NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.

Institute of Medicine (US) Committee on Health Effects Associated with Exposures During the Gulf War; Fulco CE, Liverman CT, Sox HC, editors. Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines. Washington (DC): National Academies Press (US); 2000.

Cover of Gulf War and Health

Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines.

Show details

7Vaccines

The U.S. military implements a comprehensive immunization1 program that is designed to protect the armed forces against potential disease risks (Takafuji and Russell, 1990). A standard set of vaccinations is required for each military recruit; this set varies slightly by branch of service. Additionally, when troops are assigned to specific duty stations they are given the vaccinations that are targeted to protect them from the risks found in their assigned geographic locale or that are specifically related to their assignment (IOM, 1996).

During the Gulf War, a number of different immunobiologics (e.g., cholera, meningitis, rabies, tetanus, and typhoid vaccines) were sent to protect against potential exposures to biological threats (Committee on Veterans' Affairs, 1998). Concerns prior to the Gulf War regarding Iraq's offensive biological warfare capabilities, led to decisions that available vaccines should be utilized as preventive measures against biological warfare agents. It is estimated that 310,680 doses of the anthrax vaccine licensed by the Food and Drug Administration (FDA) were distributed to the Gulf War theatre and that 150,000 U.S. troops received at least one anthrax vaccination (Christopher et al., 1997; Committee on Veterans' Affairs, 1998).

Approximately 137,850 doses of botulinum toxoid were sent to the Gulf, and it is estimated that 8,000 individuals were vaccinated (Committee on Veterans' Affairs, 1998). However, medical records from the Gulf War contain little or no information about who received vaccines, how frequently vaccines were administered, or the timing of vaccinations relative to the other putative exposures (OSAGWI, 1999). Further, existing record entries show no consistency in recording the type of vaccine (notations include “A-Vax,” “Vacc A,” “Vacc B,” and “B Vaccination”). A report by the Office of the Special Assistant for Gulf War Illnesses (OSAGWI) found that documents from the Gulf War indicate confusion about where, or whether, the vaccinations were to be recorded (OSAGWI, 1999).

Investigations since the war by the United Nations Special Commission (UNSCOM) and the International Atomic Energy Agency have found that Iraq had biological weapons prior to the Gulf War, but no evidence was found of their release. Investigators found that Iraq had produced 200 biological bombs in 1990; 100 were filled with botulinum toxin, 50 with anthrax, and 7 with aflatoxin (Zilinskas, 1997). Additionally, 13 Al Hussein (SCUD) warheads were found to have contained botulinum toxin, 10 warheads contained anthrax, and 2 contained aflatoxin (USAMRIID, 1996).

This chapter discusses several vaccine-related issues that have been of particular concern to Gulf War veterans. The chapter discusses animal and human studies that have been conducted on the safety of the anthrax vaccine and the botulinum toxoid vaccine. Additionally, the issue of multiple vaccinations is addressed. Finally, the chapter provides an overview of the scientific literature regarding squalene, an issue the committee was asked to address.

The committee issued a letter report on the safety of the anthrax vaccine in April 2000 (IOM, 2000). This letter report was issued in response to a congressional conference report (House Report 106-371). The Institute of Medicine (IOM) is currently conducting a separate two-year study on the safety and efficacy of the anthrax vaccine. That study will review some of the unpublished non-peer-reviewed information that was not available to this committee.

ISSUES IN IDENTIFYING ADVERSE EFFECTS

Vaccines are acknowledged to be one of the most effective tools in the prevention of infectious diseases. Dramatic reductions have been seen in the incidence of many diseases including pertussis, polio, rubella, measles, diphtheria, and mumps in the United States, and globally, smallpox has been eradicated (Keusch and Bart, 1998). In general, individuals experience either no adverse effects from a vaccination or mild local effects (e.g., tenderness, soreness) at the injection site. The administration of some vaccines has been determined to be associated with the potential for transient local or systemic adverse health outcomes (e.g., increased risk of fever, local pain, and/or swelling near the injection site) (Keusch and Bart, 1998). More serious reactions are rare (IOM, 1994). This section highlights some of the major issues that must be considered in determining whether an adverse health outcome is associated with receiving a vaccine. Several Institute of Medicine reports (IOM, 1991, 1994, 1997) have examined the complex issues involved in vaccine safety in greater depth.

