent with the pathology observed in pneumonic plague described in humans. The significant findings are compared across the species in .
5.3. Identification of Vaccination Regimens and Demonstration of Efficacy in Animal Models
The second requirement of the Animal Rule relates to demonstrating that the responses to the countermeasures are similar in animals and humans. The third requirement is to demonstrate the same endpoint in animals that is expected for humans given the vaccine. For the rBV A/B and rF1V vaccines, that endpoint is survival.
The rBV A/B program vaccinated NHPs with the same material administered to healthy adults in clinical trials and followed the vaccination schedule used for humans. An abbreviated schedule was used for the mice. Neutralizing antibody responses to BoNT/A1 and BoNT/B1 in CD-1 mice and rhesus macaques were evaluated across various vaccine dosages and compared to the neutralizing antibody responses observed in the clinical trial volunteers. Dosages inducing similar antibody levels were identified for both animal models and will be used in pivotal animal studies using the Phase 3 clinical material.
Initial efficacy studies in rhesus macaques demonstrated protection from aerosol challenge using the identified vaccination regimen. The protective efficacy of the antibody levels induced in humans was assessed using passive transfer studies. A guinea pig passive transfer model was developed and used to demonstrate the protective efficacy of purified immunoglobulin from human rBV A/B vaccinees [17
A similar approach was used for the rF1V program. Vaccine dosage titration studies in CMs and mice are in progress using the material used in the Phase 2b clinical trial to assess the immune responses and efficacy. The first objective is to evaluate survival across five vaccine dosages to select vaccine dosages for use in follow-on studies. The secondary objective is to collect serum from animals in all groups for evaluation in passive transfer studies and to determine the antibody titers in Bridge ELISA (described in more detail below). The follow-on study is designed to confirm the minimum protective dosage of vaccine and estimate the minimum level of antibody in rF1V-vaccinated animals that correlates with surviving aerosol exposure to Y. pestis CO92.
A mouse passive transfer model was developed to assess the ability of immune sera to provide protection from an aerosol challenge. Sera from CMs or human volunteers vaccinated with rF1V were tested in the model and the results described [18
]. A definitive correlation between survival in CMs and an antibody level remains to be determined.
The approach taken to meet the Animal Rule requirements is summarized in for the rBV A/B and rF1V vaccines and described in more detail below. Briefly, the animal models with similar disease characteristics to those observed in exposed humans are being used to assess the immune responses and efficacy induced by vaccination. The status of progress to date for the specific requirements is indicated in . The designs and statistical analysis plans for the pivotal GLP nonclinical efficacy studies will need to be prepared and discussed with the FDA. The objective of these studies will be to generate data that supports the Animal Rule requirements to demonstrate efficacy and to extrapolate a dosage likely to produce clinical benefit in humans. These studies will use the Phase 3 clinical trial material, and reproductive toxicity will be assessed concurrently.
Vaccine program status for meeting the requirements of the FDA Animal Rule.
5.4. Clinical Testing and Human Safety
The rBV A/B and rF1V vaccine candidates were (or are being) tested in the Phase 1 and the Phase 2 clinical trials. All clinical trials were evaluated by a Scientific Merit Review Board consisting of independent experts and approved by an independent Investigational Review Board and the US Army's Human Research Protections Office. The studies were managed by a contracted clinical research organization. Safety was monitored continuously by independent physicians and overseen by a Data Safety Monitoring Board (DSMB). All studies were conducted in accordance with the current Good Clinical Practice as required by applicable US federal regulations (21 CFR Parts 50, 56, and 312) and the ICH guidelines.
Male and female volunteers were recruited and assessed for eligibility after signing an informed consent form. Subjects had to be healthy, as determined by standard screening assessments including medical history, physical examination, and laboratory tests (hematology, chemistries, and urinalysis). For rBV A/B trials, subjects with a history of neurological disorders, immunological disorders or prior therapy with botulinum toxin were excluded. In trials for both vaccines, subjects with a history of use of immunosuppressive drugs, including glucocorticoids, and recent vaccinations were excluded from the study. Study vaccine was administered as a 0.5
mL IM injection in the deltoid muscle.
