4
Proposed Studies on the Efficacy of the Anthrax Vaccine

A critical aspect of the congressional mandate to the Centers for Disease Control and Prevention (CDC) concerns the efficacy of the current licensed anthrax vaccine, Anthrax Vaccine Adsorbed (AVA). The congressional charge explicitly calls for research aimed at determining immunologic correlates of protection and documenting vaccine efficacy, and at optimizing the vaccination schedule and the route of administration of the vaccine to assure efficacy while minimizing the number of doses required and the occurrence of adverse events.

Efficacy generally refers to the ability of a product to achieve its desired effect under ideal conditions, such as the human clinical trial and the controlled animal experiments planned by CDC. Efficacy is relative, not absolute. The protection provided by a vaccine can be influenced by factors that include the host response, the dose of exposure, the route of entry into the body, and the strain of the pathogen.

The set of studies being undertaken by CDC must examine immunogenicity as well as efficacy. Immunogenicity is the degree to which a substance is capable of producing immunity or evoking an immune response.1 Immunogenicity must be used as an endpoint in human studies of the anthrax vaccine because it is unethical to expose human beings to anthrax spores to directly evaluate the efficacy of an anthrax vaccine. Parallel studies of the protection provided by the vaccine in animals that are experimentally challenged with anthrax spores will be necessary to link the human immune responses with direct evidence of the vaccine’s efficacy.

OBJECTIVES AND CRITICAL RESEARCH QUESTIONS FOR CDC RESEARCH ON THE EFFICACY OF THE ANTHRAX VACCINE

CDC’s stated objectives for the efficacy component of its anthrax vaccine research program are displayed in Box 4-1. The critical research questions related to efficacy are shown in Box 4-2.

The committee found CDC’s research objectives and critical research questions regarding the efficacy of the anthrax vaccine to be generally complete and appropriate. In the committee’s view, the question concerning the level of circulating antibody that protects an unvaccinated macaque from anthrax is a crucial one. The committee is concerned that CDC assigned this question a lower priority than the others and has planned no study to address it. This issue is discussed at length later in this chapter.

Several of the other research questions listed by CDC will be only partially addressed by its planned studies. The question of how gender affects the immune response will be addressed in the human clinical trial to the extent that the enrollment of women is successful. Similarly, because of limited statistical

1  

Dorland’s Illustrated Medical Dictionary, 28th ed., s.v. “immunogenic.”



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4 Proposed Studies on the Efficacy of the Anthrax Vaccine A critical aspect of the congressional mandate to the Centers for Disease Control and Prevention (CDC) concerns the efficacy of the current licensed anthrax vaccine, Anthrax Vaccine Adsorbed (AVA). The congressional charge explicitly calls for research aimed at determining immunologic correlates of protection and documenting vaccine efficacy, and at optimizing the vaccination schedule and the route of administration of the vaccine to assure efficacy while minimizing the number of doses required and the occurrence of adverse events. Efficacy generally refers to the ability of a product to achieve its desired effect under ideal conditions, such as the human clinical trial and the controlled animal experiments planned by CDC. Efficacy is relative, not absolute. The protection provided by a vaccine can be influenced by factors that include the host response, the dose of exposure, the route of entry into the body, and the strain of the pathogen. The set of studies being undertaken by CDC must examine immunogenicity as well as efficacy. Immunogenicity is the degree to which a substance is capable of producing immunity or evoking an immune response.1 Immunogenicity must be used as an endpoint in human studies of the anthrax vaccine because it is unethical to expose human beings to anthrax spores to directly evaluate the efficacy of an anthrax vaccine. Parallel studies of the protection provided by the vaccine in animals that are experimentally challenged with anthrax spores will be necessary to link the human immune responses with direct evidence of the vaccine’s efficacy. OBJECTIVES AND CRITICAL RESEARCH QUESTIONS FOR CDC RESEARCH ON THE EFFICACY OF THE ANTHRAX VACCINE CDC’s stated objectives for the efficacy component of its anthrax vaccine research program are displayed in Box 4-1. The critical research questions related to efficacy are shown in Box 4-2. The committee found CDC’s research objectives and critical research questions regarding the efficacy of the anthrax vaccine to be generally complete and appropriate. In the committee’s view, the question concerning the level of circulating antibody that protects an unvaccinated macaque from anthrax is a crucial one. The committee is concerned that CDC assigned this question a lower priority than the others and has planned no study to address it. This issue is discussed at length later in this chapter. Several of the other research questions listed by CDC will be only partially addressed by its planned studies. The question of how gender affects the immune response will be addressed in the human clinical trial to the extent that the enrollment of women is successful. Similarly, because of limited statistical 1   Dorland’s Illustrated Medical Dictionary, 28th ed., s.v. “immunogenic.”