Surveillance

Postmarketing surveillance of licensed vaccines in the United States relies on the voluntary reporting of adverse events. In 1990, the Vaccine Adverse Event Reporting System (VAERS) became operational and is overseen jointly by the Centers for Disease Control and Prevention (CDC) and the Food and Drug Administration. VAERS reports are open-ended forms that allow for description of the symptoms, time course, laboratory tests, vaccine(s) received, and treatment provided. While health care providers and manufacturers are obligated to report specific adverse effects to vaccines covered by the National Vaccine Injury Compensation Program, anyone can file a VAERS report. For the 65,720 VAERS reports received between January 1, 1991, and December 31, 1996, the sources of reports were health care providers (47.3 percent), manufacturers (39 percent), parents (2.4 percent), and others or unknown (11.3 percent) (CDC, 1999b). There is no long-term follow-up mechanism for VAERS reports.

VAERS is a passive reporting system in that it relies on incoming reports. Adverse events are therefore likely to be underreported (IOM, 1997). Further, some reports have incomplete medical information, and the same case may be reported by different sources. VAERS data are useful in signaling potential new adverse events but are limited in their usefulness for assessing the rate or causality of adverse events (IOM, 1994). Although the number of doses distributed is usually available, the number of doses administered is not. Further, the extent of underreporting of adverse events is unknown. FDA and CDC are responsible for monitoring VAERS data to detect unusual trends and occurrences of adverse health effects. This monitoring assists FDA and CDC in responding appropriately to adverse events.

Studies of vaccine safety use either active or passive methods of surveillance in assessing the extent of adverse events. Active surveillance methods involve direct follow-up by investigators of all individuals in the study. At a minimum, active surveillance seeks to systematically contact all vaccine recipients at prespecified intervals following vaccination. Often, in addition to posing open-ended questions about possible adverse effects, active surveillance asks explicitly about specific symptoms, and sometimes specific physical or laboratory examinations are conducted. Passive surveillance methods rely on the vaccine recipient to provide information (e.g., self-reports, surveys) or use other information that may indicate adverse outcomes (e.g., days missed from work, number of visits to the clinic following vaccination). Studies on the botulinum toxoid and anthrax vaccines have relied primarily on passive surveillance approaches and have involved only relatively short periods of follow-up.

Difficulties in Detecting Adverse Events Due to Vaccinations in Humans

Detecting adverse events associated with vaccination and determining whether the health outcome is a result of the vaccination are complex tasks due to a number of factors (IOM, 1997) including the following:

