3
Anthrax Vaccine Efficacy

Evaluating the efficacy of the Anthrax Vaccine Adsorbed (AVA), particularly against inhalational exposure to anthrax, was a crucial part of this committee’s charge. The charge specifically calls for evaluation of (1) the efficacy of the anthrax vaccine (AVA) in protecting humans from inhalational anthrax, (2) the efficacy of AVA against all known strains of Bacillus anthracis, and (3) the correlation of the effectiveness of the vaccine in animal models to its ability to protect humans. This chapter presents the committee’s observations and findings regarding the efficacy of AVA against inhalational anthrax and all known strains. The committee also examines what is known and what must still be established regarding the correlation of immune protection in animal models with immune protection in humans.

The term efficacy generally refers to the ability of a product to achieve its desired effect under ideal conditions, such as a controlled clinical trial in which the product is consistently administered as prescribed. The effectiveness of a product is its ability to achieve the desired effect under real-world conditions. The charge to the committee uses both terms, and in many situations the terms are used interchangeably, with the context showing whether the effects were observed under laboratory-controlled or real-world conditions. In this report the committee is concerned primarily with evaluating efficacy.

It is important to note that efficacy is relative, not absolute. A variety of factors can play a role in determining the degree of protection from a vaccine, which can include the size of the inoculum of exposure, the strain



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The Anthrax Vaccine: Is it Safe? Does it Work? 3 Anthrax Vaccine Efficacy Evaluating the efficacy of the Anthrax Vaccine Adsorbed (AVA), particularly against inhalational exposure to anthrax, was a crucial part of this committee’s charge. The charge specifically calls for evaluation of (1) the efficacy of the anthrax vaccine (AVA) in protecting humans from inhalational anthrax, (2) the efficacy of AVA against all known strains of Bacillus anthracis, and (3) the correlation of the effectiveness of the vaccine in animal models to its ability to protect humans. This chapter presents the committee’s observations and findings regarding the efficacy of AVA against inhalational anthrax and all known strains. The committee also examines what is known and what must still be established regarding the correlation of immune protection in animal models with immune protection in humans. The term efficacy generally refers to the ability of a product to achieve its desired effect under ideal conditions, such as a controlled clinical trial in which the product is consistently administered as prescribed. The effectiveness of a product is its ability to achieve the desired effect under real-world conditions. The charge to the committee uses both terms, and in many situations the terms are used interchangeably, with the context showing whether the effects were observed under laboratory-controlled or real-world conditions. In this report the committee is concerned primarily with evaluating efficacy. It is important to note that efficacy is relative, not absolute. A variety of factors can play a role in determining the degree of protection from a vaccine, which can include the size of the inoculum of exposure, the strain

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The Anthrax Vaccine: Is it Safe? Does it Work? of the pathogen, and the host response. Even a vaccine considered highly effective may fail to protect some individuals under some circumstances. EVALUATING EFFICACY OF AVA FOR INHALATIONAL ANTHRAX The data used to evaluate the efficacy of AVA come from three sources. Studies with textile mill workers tested the efficacy of AVA and a related vaccine against occupational exposures to anthrax spores. Serological studies with humans tested the ability of AVA to elicit antibodies to protective antigen (PA), an indication of an immune response to the vaccine. Studies with animals tested the efficacy of the vaccine in protecting the animals from inhalational exposure to anthrax spores. Human Efficacy Trials Brachman and colleagues (1962) conducted the only randomized, placebo-controlled trial of the efficacy of a PA-containing anthrax vaccine. Although the safety information that it provides is reported separately in Chapter 6, here the committee describes the information on vaccine efficacy provided in that study. The vaccine studied was not AVA but was an earlier formulation produced from the R1-NP mutant of the Vollum strain of anthrax manufactured by Merck (see Chapter 7 for more details). The study was carried out from January 1955 through March 1959 in four textile mills in the northeastern United States that processed raw, imported goat hair for production into the interlinings of suit coats. The goat hair was typically contaminated with anthrax spores, and workers were exposed during handling of this material. Before receiving the vaccine, the average annual incidence of cutaneous anthrax among workers at these four mills ranged from 0.6 to 1.8 cases per 100 workers. The worker population eligible for the study included 1,249 men and women with no history of prior anthrax infection. Approximately 47 percent of employees worked in high-risk areas within the mills, and about one-half of the eligible study subjects came from one of the four mills (Mill A). Rates of refusal to participate in the study among the four mills ranged from <1 to 45 percent, and refusals were approximately equally distributed between the placebo and vaccine groups. Participating workers were randomly allocated by length of employment, age, department, and job to receive either vaccine or placebo. Inoculations of 0.5 milliliters (ml) of either vaccine or placebo (0.1 percent alum) were given; the first three inoculations were administered at 2-week intervals, followed by three injections at 6-month intervals and annual boosters thereafter. Those referred to as “complete” inoculees received at least the