Safety monitoring consisted of collection of injection site and systemic reactogenicity data in a volunteer diary via an interactive voice response system after vaccination, and assessment of treatment emergent adverse events (TEAEs) at scheduled and ad hoc visits, if needed, throughout the study. Injection site reactions (local reactions) were defined as pain, tenderness, pruritus, redness/erythema, other rash, and swelling or induration. Prespecified systemic reactions included fever, fatigue, myalgia, headache, nausea, vomiting, and diarrhea. Any other system organ manifestation was also to be recorded and evaluated as an adverse event (AE). Grading of AEs was performed by study-specific adaptation of the most up-to-date FDA guidelines for toxicity grading in preventive vaccine clinical trials. Any abnormal laboratory value, abnormal vital sign, or abnormal physical finding that was considered clinically significant by the investigator or met the grading criteria for toxicity of Grade 1 or higher was reported as an AE.
5.5. Clinical Trials for rBV A/B
The rBV A/B vaccine was evaluated in adults (18–45 years) in two Phase 1 (rBV A/B-01 and rBV A/B-01B) and one Phase 2 (rBV A/B-02) clinical trials (unpublished). Blood was collected to determine the NAC to BoNT/A1 and BoNT/B1 using the MNA at pre-determined intervals during the study and for calculation of the seroconversion rate. Blood was also collected for passive transfer studies for evaluation of efficacy in animals.
The first trial, rBV A/B-01, was a single-center, open-label, dosage-escalation study designed to evaluate the safety, tolerability, and immunogenicity of a two-dose regimen (Days 0 and 28) of rBV A/B given at three ascending dosages (10μ
g, and 40μ
g total immunizing protein with adjuvant) and an unadjuvanted antigen-only formulation at the 40μ
g total immunizing protein. Forty-four volunteers participated in this study, with 11 in each of the 4 treatment cohorts. The second study, rBV A/B-01B, was a follow-on study to evaluate formulated vaccine administered at two dosages (40μ
g and 100μ
g total immunizing protein) using four different three-dose schedules (Days 0, 28, 56, Days 0, 28, 112 or Days 0, 28, 182, or Days 0, 56, 182). The addition of a third vaccine dose in the Phase 1B study was expected to increase the level and duration of the immune response. Eighty volunteers participated in this study (10 volunteers per vaccination cohort, 40 volunteers per dosage group). Dose escalation to the higher dosage occurred after a review by the DSMB of all safety data in both studies.
The majority of volunteers experienced at least one TEAE, and most AEs were mild to moderate in intensity and self-limited in both Phase 1 clinical trials. About 30% of the TEAEs were considered related to vaccination, and these generally consisted of injection site reactions, with pain being the most prevalent. Pruritus, erythema, and swelling were reported much less frequently. The most common related systemic reactions included headache, diarrhea, and malaise. Sporadic abnormalities in laboratory test results, most commonly hemoglobin changes, were reported after vaccination in most volunteers but were not considered clinically significant, and there were no notable changes from baseline through the end of each study (6 months after last vaccination) within or across cohorts. There were no serious adverse events (SAEs). The overall incidence of TEAEs and the incidence of administration site reactions were higher in the cohorts that received rBV A/B vaccine compared to the cohorts that received antigens only in the rBV A/B-01 study. No apparent dosage relationship was seen across cohorts that received adjuvanted rBV A/B in either study.