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power, it is unlikely that the study will be able to determine other potential risk factors for any differences in immune response to AVA. Larger numbers of participants would be needed to account simultaneously for age and other immune effectors such as illness or nutritional status. The role of immune memory in protection is not likely to be sufficiently addressed by the B-cell studies planned, which focus unnecessarily on cellular as opposed to humoral factors. Finally, the proposed studies will not fully answer the question of which components of AVA contribute most significantly to protection because there is no plan to evaluate individual components in AVA; the protective effects of AVA are known to be due primarily to an immune response to protective antigen (PA). 2 BOX 4-1 CDC Objectives for Research on the Efficacy of the Anthrax Vaccine Assess AVA efficacy in humans immunized with AVA by measuring immune responses identified as protective in efficacy objective B (animal studies). Immune markers of protection will be evaluated by varying the number of priming shots and the route of administration. Assess AVA efficacy in animals immunized with serial dilutions of AVA and challenged with live, inhaled anthrax spores. Use blood samples from the subjects in the clinical trial and in animal studies to identify immune correlates of protection and validate laboratory studies to measure them. SOURCE: CDC, 2002e, p. 10. BOX 4-2 Critical Research Questions Regarding the Efficacy of the Anthrax Vaccine, as Identified by CDC What are the correlates for protection against inhalational anthrax? When is protection achieved, and how long does it last? Are enzyme-linked immunosorbent assay (ELISA) and toxin neutralizing antibody assay (TNA) the most appropriate measurements of immune response to AVA? How does gender affect immune response to AVA? What are important risk factors for lowered immune response to AVA? How can we bridge from animal challenge data to predict likelihood of survival in AVA-vaccinated humans? What is the role of circulating antibody in protection? What is the role of immune memory in protection? What is the antigenic make-up in the AVA lots used for CDC studies? What is the quantity of PA [protective antigen] in the AVA lots used for CDC studies? Which components of AVA contribute most significantly to protection against anthrax? What is the basis, if any, for the current series and can that series be reduced to a more practical number? Is the vaccine equally efficacious or immunogenic when administered intramuscularly? What level of circulating antibody protects an unvaccinated macaque from anthrax? SOURCE: CDC, 2002f. 2   Efficacy studies in laboratory animals have indicated that PA must be present in a cell-free anthrax vaccine or produced by a live vaccine to achieve protection (Ivins et al., 1986, 1992, 1998; discussed in IOM 2002).

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ANTHRAX VACCINE ADSORBED: HUMAN REACTOGENICITY AND IMMUNOGENICITY TRIAL TO ADDRESS CHANGE IN ROUTE OF ADMINISTRATION AND DOSE REDUCTION (HUMAN CLINICAL TRIAL) This study is intended to compare the immunogenicity and reactogenicity of AVA when given under the currently licensed regimen—subcutaneous (SQ) administration of six primary doses of vaccine (at 0, 2, and 4 weeks and 6, 12, and 18 months) and annual booster doses—with the immunogenicity and reactogenicity of the vaccine when a reduced number of doses are given intramuscularly (IM) (CDC, 2002a,h). The components of the study related to immunogenicity are discussed here; those related to reactogenicity are reviewed in Chapter 5. The study follows up the findings from a pilot study carried out at the U.S. Army Medical Research Institute of Infectious Diseases indicating that concentrations of anti-PA antibodies measured 2 weeks after the administration of two doses of AVA given 4 weeks apart (either IM or SQ) were comparable to those measured 2 weeks after the administration of three doses (SQ) given 2 weeks apart (the licensed dosing schedule) (Pittman, 2002). Taking into account these pilot data, CDC proposes two hypotheses related to immunogenicity for the Human Clinical Trial: AVA administered by the IM route elicits antibody responses that are not inferior3 to those achieved by the SQ administration. AVA administered by the IM route and with fewer doses elicits antibody responses that are not inferior to those achieved by the currently licensed schedule. (CDC, 2002a,h) Study Design The study is designed as a prospective, randomized, double-blind,4 placebo-controlled clinical trial to be conducted over a period of 43 months at five sites in the United States. The study population will consist of 1,560 healthy civilian adult men and women between the ages of 18 and 61 years. The study will be open to anyone meeting the eligibility criteria, but recruitment efforts will focus on groups for whom AVA vaccination for bioterrorism preparedness has been considered, including emergency first responders, federal responders, and medical practitioners. The size of the study population reflects an allowance for up to 50 percent attrition over the course of the study. Study participants will be randomly assigned to one of six study groups of 260 persons each (see Ta-ble 4-1). One study group will receive AVA under the currently licensed regimen (SQ administration of six doses over 18 months, followed by two boosters a year apart). A placebo group will receive eight injections of sterile saline; half of the group will receive SQ injections and half will receive IM injections. In the four other study groups, participants will receive either four, five, seven, or eight IM doses of AVA. The study plan calls for all vaccine doses to come from AVA Lot FAV063, manufactured by BioPort Corporation as a “post-renovation qualification lot” (CDC, 2002a, p. 61). Participants who receive fewer than eight doses of AVA will receive an injection of the saline placebo in place of an omitted dose of AVA. To evaluate the immunogenicity of AVA in each study group, each participant will have a total of 16 blood samples drawn at specified times over the course of the study, including a sample that will be drawn before the first vaccination. Serum from the blood samples will be assayed for total levels of immunoglobulin G (IgG) antibodies to PA using a standardized and validated enzyme-linked immunosorbent assay (ELISA). The study’s primary endpoints for immunogenicity will be a fourfold rise in antibody 3   For FDA to approve a modification of the AVA label regarding the route of administration or the schedule of doses, data must show that the modified route of administration and dosing schedule are at least as immunogenic as (non-inferior to) the currently licensed regimen. 4   Unblinded staff will prepare and administer the vaccine or placebo, but CDC staff, the investigators monitoring and analyzing immunogenicity and reactogenicity, and the participants will remain blinded to study group assignment.