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The Anthrax Vaccine: Is it Safe? Does it Work? first three injections and subsequent inoculations on schedule (personal communication, S. A. Plotkin, consultant to the Institute of Medicine Committee to Assess the Safety and Efficacy of the Anthrax Vaccine, January 29, 2001). Otherwise, the inoculees were referred to as “incomplete.” There were 379 complete vaccine recipients and 414 complete placebo recipients. Only data for those designated complete inoculees were included in the calculation of efficacy. Routine visits and environmental sampling were conducted throughout the study to confirm exposure and identify cases of anthrax. Over the course of the study, 26 cases of anthrax occurred, including an outbreak of 9 cases over a 10-week period at one of the mills (Mill A). Twenty-one of the 26 cases were cutaneous anthrax and 5 were cases of inhalational anthrax, all of which occurred during the outbreak at Mill A. Of the 26 cases, 3 occurred among vaccine recipients (1 complete and 2 incomplete inoculees), 17 occurred among individuals in the placebo group (15 complete and 2 incomplete inoculees), and 6 cases occurred among individuals who were not inoculated with the vaccine or the placebo. None of the cases of inhalational anthrax occurred in persons who had received the vaccine. The overall effectiveness of the vaccine against anthrax infection generally was 92.5 percent (lower 95 percent confidence interval = 65 percent): 91.4 percent in the high-risk group of workers and 100 percent in the low-risk group of workers. It was not possible to evaluate the efficacy of the vaccine against inhalational anthrax separately because of the small number of cases. (Given the definition of “complete” vaccination in the published paper, one of the cases counted in the incomplete vaccination group should have been included in the complete vaccination group. Doing so would have reduced the reported effectiveness somewhat. It is not possible to calculate the effectiveness after the addition of this case to the complete vaccination group since the necessary information is not provided in the paper.) As part of another effort, the Centers for Disease Control and Prevention (CDC) collected observational data on the occurrence of anthrax in industrial settings like textile mills between 1962 and 1974 (FDA, 1985). During this period, both the Merck vaccine and AVA, produced by the Michigan Department of Public Health, were used and 27 cases of cutaneous anthrax were identified. No cases occurred in those who had received the full course of immunizations. Three cases occurred in persons who worked in or near mills but who were not mill employees and who were not vaccinated. The remaining 24 cases occurred among mill employees; 3 of these had received one or two doses of the vaccine, and the remaining 21 persons were unvaccinated. Thus, of the 27 cases observed, 2 occurred in persons who had received two doses of vaccine, 1 occurred in a person who

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The Anthrax Vaccine: Is it Safe? Does it Work? had received one dose of the vaccine, and the other 24 occurred in persons who were completely unvaccinated. Finding: The randomized field study carried out by Brachman and colleagues (1962) provides solid evidence indicating the efficacy of a vaccine similar to AVA against B. anthracis infection. The subsequent CDC data are supportive. However, the small number of inhalational cases in those studies provides insufficient information to allow a conclusion to be made about the vaccine’s efficacy against inhalational infection. Human Antibody Response to AVA Information regarding the ability of AVA to elicit antibodies in human vaccinees is available from five studies. An indirect hemagglutination assay and an enzyme-linked immunosorbent assay (ELISA) for antibodies to PA were carried out with serum specimens from 190 vaccinees (Johnson-Winegar, 1984). Serum samples were obtained 2 weeks after the third immunization in the vaccination series. By the indirect hemagglutination assay, 83 percent of vaccinees seroconverted (titer of 1:8 or above). Other data indicated that at 2 weeks after an annual booster immunization that follows the six-dose regimen, all 85 vaccinees evaluated had seroconverted. Two retrospective serological studies evaluating the effect of the dosing interval on the human antibody response (Pittman et al., 2000) served as preliminary studies for the larger prospective study described later (Pittman et al., 2002). Increasing the interval between the first and second doses of vaccine from 2 to 4 weeks resulted in a statistically significant three- to fourfold increase in the geometric mean anti-PA immunoglobulin G (IgG) antibody titer measured several weeks after the administration of the second dose. Data on the human antibody response to AVA were also provided by a study of volunteers from Ft. Bragg, North Carolina. Pittman and colleagues (Pittman, 2001; Pittman et al., 1997, in press) conducted a study to assess the persistence of antibodies against B. anthracis 18 to 24 months after initial vaccination during Operations Desert Shield and Desert Storm and to assess the safety and immunogenicity of a vaccine booster dose. Study participants were recruited from among active-duty personnel at Fort Bragg in 1992 and 1994. The study population consisted of 495 male Desert Shield or Desert Storm veterans who received one to three primary doses of AVA in 1990 or 1991. Serological analyses were performed for a subset of participants (20 participants who had received AVA only and 259 participants who had received both AVA and pentavalent botulinum toxoid)