  • Lack of long-term follow-up. Many controlled studies are geared toward monitoring immediate reactions to the administration of the vaccine; subjects are often followed for 6 months at the most.
  • Small sample sizes. Vaccine trials to determine immunogenicity often involve sample sizes of no more than several hundred individuals. Trials of this size are unlikely to detect rare effects since large sample sizes are needed to detect rare occurrences.
  • Multiple vaccinations. Individuals often receive several vaccines at a time or over a short period, which makes it difficult to identify the culprit vaccine in the event of an adverse effect. Controlled safety trials of vaccine combinations would have to include as many study groups as there are combinations of vaccines under study, plus at least one reference group, and thus would require large sample sizes.
  • Multiple end points. The large number of symptoms potentially associated with vaccination complicates surveillance because the reporting mechanism must allow for numerous symptom categories in addition to as-yet-unreported symptom types.
  • Lack of symptoms specific to vaccination. Since there is no unique clinical syndrome or laboratory diagnosis associated with vaccination, it is difficult to differentiate whether symptoms, such as fatigue or seizures, are due to receiving the vaccine or to some unrelated factor coincident with vaccination.
  • Passive reporting systems. Passive surveillance systems are most useful as a sentinel for identifying rare or previously unrecognized side effects of newly marketed vaccines and for monitoring the safety of individual vaccine lots. However, these systems do not provide information about the rates of reactions to vaccines. As discussed above, VAERS is a passive system that relies on health care providers, those receiving vaccinations, and others to report health outcomes that may be linked to vaccine exposure in the recent or more distant past. Reporting is likely to depend on the gravity of the effect, the time lapsed since exposure, and the diligence in symptom reporting by the patient's health care workers. Thus, underreporting is an inherent issue. Furthermore, supporting information (e.g., laboratory results) to infer causality may be inaccurate or missing.
  • High vaccination rates. For widely administered vaccines, it is difficult to find a comparable control group that has not received the vaccine. Unvaccinated individuals constitute a small, highly selected group that may differ from those vaccinated in other aspects and, thus, are not generally suitable as a control group. Further, their small number is unlikely to allow for the study of background rates of rare medical events.
  • Restricted population. The large majority of controlled vaccine trials are geared toward investigating childhood vaccines. Adverse effects in children may not be generalizable to adults.
  • Progress in vaccine technology. Earlier vaccines against a particular infectious agent that have been subjected to considerable animal and human study may be substantially different from vaccines currently in use against the same infectious agent. Thus, even careful and extensive earlier studies may not be generalizable to current experience.

Difficulties in Detecting Adverse Events Due to Vaccinations in Animals

Focus on Vaccine Efficacy

Most animal studies focus on the efficacy of the vaccine and do not examine adverse effects. Further, adverse effects that produce symptoms, rather than objectively measurable pathology, are difficult or impossible to study in animals (some studies use animal behavior to infer animal symptoms such as fatigue). The lack of data on adverse events in animal studies can indicate that no adverse events occurred, that adverse events were not monitored, or that adverse events were not sufficiently severe to warrant termination of the experiment. Additionally, most animal studies are concerned with monitoring immediate toxicity to the administration of the vaccine. Animals are most often followed for short periods of time (i.e., weeks to months), and the long-term effects of vaccination are not considered.

Possibility of Immune Stimulation

Studies in animals have generally not considered the mechanism responsible for adverse health effects. In some cases, adverse effects of the vaccination could be due to the toxicity of the antigen in the vaccine, the preservatives or contaminants in the vaccine, or the vaccine adjuvant.2 Adverse effects may also result from the intended goal of immunization (i.e., stimulation of the immune system). Immune stimulation may result in a state of immune enhancement, hypersensitivity, or an immune-mediated pathological response. The pathological immune response may be directed toward the antigens administered in the vaccine or to self-antigens (i.e., autoimmunity). Immune-mediated tissue damage requires an initial exposure to the antigen to sensitize the animal. The symptoms of immune-mediated tissue damage may occur on subsequent exposures.

A discussion of the immunological reactions that can cause disease has been included in a previous IOM (1994) report and is summarized only briefly here. Classically, such immune-mediated pathology is divided into Types I–IV hypersensitivity as proposed by Coombs and Gell (1968). However, the response to any one antigen may involve a combination of types of hypersensitivity, depending on the antigen dose, site of exposure, and duration of antigen stimulation. Type I hypersensitivity is a response to the antigen that occurs within minutes; symptoms range from a mild rash or urticaria to airway obstruction or acute life-threatening anaphylactic shock. In Type II reactions, antibodies combine with a tissue antigen, resulting in complement system activation and damage to the tissue by the inflammatory process. Drug-induced hemolytic anemia is an example of a Type II hypersensitivity reaction. Type III hypersensitivity involves the interaction of circulating antibody and antigen to form immune complexes that deposit on the walls of blood vessels. The resultant fixation of complement and neutrophil recruitment leads to tissue destruction. The pathology of Type III hypersensitivity tends to be seen in the lung, kidney, joints, and brain in animal studies. A localized reaction in the skin can lead to pain, swelling, induration, and edema. Type IV hypersensitivity or delayed-type hypersensitivity is dependent on the stimulation of antigen-specific lymphocytes and recruitment of macrophages by cytokines. The resultant inflammation leads to tissue destruction. Contact dermatitis to poison ivy is an example of a Type IV hypersensitivity reaction. Animal studies have limitations in detecting adverse effects due to Types II through IV hypersensitivity because the time course of such responses may involve months or years to become clinically apparent in an animal, which is beyond the time frame monitored in most animal studies.