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TABLE 4-1 Schedule of Injections for Study Groups in the Human Clinical Trial of Alternative Routes of Vaccine Administration and Schedules of Vaccine Doses   Timing and Content of Injections Study Group No. and Route of Injections   Week Month n 0 2 4 6 12 18 30 42 1 8 SQ 260 AVA AVA AVA AVA AVA AVA AVA AVA 2 8 IM 260 AVA AVA AVA AVA AVA AVA AVA AVA 3 7 IM 260 AVA S AVA AVA AVA AVA AVA AVA 4 5 IM 260 AVA S AVA AVA S AVA S AVA 5 4 IM 260 AVA S AVA AVA S S S AVA 6a 8 IM 130 S S S S S S S S 6b 8 SQ 130 S S S S S S S S NOTE: AVA: Anthrax Vaccine Adsorbed; S: saline placebo; SQ: subcutaneous; IM: intramuscular SOURCE: Adapted from CDC, 2002a, p. 56. titer and in antibody concentration. In a subset of the serum samples, an in vitro toxin neutralization assay (TNA) will be used to measure the functional activity of anti-AVA antibodies. These assays are also the focus of effort in the studies of the immune correlates of protection (ICP). Samples from three time points in the clinical trial will be used to examine the kinetics of the immune response to AVA, and samples from a subset of participants will also be used for additional tests in studies of the correlates of protection and of immunogenetics. For the study of immune kinetics, blood drawn 3 to 15 days following the vaccinations at 6 months, 30 months, and 42 months will be assessed for PA-specific antibody titer, concentration of anti-PA IgG, and TNA titer. (Participants will be randomly assigned to return to the study site in a manner that distributes their blood sampling evenly over the 3–15 day period.) The rate of increase in the geometric mean concentration (GMC) of anti-PA IgG will be compared among the groups, based on the first post-injection day on which a fourfold rise in antibodies and TNA titers occurs. A complementary study of antibody kinetics in nonhuman primates (NHPs) will take place in parallel as part of the NHP Vaccine Dose Ranging Study. The study of immunogenetics is planned to test the hypothesis that genetic polymorphisms of the human leukocyte antigen system (HLA) significantly influence the immune response to AVA (CDC, 2002h). The substudy will be carried out by the Mayo Clinic and Foundation. A random sample of 344 participants from the clinical trial will serve as the population for both this study and the Immune Correlates of Protection Study described later. Approximately 275 of these subjects are expected to receive AVA; the remainder will receive only the saline placebo. HLA typing will be carried out on blood samples drawn at the start of the study before vaccination. This substudy has the following aims: To estimate the association between specific alleles of class I HLA alleles (A, B, C) and the immune response following anthrax immunization in human subjects To estimate the association between specific alleles of class II HLA alleles (DRB, DQA, DQB, DPA, and DPB) and the immune response following anthrax immunization in human subjects To estimate the effects of genetic variation across the class I and class II HLA alleles and the immune response (circulating antibody level and anthrax-specific lymphoproliferative responses) following anthrax immunization in human subjects (CDC, 2002h, pp. 5–6)