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The Anthrax Vaccine: Is it Safe? Does it Work? whose blood had been drawn prior to and 24 to 36 days after receipt of booster doses of vaccine. Evaluation of anti-PA IgG levels indicated that roughly 2 years after their initial vaccinations, the proportion of volunteers (20 to 50 percent) with detectable anti-B. anthracis antibodies was low, and among those with detectable antibodies, persisting antibody levels were low. After receipt of booster doses, all but two volunteers had detectable anti-PA antibody responses. The geometric mean titers (ELISA) in those with detectable antibody responses ranged from roughly 4,500 in those who had received only one initial dose to 10,000 in those who had received three priming doses. Data on reactogenicity are reported in Chapter 6. A pilot study carried out at the U.S. Army Medical Research Institute of Infectious Diseases examined immune responses to alternative AVA dosing schedules and routes of administration (Pittman et al., 2002). In that study, 173 U.S. military and civilian volunteers (109 men and 64 women) were randomized to one of seven groups, defined on the basis of dosing schedule and route of administration. Three experimental dosing schedules were tested: a single injection on day 0, injections on days 0 and 14, and injections on days 0 and 28. For each experimental dosing schedule, two groups were established: one group was inoculated subcutaneously and the other group was inoculated intramuscularly. A control group was administered AVA by the licensed six-dose schedule and subcutaneous administration. The anti-PA IgG concentrations measured 2 weeks after the administration of two doses of AVA 4 weeks apart (either intramuscularly or subcutaneously) were comparable to those measured 2 weeks after the administration of three doses (given subcutaneously) 2 weeks apart (the licensed dosing schedule). The distribution of peak anti-PA IgG antibody concentration did not differ among those who received two doses (either intramuscularly or subcutaneously) 4 weeks apart and those who received three doses (subcutaneously) 2 weeks apart. Antibody response rates (>25 micrograms per milliliter [µg/ml]) for these groups were 96 to 100 percent (the reasons for the insensitivity of the ELISA described in that study were unclear to the committee). Similar results were obtained by a toxin neutralization antibody (TNA) assay. A single dose of AVA was not sufficient to elicit peak anti-PA antibody concentrations or seroconversion rates comparable to those achieved by the licensed dosing schedule. These findings indicate that AVA administered by its licensed dosing schedule as well as by schedules that omit the dose administered at 2 weeks generates substantial antibody responses (at least 25 µg/ml) in 96 to 100 percent of recipients. CDC plans to conduct a larger randomized, controlled, multicenter trial to test the immunogenicity of AVA using a reduced number of doses administered intramuscularly. The data presented above regarding the antibody responses generated

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The Anthrax Vaccine: Is it Safe? Does it Work? by AVA in humans will be useful when future studies (passive protection studies, discussed later in this chapter) determine a likely protective level of anti-PA antibody on the basis of the results obtained from animal studies. Efficacy Data from Animal Models Extrapolation of Results from Animal Studies to Humans Food and Drug Administration (FDA) regulations spell out the criteria required to prove “effectiveness” (meaning efficacy in the context of this report) through controlled clinical investigations and field trials.1 However, for potentially lethal exposures such as inhalational exposure to anthrax spores, there is a serious problem in meeting these criteria. Field studies that rely on natural exposure to disease are not feasible as a means of evaluating the efficacy of the anthrax vaccine because inhalational anthrax is very rare, even in areas where anthrax occurs naturally or where it is an occupational hazard. Moreover, the particular concern regarding inhalational anthrax is exposure to anthrax spores processed for use as biological weapons. Controlled trials in which subjects are exposed to potentially lethal agents such as anthrax spores are simply not ethical. They would involve the administration of a potentially lethal substance to healthy human volunteers without a proven treatment that could be used if the vaccine or some other protective agent being tested failed. Recognizing the need to provide for circumstances in which efficacy studies with humans cannot be ethically conducted, FDA published a proposed rule in October 1999 regarding the use of data from animal studies (FDA, 1999). In it FDA recommended the evidence needed to demonstrate the efficacy of new drugs for use against lethal or permanently disabling toxic substances. Although AVA is already a licensed vaccine, noting FDA’s proposed requirements for the approval of such vaccines in the future can be helpful in evaluations of data from animal studies regarding the efficacy of AVA.2 At the time this report was completed, the proposed rule for using data from animal studies had not yet been finalized. The committee 1   The regulations are described in (a) Review Procedures to Determine that Licensed Biological Products Are Safe, Effective, and Not Misbranded Under Prescribed, Recommended, or Suggested Conditions of Use (21 C.F.R. § 601.25 [2001]) and (b) FDA Action on Applications and Abbreviated Applications: Adequate and Well-Controlled Studies (21 C.F.R. § 314.126 [2001]). 2   In its proposed rule, FDA proposed that evidence from studies with animals be relied upon when controlled trials with humans cannot ethically be carried out. It would do so, however, only when “there is a reasonably well-understood pathophysiological mechanism of the toxicity of the substance and its prevention or substantial reduction by the product; the