In the rBV A/B-01 trial, at least 80% of volunteers vaccinated with the two highest dosages (20μ
g and 40μ
g total immunizing protein) of rBV A/B developed sustained NAC above the lower limit of quantitation for anti-BoNT/A1 and anti-BoNT/B1 antibodies. The antigen-only formulation was not immunogenic. In the rBV A/B-01B trial, administration of three doses of either 40μ
g or 100μ
g of rBV A/B vaccine elicited detectable levels of neutralizing antibody for both BoNT/A1 and BoNT/B1 in all volunteers. Longer vaccination schedules (third vaccination given at Day 182) elicited a greater NAC than shorter schedules (third vaccination given at Day 56 or Day 112). Based on maximum NAC and antibody kinetics, the vaccination schedule of Days 0, 28, and 182 elicited the highest NAC levels, and there were no significant differences among dosages.
The rBV A/B-02 Phase 2 trial was a multicenter, blinded, randomized study designed to evaluate the safety, tolerability, and immunogenicity of a three-dose regimen (Days 0, 28, 182 and Days 0, 56, 182) of rBV A/B given at a single dosage of 40μ
g total immunizing protein compared to saline placebo. There were 440 volunteers in this study allocated to 2 cohorts of 165 subjects each that received rBV A/B and 2 cohorts of 55 subjects each that received saline. Subjects were followed up to 12 months after the last vaccination. Interim data to 4 weeks after the last vaccination were analyzed; final data analysis is not yet complete.
Nearly all volunteers experienced at least one AE, with approximately the same number among vaccine-treated and placebo-treated volunteers. The majority of AEs were mild or moderate in intensity, and there was no difference in the overall incidence of TEAEs among treatment cohorts. Three subjects were discontinued because of AEs that could be related to vaccination (allergic dermatitis, erythema, and swelling, all at the injection site). There were no SAEs related to study vaccine. More volunteers treated with rBV A/B reported injection site reactions compared to those treated with placebo, the most common being pain, tenderness, swelling, erythema, pruritus, and axillary pain. The most common systemic reactions were headache, myalgia, arthralgia, feeling abnormal, fever, anxiety, malaise, and nausea. Most TEAEs, however, were laboratory values outside the normal range reported as AEs per the protocol. These occurred in about 96% of subjects treated either with rBV A/B or placebo. The most frequently reported laboratory AEs were hemoglobin decrease from baseline or increase from baseline, with no significant difference between treatment cohorts. Most laboratory-related TEAEs were considered mild or moderate in severity and not clinically significant and resolved without treatment.
The highest neutralizing antibody rates for both anti-BoNT/A1 and anti-BoNT/B1 were observed at Day 210, 28 days after the last vaccine dose and were similar for both vaccination schedules. Final data will evaluate the immune response to one year after last vaccination.
5.6. Clinical Trials for rF1V
The rF1V vaccine was evaluated in one Phase 1 (rF1V-01) and one Phase 2 (rF1V-02a) clinical trial. A second Phase 2 (rF1V-02b) clinical trial is ongoing. Male and female volunteers, age 18 to 40 years in the rF1V-01 trial and 18 to 55 years in the rF1V-02a trial, were recruited and assessed for eligibility after signing an informed consent form. These studies were conducted, monitored, and reviewed as described for rBV A/B. The immune response to the vaccine was evaluated by measurement of the concentration of antibodies to rF1, rV, and rF1V by the Bridge ELISA at predetermined intervals during the study and calculation of the seroconversion rate. Blood was also collected for passive transfer studies for evaluation of efficacy in animals.
The first trial, rF1V-01, was a single-center, open-label, dosage-escalation study designed to evaluate the safety, tolerability, and immunogenicity of a two-dose regimen (Days 0 and 28) of rF1V given at four ascending dosages (20μ
g, and 160μ
g total immunizing protein). Forty-four subjects participated in the study, with 11 per cohort. Based on analysis of the immunogenicity data, an extension study evaluated the effect of a third dose of 160μ
g administered about 230 days following the first dose in 8 of 11 subjects who had previously received the same vaccine dosage. All volunteers were followed for 180 days after the last vaccination.