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Planned Analyses The study protocol specifies that the primary analyses for immunogenicity will be conducted using the participants who can be evaluated “according to protocol,”5 based on adherence to the schedules for injections and blood sampling. An intent-to-treat analysis will also be conducted, using all available data for each participant regardless of compliance with the study protocol. Missing data will be assumed to be missing at random. With the intent-to-treat analysis, investigators can assess whether deviations from the protocol were vaccine group-related and led to bias in the results. A third set of immunogenicity analyses will be done to complement the according-to-protocol and intent-to-treat analyses to assess responses among participants who receive all doses of vaccine, regardless of whether a protocol violation has occurred. For each blood sampling, the proportion of participants with a fourfold rise in anti-PA IgG antibody titer will be summarized. The proportion of participants with a fourfold rise in antibody titers in each of the study groups under the alternative regimens will be compared with the proportion in the study group receiving the vaccine under the licensed regimen to determine if the alternative regimens are at least as immunogenic as (i.e., non-inferior to) the licensed regimen. GMCs of anti-PA antibody for each study group will also be calculated and summarized for each blood draw. The significance of differences in GMCs between the licensed and alternative regimens will be assessed using mixed-model analysis of variance (ANOVA) procedures. The null hypothesis that a modified route and dose schedule is inferior will be rejected in one-sided tests if the upper 97.5 percent confidence limit for the GMC ratio (reference group:study group) is <1.5 and if the ratio of fourfold responders is <1.12. Sample sizes to achieve 80 percent power with a 95 percent one-sided hypothesis test were calculated using variance estimates from a pilot study of changes in the route of administration and dosing schedule and allowance for a 50 percent attrition rate. CDC will contract with statistical experts to examine deviations from the protocol in the form of dropouts, noncompliance, and loss to follow-up and to devise appropriate analyses (CDC, 2002h). Committee Comments On the whole, the committee found that the study, as described in the protocol, provides an appropriate basis for comparing the immunogenicity of SQ and IM administration of AVA, and for comparing the immunogenicity of the licensed schedule of SQ doses with regimens that use fewer IM doses. The criteria for analysis of non-inferiority appear to be appropriate. The study should provide information that is valuable both for optimizing the administration of the currently licensed anthrax vaccine and, when carried out in conjunction with the NHP challenge studies, for the development and licensure of new anthrax vaccines. Both of these needs were emphasized by the Institute of Medicine (IOM) committee that recently reviewed the efficacy and safety of AVA (IOM, 2002), and they are of greater public concern following the deaths and nonfatal cases of anthrax that occurred as a result of the bioterrorist incidents in the fall of 2001. The committee also notes that the use of vaccine from a lot manufactured following the completion of the renovation of the BioPort facilities will provide an opportunity to evaluate the immunogenicity of the newly manufactured vaccine, as was recommended in the recent IOM report (IOM, 2002). Finding: As described in the study protocol, the human clinical trial is generally responsive to the congressional mandate and to important research needs for determining immunologic 5   According-to-protocol analysis includes in its calculations only those study participants who fulfill all entry criteria and complete the trial according to its protocol. Intent-to-treat analysis includes data from all study participants regardless of whether every person enrolled or randomly assigned to a group completed the trial. In randomized controlled trials, the intent-to-treat approach preserves the similarity between different treatment groups that randomization provides. Intent-to-treat analysis accounts for participants who are lost to follow-up or unable to complete the study protocol. Failure to include all participants may permit a bias in the results if non-completion of the trial is related in any way to the treatment or the vaccine tested (Altman et al., 2001; Health Technology Assessment News, 1999).

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correlates of protection, documenting the immunogenicity of AVA, and optimizing the vaccination schedule and routes of administration of AVA. Since the release of its interim report in July 2001 (IOM, 2001), the committee has received more detailed plans regarding the substudy to assess HLA genetic polymorphisms. CDC’s rationale for the inclusion of this effort is that it will extend work previously carried out with measles vaccine and other vaccines. The earlier work indicated that HLA genetic polymorphisms significantly influence antibody levels following receipt of live measles vaccine (Hayney et al., 1996, 1998; Poland et al., 1998; St. Sauver et al., 2002). With analysis of samples from this prospective clinical trial of AVA, the investigators propose to extend their work to a vaccine against a toxin-producing pathogen to further evaluate the generalizability of their earlier findings. The committee is not persuaded that this substudy contributes meaningfully to the research plan for AVA. There is good evidence that immune response is directed in part by genetic background, but large sample sizes are usually needed to associate differences in response with differences in genetic characteristics. The substudy appears adequately powered to address its specific aims of estimating the association between specific alleles of class I or II HLA alleles and the immune response to AVA or of estimating the associations between genetic variation across class I and class II alleles and changes in the immune response. However, it does not appear to be adequately powered to be able to take into account demographic variables such as sex and race, which also affect immune response. Further, at least two different endpoints are proposed: (1) antibody levels to assess the humoral response, and (2) a lymphoproliferative assay and perhaps other measures to assess the cellular response. The adjustments that would be necessary to allow for multiple comparisons are not described. While the question of the relationship between HLA alleles and immune response may have some broad scientific interest, it warrants only low priority among CDC’s studies of the safety and efficacy of AVA. Finding: The HLA substudy experiments as described are not critical to resolving the concerns regarding the safety and efficacy of AVA. As part of the CDC anthrax vaccine safety and efficacy research program, the studies should be considered of low priority. The success of the study will depend, in part, on recruiting and retaining an adequate number of participants. The study protocol comments on plans for recruiting participants and notes that up to 50 percent of those who begin the study might be lost. The committee urges that CDC and the participating centers ensure that those interested in participating in the trial fully understand the demands of the study, in terms of both the vaccination schedule and the time commitment involved. The plan to assume that missing data are missing at random should be supported by the collection during the course of the study of the information necessary to test that assumption. Even if efforts are made to minimize loss to follow-up and noncompliance, CDC should be taking steps to ensure appropriate analysis of incomplete data. In the interim report, the committee urged careful consideration of statistical methodologies for analysis of the data from the human clinical trial, noting that the intent-to-treat analysis may be less appropriate for a clinical trial of a vaccine to be used in a military setting than for vaccines intended for general civilian use (IOM, 2001). Appropriate analysis of the clinical trial data is crucial to gaining approval from FDA for any change in the route of administration of AVA or in the schedule of doses required. The committee urges continued consultation with experts in the analysis of data from clinical trials with significant loss to follow-up or noncompliance. In addition, CDC should be consulting with FDA on this matter. Although some sections of the protocol reviewed by the committee indicate that consultation with FDA has taken place, no specific mention is made of consultation with FDA on the appropriateness and acceptability of these particular analyses. The committee wishes to emphasize that concurrence from FDA on the appropriateness of the analysis is imperative. Recommendation: CDC should consult with FDA and receive their approval regarding the type of analysis (according to protocol, intent to treat, or other) that will provide appropriate