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The Anthrax Vaccine: Is it Safe? Does it Work? hopes that all parties involved continue to work toward finalization of the rule as quickly as possible. Finding: Because additional clinical trials to test the efficacy of AVA in humans are not feasible and challenge trials with volunteers are unethical, by necessity animal models represent the only sources of the supplementary data needed to evaluate AVA’s efficacy. Choice Among Animal Models of Human Disease Animal models inevitably have different strengths and weaknesses in representing human disease and immunity. Therefore, different models may be appropriate for different applications. Animal models with pathological and immunological characteristics similar to those of humans could be considered the most appropriate ones for evaluations of vaccine efficacy. This is not to say that other animal models might not be appropriate for certain screening purposes, but it is important to weigh the data obtained with those models accordingly. The sections below review and compare the pathological and immunological features of different animal models in relation to human anthrax disease and immunity. Comparison of Anthrax Pathology in Humans and Animals As described in Chapter 2, anthrax is a zoonotic disease (a disease in which the same organism infects and causes disease in both humans and animals) caused by B. anthracis. B. anthracis most commonly infects grazing animals, as B. anthracis spores are stable for long periods in soil and grazing animals are the most readily available hosts. Humans can also be infected, however, generally from contact with the products of infected animals such as hides, hair or wool, meat, or by-products. Also, as discussed earlier in this report, the same organism produces diseases with different clinical manifestations (cutaneous, gastrointestinal, or inhalational), depending on the site of exposure and infection. Pathology of Inhalational Anthrax in Humans As discussed in Chapter 2, inhaled anthrax spores are phagocytosed (taken up) by macrophages and transported from the lungs to the nearby peribronchial and mediastinal     effect is independently substantiated in multiple animal species, including species expected to react with a response predictive for humans; the animal study endpoint is clearly related to the desired benefit in humans, generally the enhancement of survival or prevention of major morbidity; and the data or information on the kinetics and pharmacodynamics of the product or other relevant data or information, in animals and humans, allows selection of an effective dose in humans.”

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The Anthrax Vaccine: Is it Safe? Does it Work? lymph nodes (Albrink, 1961; Ross, 1957). The spores germinate in the lymph nodes and produce the three toxin components: PA, edema factor (EF), and lethal factor (LF). The toxins damage the lymph nodes, with subsequent dissemination via the bloodstream to many distant sites. Information about the characteristic features of inhalational anthrax has primarily been gained from autopsy studies. In 1957, five cases of inhalational anthrax occurred at a goat hair-processing mill in New Hampshire. Four cases were fatal, and three of these were examined by autopsy. Pathology findings in the three cases included hemorrhagic edema in the mediastinum, hemorrhagic lymph nodes, and pleural effusions (leaking of fluid from the lining of the lungs). Two of the three cases demonstrated enlarged spleen, and microscopic hemorrhages and inflammation of the meninges (the lining of the brain; Albrink et al., 1960; Plotkin et al., 1960). In a 1966 case of human inhalational anthrax, the patient also had a hemorrhagic edematous mediastinum and mediastinal lymph nodes, as well as a pleural effusion. The outer membrane of the brain had microscopic hemorrhages and inflammation (LaForce et al., 1969). In a case of anthrax described in a weaver in 1978, the patient similarly had pleural effusion, hemorrhagic mediastinitis, and leptomeningitis, as well as an enlarged spleen (Suffin et al., 1978). The 1979 release of anthrax spores from a military facility in the town of Sverdlovsk in the former USSR led to 68 deaths (Guillemin, 1999). Necropsies were performed and tissue samples and microscopic slides were preserved for 42 cases, providing a wealth of additional information demonstrating the pathological changes in human tissue that occur as a result of inhalational exposure to anthrax spores. The most striking pathological features in the anthrax cases from Sverdlovsk were prominent and consistent lesions of hemorrhagic thoracic lymphadenitis and hemorrhagic mediastinitis (Abramova et al., 1993; Walker, 2001). These consist of bloody, inflamed lymph nodes in the chest and bleeding and inflammation of the tissues in the area between the lungs. In addition, disseminated infection was often noted, with bacteremia, meningitis, and involvement of the submucosa of the gastrointestinal tract, particularly the small intestine, stomach, and colon. Another noteworthy aspect was edema, particularly in the lungs, mediastinum, pleurae (lining of the lung), and brain. The vascular damage that led to the edema was thought to be consistent with the effects of the toxins secreted by B. anthracis (Abramova et al., 1993; Walker, 2001). As noted in Chapter 2, review of the first 10 reported cases of inhalational anthrax resulting from the bioterrorism release of anthrax spores in the autumn of 2001 indicated that all 10 patients had abnormal chest X rays (Jernigan et al., 2001). Abnormalities included infiltrates, pleural effu-