Genetic inheritance strongly influences the immune response, both to immunization and to actual infection (Box 7.1), in animals and humans, which explains why immunologically mediated adverse reactions to vaccination are so variable from one animal, or person, to the next.

Box Icon

Box 7.1

Genetics and the Immune Response. Genetic factors can influence the host's response, including the immune system's response, to foreign antigens in many ways—for example, metabolism of the antigen, antigen processing, alteration of self-antigens, (more...)

ANTHRAX VACCINE

Work on a vaccine to provide protection against the zoonotic disease anthrax3 began with the work of Pasteur and Greenfield who developed heat-attenuated anthrax vaccines in the 1880s (Turnbull, 1991). In the 1930s, Sterne developed a live attenuated spore vaccine, and versions of this vaccine continue to be used effectively to immunize livestock. The primary use of the anthrax vaccine in humans was initially to protect persons working with animal hair or hides, including goat hair mill workers, tannery workers, and veterinarians.

Currently three anthrax vaccines are commercially available for human use. A live attenuated spore vaccine for humans was developed in the 1940s from a Sterne strain derivative and has been tested and used on a large scale in humans in the countries of the former Soviet Union (Shlyakhov and Rubinstein, 1994a). The British and U.S. anthrax vaccines were developed in the 1950s using filtrates of anthrax strains. Protective antigen, one of the three toxin proteins (discussed below), produced by the anthrax bacillus is the immunogenic component of both the U.S. and the U.K. vaccines. The British vaccine is an alum-precipitated cell-free filtrate of an attenuated Sterne strain culture and was licensed in 1979 (Pile et al., 1998).4 The U.S. vaccine is an aluminum hydroxide-adsorbed cell-free culture filtrate of an unencapsulated strain (Pile et al., 1998).

The anthrax vaccine was first produced on a large scale in the United States by Merck, Sharp, and Dohme in the 1950s for Fort Detrick (GAO, 1999c). Production was turned over to the Michigan Department of Public Health (MDPH) in the 1960s, and some changes were made in the manufacturing process; a different strain of anthrax was used in the MDPH vaccine, and the yield of protective antigen was increased (GAO, 1999c). In 1966, the Investigational New Drug (IND) application was submitted to the Division of Biologic Standards (DBS), formerly in the National Institutes of Health (NIH). Product licensure for Anthrax Vaccine Adsorbed was granted on November 10, 1970. The safety study of the anthrax vaccine submitted to the DBS contained information on the administration of approximately 16,000 doses. In 1985, an FDA advisory panel reviewing the status of bacterial vaccines and toxoids categorized the anthrax vaccine in Category 1 (safe, effective, and not misbranded) (FDA, 1985).

In December 1997, the Secretary of Defense announced that all U.S. military forces would receive anthrax vaccinations for protection against the threat of biological warfare. The Anthrax Vaccine Immunization Program (AVIP) began vaccinations in March 1998; the first personnel vaccinated were members of units deployed or scheduled to deploy to high-threat areas (Claypool, 1999).