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support for a change in the labeling of AVA regarding the route of administration and the number of doses required. NONHUMAN PRIMATE VACCINE DOSE RANGING, IMMUNOGENICITY, AND CHALLENGE TRIAL The NHP study is intended to provide information from experiments with rhesus macaques about the relationship between immune responses developed from vaccination with AVA and protection from aerosol challenge with anthrax spores (CDC, 2002b,i). Based upon the assumption that similar immune responses in human and nonhuman primates will be similarly protective, the study will help provide information about the protection that AVA (and future anthrax vaccines) provides to humans. CDC has planned the study to address the following objectives: Using dilutions of AVA to induce a spectrum of immune responses, identify immune correlates of protection against challenge at 12, 30, and 42 months after initial immunization of rhesus macaques (Macaca mulatta) Bridge these data to immune response data from similar time points in the human trial to potentially identify surrogates of protection that reflect the correlates of protection in macaques Provide survival (and potentially immunological) data from macaques immunized intramuscularly with three doses of AVA that will support the objectives of dropping doses and changing the route of administration of AVA of the human clinical trial (CDC, 2002b, p. 14; 2002i, p. 1) Study Design Rhesus macaques randomly assigned to receive a three-dose series (0, 4 weeks, and 26 weeks) of AVA at full dose or fixed dilutions (1:5, 1:10, 1:20 or 1:40) of the full dose will be challenged at 12, 30, or 42 months after vaccination with approximately 200 times the amount of anthrax spores that would be expected to kill half the animals. Vaccinations with different dilutions of AVA are expected to induce different levels of immune response in the macaques. Rates of survival after lethal challenge of these animals will provide data to describe a relationship between immune response and survival. The current study design represents a modification of the original plan, which had as its objective to identify an “appropriate” dose of AVA for use in macaques. In Phase I of the study, begun March 2001, five groups of macaques (10 AVA-vaccinated animals and two saline-vaccinated controls per group) received IM injections of either a full dose of AVA or of dilutions of 1:5, 1:10, 1:20 or 1:40 at 0, 4, and 26 weeks. Preliminary data indicate that the vaccine dose dilutions elicit dose-dependent gradations in humoral and cellular immune factors (anti-PA IgG, toxin neutralization, and T-cell proliferation) that are expected to be important for protection (CDC, 2002b). To minimize unnecessary animal deaths, investigators will use the results of an aerosol challenge of the animals in the 1:20 dilution group at 54 weeks (to have taken place March/April 2002) as the basis for deciding which challenges to administer to additional groups of macaques (see CDC, 2002b for details, pp. 16–22, p. 42). Blood samples taken at specified intervals following vaccination and following challenge will provide material for analysis of immune factors that may play a role in protection from challenge. The study will be carried out at Battelle Memorial Institute, Emory University Vaccine Center, and the Meningitis and Special Pathogens Branch of the National Center for Infectious Diseases at CDC. Most of the macaques involved in the study (132 for the entire study) are located at Battelle, but 33 animals, divided among three vaccine-dilution groups (undiluted, 1:10, and 1:20) of 10 animals each and 3 control animals, were to be vaccinated at Emory University in June 2002. Blood specimens from these animals will be collected at Emory during the first 30 months following the start of vaccination. The animals will then be transported to Battelle for an aerosol anthrax spore challenge at 30 months. Under some