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The Anthrax Vaccine: Is it Safe? Does it Work? sion, and mediastinal widening. Mediastinal lymphadenopathy was observed in seven patients. Pathophysiology of Anthrax in Potential Animal Models The pathophysiology of anthrax infections varies in different animal models, making the disease in some species more similar than that in others to the disease in humans. Also, the organism characteristics associated with virulence in various animal species differ. Mice and Rats Mice are considered among the laboratory animals most susceptible to infection with B. anthracis. However, inbred mouse strains differ in their susceptibilities to lethal infection with different strains of B. anthracis. In contrast to humans, the pathophysiology of anthrax in mice depends upon the encapsulation of the bacillus rather than the toxins (Welkos, 1991). The degree of virulence in the mouse model is correlated with the presence of the polyglutamic acid capsule on the bacillus (Welkos, 1991). In contrast, rats are extremely sensitive to the effects of the toxins but are relatively resistant to infection (Young et al., 1946). Thus, neither mice nor rats are considered good models of human anthrax. Guinea Pigs Guinea pigs are also highly sensitive to anthrax and have been used for many years to study the pathogenesis of B. anthracis infection (Ross, 1957). The pathological changes observed in guinea pigs after inhalational exposure to B. anthracis are characterized by widespread edema and hemorrhage, particularly in the spleen, lungs, and lymph nodes. As seen in other animal models, the cellular inflammatory response observed after inhalational exposure is limited, consistent with a fulminating septicemia rather than a primary pulmonary infection (Albrink and Goodlow, 1959; Ross, 1957). Rabbits In rabbits the effects observed from inhalational infection with B. anthracis are similar to those seen in humans and rhesus monkeys. Substantial pathological lesions are consistently observed in the spleen, lymph nodes, lungs, gastrointestinal tract, and adrenal glands (Zaucha et al., 1998). Inflammation, hemorrhage, and edema are frequently observed in the mediastinum and the intrathoracic lymph nodes. Hemorrhage and edema are sometimes found in the meninges of the brain, but with less inflammation than that seen in humans and rhesus monkeys (Zaucha, 2001). Other differences between rabbits and humans or rhesus monkeys include milder mediastinal lesions and a lower incidence of lung lesions. The weaker inflammatory response in rabbits may be the result of the shorter observed survival time, limiting development of leukocytic infiltration (Zaucha et al.,

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The Anthrax Vaccine: Is it Safe? Does it Work? 1998). Primary pneumonic foci were not observed in rabbits, although they have been observed in humans, perhaps as the result of preexisting pulmonary disease. Nonhuman Primates Chimpanzees. At least one paper describes the effects of experimentally induced inhalational anthrax in chimpanzees (Albrink and Goodlow, 1959). Two of the four test animals in that study survived, despite evident bacteremia. They were later rechallenged with anthrax spore aerosols (only one animal survived rechallenge with a larger dose). The pathological changes resembled those observed in guinea pigs, mice, and monkeys, with widespread edema and hemorrhage, particularly in the spleen, lungs, and lymph nodes. Death appeared to result from fulminating sepsis rather than a primary pulmonary infection. Rhesus monkeys. Most information about the pathology of inhalational anthrax in nonhuman primates is gleaned from studies carried out with rhesus monkeys (Macaca mulatta; also called macaques). The pathophysiology of anthrax in rhesus monkeys is similar to that in humans. Several studies have reported on the gross pathological changes observed in rhesus monkeys exposed to inhaled aerosolized anthrax spores (Berdjis et al., 1962; Fritz et al., 1995; Gleiser et al., 1963). Berdjis and colleagues (1962) described their findings from serial postmortem observation of young M. mulatta monkeys exposed to low and high doses of inhaled spores. They observed edema, hemorrhage, necrosis, and inflammatory infiltrates in the lungs, lymph nodes, spleen, and liver. Two autopsy studies of animals that died from inhalational exposure to anthrax (Fritz et al., 1995; Gleiser et al., 1963) had somewhat different findings. Both reported edema, hemorrhage, and necrosis of the lymph nodes in the monkeys; but in the first study (Gleiser et al., 1963), the affected lymph nodes were predominantly intrathoracic (hilar, mediastinal, and tracheobronchial), whereas in the more recent study (Fritz et al., 1995) the mesenteric lymph nodes were more commonly involved. Enlargement of the spleen was common in the study by Gleiser and colleagues but not in the study by Fritz and colleagues. Hemorrhagic meningitis was observed in a third of the animals in the study by Gleiser and colleagues and in half of the animals in the study by Fritz and colleagues. In addition, Dalldorf and colleagues (1971) described similar pathological effects of inhalational anthrax on cynomolgus monkeys (Macaca fascicularis), another type of macaque. Dalldorf and colleagues observed involvement of the mediastinal lymph nodes in all infected subjects, which led to edema and hemorrhage and which was accompanied by bacteremia.