All volunteers experienced at least one TEAE, and the majority were either mild or moderate in intensity. Injection site reactions were the most frequent related TEAEs and were generally mild or moderate and more frequent at the two highest dosages after two or three vaccinations. The most common injection reactions were pain, swelling, and erythema. Systemic reactions considered related to vaccination were headache, fatigue, nausea, and diarrhea. Most of these reactions were also mild or moderate and were not considered clinically significant. There were no clinically significant or related laboratory changes. After allowance for the different length of time between the vaccinations, there was no apparent increase in the frequency of TEAEs after the second or third vaccination compared to the first vaccination. Serial electrocardiograms were recorded after the first two doses in all cohorts, and no clinically significant abnormalities were observed.
The rF1V vaccine was immunogenic after two doses of 20μ
g, or 160μ
g of vaccine. The antibody response was markedly increased in volunteers who received three doses of 160μ
g of vaccine, compared to their response after two 160μ
g doses. Peak GMCs of all three antibodies tested (anti-rF1, anti-rV and anti-rF1V) occurred 14 days after the third dose. The administration of a third dose also increased the rate of detectable antibody to all three antigens. Based on this study, the two highest dosages (80μ
g and 160μ
g) were selected for evaluation in the Phase 2a study in a three-dose regimen.
The rF1V-02a Phase 2 trial was a multicenter, blinded study designed to evaluate the safety, tolerability, and immunogenicity of a three-dose regimen (Days 0, 28, and 182 and Days 0, 56, and 182) of rF1V given at two dosages (80μ
g and 160μ
g total immunizing protein). There were 400 subjects (100 per cohort) in the study. Vaccinated subjects were followed for 12 months after the last vaccination.
All subjects experienced at least one TEAE, and no statistically significant difference in overall incidence across groups was observed. A total of six volunteers discontinued due to a TEAE, two because of injection site reactions. The majority of TEAEs were mild or moderate in intensity. There were no SAEs related to study vaccine. Most volunteers had TEAEs that occurred within 28 days following a vaccination, and these were primarily injection site reactions. Most of these reactions were mild or moderate in intensity, and the most common were pain, swelling, erythema, and pruritus. The most common related systemic reactions were headache, malaise, nausea, and diarrhea. Most of these reactions also were mild or moderate. The most common laboratory abnormalities reported as AEs were increased blood glucose and protein present in the urine and decreased hemoglobin. These TEAEs were sporadic, not associated with other clinical abnormalities, and resolved without treatment. In general, no clinically meaningful trends were noted in changes to laboratory parameters in any vaccination group, dosage, or schedule. Overall, the 80μ
g dosage had a slightly better safety and tolerability profile than the 160μ
g dosage, and the Days 0, 56, and 182 schedule had a slightly better safety and tolerability profile than the Days 0, 28, and 182 schedule. In addition, there did not appear to be an increase in the rate of either local or general TEAEs within 28 days after vaccination with subsequent vaccinations.
The immunogenicity data indicate that GMCs for anti-rF1, anti-rV, and anti-rF1V antibodies were much higher after the third vaccination than after the second vaccination, and almost all subjects had evidence of seroconversion 7 to 14 days after the last vaccination. The seroconversion rate was indistinguishable among the selected dosage and schedules following vaccination 3. Both groups that received vaccination on the Days 0, 56, and 182 schedule showed higher anti-rF1, anti-rV, and anti-rF1V GMCs and seroconversion rates from 7 days after the second vaccination to the prevaccination 3 assessment than groups that received vaccination on the Days 0, 28, and 182 schedule. Based on the results of this study, the 80μ
g dosage and the Days 0, 56, 182 vaccination schedule were selected for further testing in the Phase 2b clinical trial. In addition, a shorter vaccination schedule is being evaluated in the Phase 2b clinical trial to assess whether or not equivalent immunogenicity is achieved earlier by administering the third vaccination sooner.
In completed human trials, the rF1V vaccine was safe and well tolerated in the dosages and schedules used and elicited an immunological response to the vaccine recombinant antigen (rF1V) and to each of its components (rF1 and rV).