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of the scenarios for survival and challenge, results from challenges of animals based at Battelle will lead to certain of the animals housed at Emory being removed from the study rather than being challenged (CDC, 2002b). CDC plans to use logistic regression for its primary analyses, as well as alternative approaches such as generalized additive models, classification and regression trees, and multiple adaptive regression trees. Genuine predictive performance measures will be used to assess the predictive performance of methods used for the study. Power calculations awaited additional refinement at the time the protocols were provided to the committee. The NHP studies will require the use of two different lots of AVA. The first 60 animals were vaccinated in March 2001 with AVA from Lot FAV048B. Future doses will come from Lot 063 to be consistent with the “postrenovation” lot of vaccine that will be used in the human clinical trial. While it would have been desirable to use only one lot for all of the studies, the postrenovation lot was not yet available at the start of the NHP studies. The laboratory assays for this study as well as those for the human clinical trial and the study of immune correlates of protection have all been reviewed by the Laboratory Issues panel, one of the expert consultation panels convened by CDC in response to recommendations from this committee in the interim report. Committee Comments As described in the summaries and research protocols provided to the committee, the NHP study is an appropriate and crucial aspect of the congressionally mandated research to document the efficacy of AVA and to determine immune correlates of protection. Because challenge of humans with lethal agents such as anthrax is not ethical, animal experiments are necessary, and the rhesus macaque is an appropriate model for such studies (discussed in IOM, 2002). The current approach to the NHP study, significantly modified since it was first presented to the committee, promises to meet the need for additional information about protective levels of antibody or other immune factors. Such information can be useful not only in optimizing the dose schedule for AVA, but also in evaluating the efficacy of new anthrax vaccines under development. Finding: The committee finds that the nonhuman primate studies that have been proposed as a means to provide information about the efficacy of AVA are well designed and responsive to the congressional mandate and to important research needs. The information gathered will inform and influence the development and licensure of new protective antigen-based anthrax vaccines. Two lots of vaccine, one of them produced before the renovations of the manufacturing facility and one produced “postrenovation,” will be used in the NHP study. The research plan indicates that detailed analyses of the vaccine composition are to be carried out by a contract laboratory. It is important that there be a detailed comparison of the antigen content of the vaccine from the two lots used in the study. The characterization of the vaccine lots should be described in formal protocols, which should be reviewed by the Laboratory Issues Panel. Recommendation: Careful characterization of the vaccine lots used in the clinical trial and nonhuman primate studies is crucial. Protocols for this work should undergo review by the expert consultative panel convened for laboratory issues. The protocol describes an approach of “retiring” some animals without anthrax aerosol spore challenge based upon results of challenges of animals immunized with AVA at 1:20 dilution. The protocol provided by CDC in February 2002 does not describe the analytical approaches planned to account for the issues of missing data that will be raised by retiring selected animals. Investigators should consult with

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the expert consultation panel on statistics to arrive at appropriate methods for handling missing data due to retiring animals or other causes. The protocol provided to the committee lacked detail regarding plans for statistical analysis for the NHP studies. Given the complex experimental design, it is imperative that CDC receives expert input in devising their analytical plan. Recommendation: CDC should consult with the Statistics Panel for expert guidance on analyses of data from the nonhuman primate studies, including devising appropriate methods for handling missing data. The research protocols and other materials submitted to the committee for review do not describe plans for any passive protection studies. Passive protection studies are experiments in which serum from immunized animals or humans (i.e., immunoglobulin) is administered to naïve animals who are then challenged so that the level of protection provided by the circulating antibody from the immunized animal or human can be evaluated. Because immunoglobulin has a limited half-life in animals, it is necessary to evaluate the protection it affords during a limited period of observation appropriate to this half-life. Although CDC cites identifying the level of circulating antibody required to protect an unvaccinated macaque from anthrax as a critical research question, it assigns the question a low priority. The committee, however, considers passive protection studies to be an essential component of a research program on AVA. While studies of active protection can be extremely useful, they do not permit identification of a specific protective factor that might be used as a proxy for protection in clinical circumstances. Passive protection studies would make it possible to demonstrate unequivocally that the immune factors (e.g., antibody to PA) present in the serum of vaccinated animals afford protection. They can also show whether immune factors generated in humans are protective in animals, providing additional information for bridging data from animals to humans. The studies can be used to directly determine the amount of circulating antibody required for protection from inhalational challenge in the absence of immunologic memory. They can also be used to estimate the level of circulating antibody to PA that must be reached before antibiotic prophylaxis can be discontinued. In addition, passive protection studies can provide information about the expected time frame (how quickly and for how long) for protection following immunization with AVA or more modern PA-based vaccines. These experiments are also essential to better characterize the dosage of therapeutic immune globulin necessary to prevent disease in people who may have been exposed to anthrax spores or to treat patients with anthrax disease. Finding: Passive protection studies are important for improving understanding of the mechanism(s) of the efficacy of AVA and can help to address practical issues related to the management of anthrax disease. Recommendation: CDC should conduct passive protection studies as part of its anthrax vaccine safety and efficacy research program. The distribution of anthrax spores through the postal system in the fall of 2001 made it clear that additional information is needed about the protection afforded by vaccination with AVA against different challenge doses of aerosolized spores of B. anthracis. CDC reported that different doses of anthrax challenge were not included in the protocol for the NHP studies because doing so would introduce more variables than could be accommodated with a study of the size they have planned (CDC, 2002g). The committee urges CDC to help address this research gap. The passive protection studies recommended above will help determine an optimal amount of antibody or other correlate of protection to protect against a challenge dose of a particular size. Once a protective level of antibody (or other correlate of protection) has been established, the effect of varying the size of the inoculum should be evaluated. Finding: Research is needed to understand better the effect of the size of the challenge dose on the protection afforded by AVA.