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The Anthrax Vaccine: Is it Safe? Does it Work? Conclusion From what is known and from the information described above, it appears that the pathology of anthrax in nonhuman primates such as macaques best mimics that seen in humans after inhalational exposure to B. anthracis. However, nonhuman primates are available in only limited numbers and are very costly to study. Although guinea pigs and, to some extent, rabbits are more susceptible to the disease than monkeys and humans, they are much more readily available and therefore may be helpful for use in the screening of various interventions. Efficacy of AVA Against Anthrax in Animal Models Mice It is difficult to immunize mice against anthrax. Different strains of mice differ dramatically in their susceptibilities to different B. anthracis strains and in the protection from challenge afforded by AVA and other anthrax vaccines (Welkos and Friedlander, 1988). In fact, the bacterial capsule rather than the toxin appears to be the primary virulence factor for mice, so that vaccines such as AVA based on the PA aspect of anthrax toxins (LF or EF) are of reduced efficacy (Welkos, 1991; Welkos et al., 1993). Only when PA was combined with a potent adjuvant was protection conferred (Welkos et al., 1990). Protection against strains fully virulent in the mouse may involve mechanisms in addition to humoral immunity (Welkos and Friedlander, 1988). Hamsters Few data describing the efficacy of AVA in hamsters are available. However, Fellows and colleagues (2002) have demonstrated that AVA failed to protect Golden Syrian hamsters against parenteral challenge with virulent B. anthracis spores. Guinea Pigs Guinea pigs have been used extensively in anthrax vaccine development and serve as the standard test system for evaluations of anthrax vaccine potency. In the potency test, guinea pigs are immunized parenterally and are challenged with 1,000 spores of the Vollum strain administered subcutaneously (FDA, 1973). However, guinea pigs are difficult to protect by immunization with

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The Anthrax Vaccine: Is it Safe? Does it Work? CORRELATION OF PROTECTION: ANIMAL MODELS AND HUMAN IMMUNITY Establishing Animal Model Correlates of Anthrax Vaccine Efficacy Reuveny and colleagues (2001) recently published findings from studies with guinea pigs evaluating anti-PA antibody and TNA as correlates of protection from B. anthracis. Guinea pigs were immunized with single injections of various dilutions of PA vaccine or with PA vaccine inactivated to various extents by heat and were then challenged with an intradermal injection of Vollum strain B. anthracis spores. An additional study used passive immunization of the guinea pigs with various amounts of hyperimmune sera before challenge. The investigators reported associations between percent survival of the guinea pigs and both anti-PA IgG antibody titers and TNA titers. However, the TNA titers appeared to be a better correlate of protection in this model system, whereas the anti-PA IgG anti-body titers determined by ELISA had a limited value in predicting protective immunity. The passive transfer studies showed similar results, and the fact that hyperimmune sera could protect the animals indicates that a humoral response alone may be sufficient to confer protection. It must be noted that immunity to B. anthracis is complex. While anti-PA antibodies have been shown to be necessary and sufficient for protection in the studies reviewed below, other studies, while confirming the central role of anti-PA antibodies, suggest that other antigens may also contribute to the ability to confer protection from the disease in some animal models (Brossier et al., 2002; Cohen et al., 2000; Pezard et al., 1995). Indeed several studies indicate difficulty in establishing a direct correlation between PA-specific antibody titers and protection (Ivins et al., 1990, 1995; Turnbull, 1991; Turnbull et al., 1986, 1988) whereas others have succeeded (see Reuveny et al. [2001] discussed above and Barnard and Friedlander [1999] discussed below). Efficacy studies 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). Little and colleagues (1997) showed by passive transfer between guinea pigs that the serum of animals immunized against PA was protective for naïve recipients (but that anti-EF and anti-LF anti-bodies were not). McBride and colleagues (1998) showed that recombinant PA administered with an appropriate adjuvant would protect guinea pigs from aerosol challenge. Barnard and Friedlander (1999) demonstrated that the protective efficacy of recombinant PA vaccines correlated with the anti-PA antibody titers they elicited in vivo and the level of PA they produced in vitro. Beedham and colleagues (2001) showed that several susceptible strains of mice could be protected from challenge by immunization with recombi-

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The Anthrax Vaccine: Is it Safe? Does it Work? nant PA with adjuvant. Furthermore, their study indicated that protection resulted from circulating antibody, as passively transferred lymphocytes were not protective. As described earlier, Pitt and colleagues (2001) reported serological correlates of immunity against inhalational anthrax in rabbits. The animals were immunized with AVA, and the levels of antibody to PA and TNA associated with protection were quantified. The levels of either antibody at 6 and 10 weeks after immunization proved to be predictive of survival. Efforts have been made to evaluate the relationship between levels of anti-PA antibody and protection from B. anthracis challenge in nonhuman primates. A study by Ivins and colleagues (1998) indicated that rhesus monkeys have a strong immune response to PA, as evidenced by high titers of antibody to PA and high TNA titers. A single dose of AVA protected the monkeys from aerosol challenge with anthrax spores (roughly 5 × 106 spores). The high level of protection provided made it difficult to correlate anti-PA IgG antibody titers or other measures with levels of protection. Fellows and colleagues (2001) showed that rhesus monkeys had strong anti-PA antibody responses to two doses of 0.5 ml of AVA and were protected (80 to 100 percent survival) from aerosol challenge with spores from two virulent isolates. Finding: The available data indicate that immunity to anthrax is associated with the presence of antibody to protective antigen. The information reviewed by the committee shows that humans and certain laboratory animals both manifest the same disease after infection with B. anthracis and that both are protected by immunization with AVA, which elicits the production of antibodies to PA. This information establishes a qualitative correlation between antibodies to PA and protection in animal models and in humans. To move forward with research on the current anthrax vaccine or any new vaccines, however, a quantitative correlation of the protective levels of antibodies in animals with the antibody titers obtained after full immunization in humans is needed. Those correlates in animal models can then be used to test new vaccines for efficacy with confidence that the data from animal studies will be predictive of the clinical results obtained with immunized humans. Correlating the protective level of antibody can, in principle, be accomplished either by active immunization or by passive immunization. In the first instance, animals would be vaccinated with various doses of vaccine and their antibody levels would be measured. They would then be challenged to determine the level of circulating antibody at which protection would no longer be reliable. For passive immunization studies, it is necessary to immunize animals of the model species, collect the serum of the immunized animals, and

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The Anthrax Vaccine: Is it Safe? Does it Work? administer different concentrations of that antibody-containing serum to naïve animal hosts. The latter procedure provides passive immunity to the hosts. The hosts must then be challenged with aerosolized B. anthracis spores to determine which animals survive during the period of observation and hence what concentration of antibody must be present to protect the host. The period of observation of the animals will need to be limited (on the order of 2 weeks after challenge), in keeping with the half-life of immunoglobulin. It would be useful to determine a correlation between the inoculum size (the number of spores) and the antibody level associated with protection. An inoculum size sufficient to simulate or exceed the quantity of spores likely to be encountered during exposure must be established. After the intraspecies passive transfer assay in animal models, the next step extends to the human vaccinee. Here, appropriate laboratory animals would receive different concentrations of antibody-containing serum from humans vaccinated with anthrax vaccine. Again, the passively immunized animals would be challenged with aerosolized spores of an appropriate inoculum size to determine the extent of survival. In the case of immunity to anthrax, the concentration of antibody to PA is of particular interest because PA is necessary for the virulence of the different strains in both humans and animal models. A similar strategy might be applied by using toxin neutralization. The TNA assay is a functional test that evaluates the amount of antibody needed to inactivate the lethal B. anthracis toxin complex of LF and PA (together, lethal toxin). The ability of test serum samples to neutralize lethal toxin in vitro is compared with that of a standard serum sample by using cytotoxicity as the endpoint of the assay (Pittman et al., 2002). Antibodies to either protein might be expected to neutralize cytotoxicity, but the assay is principally used to quantify antibody to PA. Once quantitative correlates (the amount of antibody or toxin neutralization activity necessary for full protection from B. anthracis challenge) are established, a new vaccine could be administered to naïve animals over a range of concentrations to determine the dose required to obtain a level of antibody known to be protective. Finally, the investigational product could be administered to healthy human volunteers to determine its ability to stimulate production of comparable quantities of antibody and thereby choose the appropriate dose regimen required for protection. Furthermore, the resulting human antibody would be collected and the serum would be administered to additional naïve laboratory animals to confirm transferable passive immunity by showing that the antibodies produced would protect the host animal. The data from studies with animals already developed suggest that serological correlates of human immunity can be developed in appropriate

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The Anthrax Vaccine: Is it Safe? Does it Work? animal models. The committee commends this work and encourages its further development. Recommendation: Additional passive protection studies with rabbits and monkeys, including the transfer of animal and human sera, are urgently needed to quantify the protective levels of antibody in vivo against different challenge doses of anthrax spores. Recommendation: Additional active protection studies should be conducted or supported to develop data that describe the relationship between immunity and both specific and functional quantitative antibody levels, including studies of the relationship between the vaccine dose and the resulting level of antibody in the blood of test animals that protects the animals from challenge; the relationship between the level of antibody that protects animals from challenge and the level of antibody present in humans vaccinated by the regimen currently recommended for the licensed product; and the vaccine dose that results in a level of antibody in the blood of human volunteers similar to that in the blood of protected animals. Measurements of anti-PA antibody titers will be crucial to the success of the research described above. Progress will be hampered if assay results are not comparable across laboratories. Recommendation: The Department of Defense should support efforts to standardize an assay for quantitation of antibody levels that can be used across laboratories carrying out research on anthrax vaccines. POSTEXPOSURE USE OF ANTHRAX VACCINE Evidence that postal workers and congressional staff were exposed to aerosolized anthrax spores from anthrax-laden letters sent through the U.S. mail in the autumn of 2001 resulted in questions about the efficacy of AVA in protecting people from infection when the vaccine is administered after exposure to anthrax spores. No data from studies with humans are available to evaluate the efficacy of AVA in these circumstances. However, two papers provide information from studies with rhesus monkeys. Henderson and colleagues (1956) carried out studies in which penicillin, immune serum, and a PA-based vaccine (a predecessor of AVA) were administered after animals had been exposed to aerosolized anthrax spores. Their findings suggested that postexposure vaccination together with penicillin successfully protected animals after challenge with roughly 7.5 × 105