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Recommendation: CDC should support or conduct research on the effect of the size of the challenge dose on immunity provided by vaccination with AVA. IMMUNE CORRELATES OF PROTECTION AGAINST INHALATIONAL ANTHRAX STUDIES Closely related to the NHP studies described above are studies of the immune correlates of protection against anthrax. These studies are planned to identify components of the rhesus macaque humoral and cell-mediated immune responses to AVA that correlate with protection against aerosol challenge by virulent B. anthracis. The ICP studies will develop and apply a panel of immunologic assays to test the hypothesis that “one or more measurable immunological markers of protection can be identified in a nonhuman primate model of inhalation infection with B. anthracis spores and that one or more of these measurable immunological markers are identifiable in AVA vaccinated humans” (CDC, 2002j, p. 1). The goals of the ICP studies are listed in Box 4-3. Planned Analyses The ICP studies will be carried out at three main sites: Battelle Memorial Institute, Emory University Vaccine Center, and the Meningitis and Special Pathogens Branch of the National Center for Infectious Diseases at CDC. In addition, the Centre for Applied Microbiology and Research (CAMR) at Porton Down, United Kingdom, will develop and apply a range of standardized ELISA and other assays under subcontract to Battelle (CDC, 2002d, Appendix 8.2). Experience with aluminum adjuvants suggests that “vaccination with AVA will favor a Th-2 type immune bias manifest in part as a strong humoral IgG1 antibody response” (CDC, 2002j, p.7). Therefore the emphasis in the ICP studies is on the description and quantification of antibody responses to PA, lethal factor (LF), and edema factor (EF), using quantitative anti-PA IgG ELISA and TNA. The same assays are also being used in the Human Clinical Trial as primary endpoints for evaluating immunogenicity. Sera from all of the NHPs and from a subset of the participants from the human study will also be evaluated with more detailed analyses of the anti-PA IgG antibody subclasses; anti-PA IgM, anti-PA IgA and anti-PA IgE; as well as anti-LF IgG and anti-EF IgG. Additional assays will evaluate neutralization of the enzymatic properties of LF and EF (an endopeptidase and adenylate cyclase, respectively), neutralization of anthrax lethal toxin at different stages in the anthrax toxin complex formation and the ability of anti-AVA antiserum to promote opsonophagocytosis. To learn as much as possible about the wider immunologic response to AVA, the ICP studies will also include an analysis of factors related to cellular immune response. Cell-mediated immune response (CMI) to AVA is important because without CD4+ T-cell help, protective humoral immunity against toxemia will not be generated (CDC, 2002j). The study will therefore include comprehensive descriptive and quantitative analyses of immune-cell responses to AVA administered at different dilutions. Plans call for the analysis of NHP samples at both the Battelle and Emory sites; some of the samples from the human trial may be analyzed as well (CDC, 2002j). Committee Comments In general, the committee found the ICP studies to be an appropriate and important component of the effort to evaluate the efficacy of the anthrax vaccine. While qualitative correlations have been established between antibodies to PA and protection in animal models, quantitative correlations remain to be determined. The proposed studies should provide the additional information needed to do so. This information can be useful not only in gaining a better understanding of the mechanism and duration of protection provided by AVA, but also for licensing newer PA-based vaccines under development. One important contribution anticipated from the ICP studies is the development and standardization of assays necessary for research in this area. The recent IOM report on the anthrax vaccine (IOM, 2002)