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The Anthrax Vaccine: Is it Safe? Does it Work? spores (15 times the amount expected to kill half of the animals), whereas penicillin given alone for 5 or 10 days did not. Postexposure vaccination alone was not evaluated. The investigators estimated that 15 to 50 percent of the spores were still in the lungs of the animals 42 days after exposure and that traces of the spores remained even 100 days after exposure. Friedlander and colleagues (1993) carried out similar studies with penicillin, ciprofloxacin, doxycycline, and AVA. Rhesus monkeys were exposed by inhalation to an initial challenge of roughly 4 × 105 spores. Animals given vaccine alone were not protected. Animals given antibiotics for 30 days were well protected during the time of treatment, but after antibiotics were discontinued in the group receiving antibiotics alone, 10 to 30 percent of the animals succumbed to anthrax. None of the animals treated with doxycycline for 30 days plus vaccination died of anthrax. The surviving animals treated with antibiotics alone did not have evidence of an antibody response to PA, whereas vaccinated animals made antibodies. The survivors from the first experiment were rechallenged with roughly 3 × 106 spores (about 50 times the amount expected to kill half of the animals). Only those that had been vaccinated were significantly protected. Administration of antibiotics for 30 days after exposure to aerosolized spores provided protection (70 to 90 percent survival) after antibiotics were discontinued. However, the data also indicated that spores can persist for long periods (up to 58 days in one animal). The protection offered by use of the combination of vaccination and antibiotics was complete but was not statistically different from that offered by the use of antibiotics only. Taken together, these limited data suggest that the use of vaccine in combination with an appropriate antibiotic for 30 days could provide excellent postexposure protection against inhalational anthrax. Although the additional benefit from receiving the vaccine after a prolonged period of antibiotic use is not proven, reliance on the vaccine alone after exposure is clearly insufficient, as some protection is needed during the time required for an immune response to develop. Additional studies on the postexposure use of AVA with antibiotics are needed. Recommendation: The Department of Defense should pursue or support additional research with laboratory animals on the efficacy of AVA in combination with antibiotics administered following inhalational exposure to anthrax spores. Studies should focus on establishment of an appropriate duration for antibiotic prophylaxis after vaccine administration. CONCLUSIONS REGARDING EFFICACY A vaccine similar to the licensed vaccine, AVA, was shown to be effective against cutaneous anthrax in humans in the field trial supporting the

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The Anthrax Vaccine: Is it Safe? Does it Work? original application for licensure of AVA (Brachman et al., 1962). Although that study had too few cases to evaluate the vaccine’s efficacy for the prevention of inhalational disease, the five inhalational cases observed occurred only among nonvaccinated or placebo recipients, whereas none occurred among vaccinated workers. Data from CDC on cases reported between 1962 and 1974 also indicated that the vaccine offered protection against the cutaneous form of the disease (FDA, 1985). Furthermore, laboratory experiments indicate that AVA provides effective protection against inhalational challenge in rabbits and macaques, the animal models in which the disease is most reflective of the disease in humans (Fellows et al., 2001; Ivins et al., 1996, 1998; Pitt et al., 2001). Again, such efficacy is relative. When macaques were exposed experimentally to doses of up to about 900 times the amount expected to kill half of the animals, 88 to 100 percent of the animals were protected, as described earlier. This can be considered very effective protection. However, simulation studies conducted by Canadian researchers suggest that a person opening a letter filled with anthrax spores and standing over it for 10 minutes could inhale up to 3,000 times, and perhaps as much as 9,000 times, the amount of spores expected to kill half of a group of exposed people (Brown, 2001, 2002). Exposures to anthrax spores released in public places or in most military encounters might be expected to be lower. Without experimental data from extremely high challenge doses, it is difficult to anticipate the potential limits of the vaccine’s efficacy. For this reason the committee has recommended studies of vaccine protection as a function of challenge dose. Because PA is critical to the virulence of B. anthracis and because PA’s structure is so highly conserved, it appears likely that changing its structure would alter and thus eliminate its toxic action. Thus, it is unlikely that either naturally occurring anthrax strains or strains with bioengineered PA could both evade AVA and cause the toxicity associated with anthrax. Data from studies with animals suggest that AVA will offer protection against strains with PA-based toxicity. Finally, the available data indicate that immunity to anthrax is associated with the presence of antibodies to PA, such as those stimulated by the anthrax vaccine. Finding: The committee finds that the available evidence from studies with humans and animals, coupled with reasonable assumptions of analogy, shows that AVA as licensed is an effective vaccine for the protection of humans against anthrax, including inhalational anthrax, caused by any known or plausible engineered strains of B. anthracis.

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