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included a recommendation for efforts to standardize an assay for quantitation of antibody levels that can be used across laboratories carrying out research on anthrax vaccines. The ICP studies will use anti-PA ELISA and TNA assays developed at the National Center for Infectious Diseases at CDC, as well as additional assays to be developed and validated at CAMR as part of the research project. Finding: The committee strongly supports the use of validated assays that can be standardized across the field of anthrax vaccine research. CDC’s development and validation of such assays will provide an important contribution in this regard. Recommendation: CDC should give high priority to standardization of assays that can be used across laboratories conducting research with anthrax vaccine. The committee found the many goals outlined by CDC for the ICP studies (Box 4-3) to vary widely in scientific value. The goals of quantifying the IgG response to PA and other anthrax antigens, quantifying the lethal toxin response, describing the anti-PA IgG subclass profiles in vaccinees, and describing the maturation of anti-PA IgG avidity in AVA vaccinees are, in the committee’s view, important and necessary. The remaining goals described by CDC are viewed as of lower priority. While examination of a panel of immunologic responses is interesting, it is not likely to provide important new insights regarding AVA’s mechanism of protection. The committee is particularly dubious of the value of the proposed efforts to characterize a role for AVA-specific CD4+ T helper cells in the anamnestic anti-PA antibody response to in vitro challenge. The protocol does not make clear how the in vitro data could be correlated with responses in vivo. Similarly, there is no clear plan for correlating the count of bone marrow plasma cells to anamnestic immune re BOX 4-3 Goals of Assays of Immune Correlates of Protection, as Specified by CDC Quantify the IgG response to anthrax toxin PA and other selected anthrax antigens. Quantify the anthrax lethal toxin (PA+LF) neutralization response. Describe the anti-PA IgG subclass profiles in AVA vaccinees. Describe the maturation of anti-PA IgG avidity in AVA vaccinees. Determine the ability of anti-AVA antisera to promote opsonophagocytosis. Quantify the PA-specific proliferative responses of circulating T-cells. Describe and quantify selected cytokine mRNA synthesis and protein secretion patterns in circulating T-cells following in vitro stimulation with PA. Characterize the magnitude and duration of T-cell (CD4+) and B-cell components of immune memory to AVA vaccination. Enumerate the circulating PA-specific memory CD4+ T-cells at selected time points during and post immunization. Enumerate the T-cell-dependent, PA-specific, bone-marrow plasma cells (NHPs only) and circulating antibody-secreting memory B-cells (NHPs and humans) at selected time points during and post immunization. Identify CD4+ T-cell-stimulating epitopes in the PA protein in order to track PA-specific memory T-cells and a protective immune response against anthrax. This may help with the longer-term goal of identifying candidate DNA vaccine epitopes. SOURCE: CDC, 2002j, pp. 2–3.

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sponse outcome. Finally, identification of the CD4+ T-cell-specific epitopes on the PA molecule is a detail of the immunologic response mechanism that does not directly apply to safety or efficacy and is not appropriate to pursue as part of CDC’s research program. The committee also had concerns about CDC’s plans for certain ICP studies to be done on the 33 macaques housed at Emory. The studies to be carried out exclusively at Emory are semi-quantitative analysis of in vitro PA-specific antibody and cytokine-secreting peripheral blood mononuclear cells (PBMC). Two other assays—the quantification of the PA-specific T-cell proliferative capability in peripheral blood cells and the identification of CD4+ T-cell-stimulating epitopes and linear humoral epitopes on the PA protein—are being done in conjunction with Battelle and CDC, respectively. Investigators at Emory also plan to carry out lymph node biopsies, bone marrow biopsies, and bronchoalveolar lavage on the 33 rhesus macaques housed there. The researchers hope to use the results of those tests to correlate immune responses occurring in tissues or organs of the macaques with their PBMC and to infer the type and magnitude of immune responses to AVA that are occurring systemically in human vaccinees (CDC, 2002c). The hypotheses to be tested are that the generation and persistence of long-lived plasma cells in the bone marrow are key determinants of long-term humoral immunity and that sustained serum antibody to vaccine antigens will correlate with the presence of antigen-specific plasma cells in the bone marrow (CDC, 2002c). An ELISPOT assay will be used to test bone marrow samples for the presence of plasma cells specific for PA, LF, and PA-LF. According to the protocol, the analysis may provide new guidelines for the minimum number of immunizations necessary to generate long-term humoral immunity to anthrax. As noted above, however, the committee questions the extent to which these studies can contribute to addressing this research question. Studying the duration of the immune memory response is important and necessary, but such studies should focus on the duration of anti-PA antibodies and other antibodies. Thus far, only anti-PA antibodies have been shown to be related to protection in rhesus macaques. The protocols call for the lymph node biopsies, bone marrow biopsies, and bronchoalveolar lavage to be carried out at least 12 times (CDC, 2002c). The committee acknowledges that the aim of the study is to gather as much information as possible from the macaques to be able to assist in understanding mechanisms in humans. But the tremendous number of invasive procedures to be carried out on the animals is of concern. In the absence of specific, compelling hypotheses to be tested, the extensive procedures do not appear to be adequately justified or even likely to be feasible, since the frequent administration of anesthesia and the repeated biopsies may alter the responses of interest. The proposed research is descriptive rather than hypothesis-driven and should be of a low priority. Finding: The biopsies of lymph nodes and bone marrow and the bronchoalveolar lavage planned as part of the Immune Correlates of Protection Study require multiple invasive procedures that do not appear to be adequately justified. Recommendation: On the basis of the information provided to the committee for evaluation, the committee recommends that the NHP studies requiring multiple samplings from biopsies of lymph nodes and bone marrow and from bronchoalveolar lavage should not be continued in their current form. If such studies can be adequately justified, they should be modified to require fewer invasive procedures. Finding: With the exception of the biopsy and bronchoalveolar lavage studies noted above, the committee finds that the ICP studies that have been proposed as a means to provide information about the efficacy and immunogenicity of AVA are responsive to the congressional mandate and to important research needs. The information gathered will inform and influence the development and licensure of new PA-based anthrax vaccines.

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