It is estimated that 68,000 doses of the U.S. anthrax vaccine were distributed from 1974 to 1989; 268,000 doses in 1990; and 1.2 million doses from 1991 to July 1999 (Ellenberg, 1999). The exact number of people who received the vaccine is not known. The current dosing schedule is 0.5 ml administered subcutaneously at 0, 2, and 4 weeks and 6, 12, and 18 months, followed by yearly boosters. BioPort Corporation (previously Michigan Biologic Products Institute, formerly MDPH) manufactures the U.S. vaccine, approved for use in men and women age 18 to 65 years. The vaccine contains no more than 2.4 mg aluminum hydroxide per 0.5-ml dose as an adjuvant, formaldehyde as a stabilizer (final concentration ≤ 0.02 percent), and benzethonium chloride (0.0025 percent) as a stabilizer (BioPort, 1999; Friedlander et al., 1999).

The length of the dosage schedule, along with questions about the extent of the efficacy of the current vaccine against newly engineered strains of anthrax, has led to ongoing research efforts to produce a second-generation recombinant vaccine (Ibrahim et al., 1999; Nass, 1999). Additionally, researchers hope that new processes will be designed to ensure a more precise amount and a more highly purified component of protective antigen in the vaccine (GAO, 1999b; Russell, 1999).

Toxicology

Anthrax disease results from exposure to the bacterium Bacillus anthracis through three primary routes: cutaneous, inhalation, and gastrointestinal. Regardless of the route of exposure, the presence of the organism provokes an immune response. Both humoral and cell-mediated immunity play a role in defending against B. anthracis (Turnbull et al., 1986; Shlyakhov and Rubinstein, 1994b). An individual who has recovered from B. anthracis infection is protected against a subsequent infection with the same organism. Some studies have correlated protective immunity in animals with the antibody response to B. anthracis (Barnard and Friedlander, 1999), but other studies have not confirmed this finding (Little and Knudson, 1986; Turnbull et al., 1986).

Knowledge of the pathogenic mechanisms of Bacillus anthracis can provide insight into the potential adverse effects associated with administration of the various anthrax vaccines (Friedlander, 1997; Ibrahim et al., 1999). B. anthracis is pathogenic by virtue of its capsule and protein exotoxins. The capsule of the bacillus is encoded by an extrachromosomal plasmid pX02 (Little and Knudson, 1986). Another plasmid (pX01) encodes for all three toxin proteins: edema factor (EF), lethal factor (LF), and protective antigen (PA). PA, the transport protein, is required for transport of the enzymatic proteins EF and LF into the target cells of the host; PA must be present for the toxins to confer virulence (Ibrahim et al., 1999). In vitro studies of the toxins have revealed that PA binds to cells and undergoes limited proteolysis, which exposes a potential binding site for LF and EF. The LF–PA and EF–PA complexes enter the target cell by receptor-mediated endocytosis, followed by translocation of LF or EF to the cytosol (Friedlander, 1986; Leppla et al., 1990). The edema toxin complex, composed of EF and PA, acts through calmodulin-dependent adenylate cyclase activity to cause the excessive fluid accumulation that is associated with anthrax infection (Leppla, 1982; Ibrahim et al., 1999). The lethal toxin complex, composed of LF and PA, is the primary cause of shock and death (Ibrahim et al., 1999). Lethal toxin is a zinc metallopeptidase that is rapidly cytolytic for macrophages in vitro and induces the release of the cytokine tumor necrosis factor (TNF) from macrophages (Hanna et al., 1993). Studies in mice indicate that TNF and interleukin-1, in particular, contribute to the death induced by injection of lethal toxin (Friedlander, 1986).

Mechanism of Action

Live attenuated spore vaccines. Live spore vaccines used in veterinary practice, as well as the Soviet Sterne live spore vaccine used in humans, are (pX01+, pX02−) unencapsulated strains of B. anthracis. These vaccines are administered intramuscularly, subcutaneously, or by scarification. The host mounts an immune response to the organism and its toxin proteins. Live spore vaccines induce a humoral immune response. However, the live spore vaccine also elicits a cell-mediated immune response (Shlyakhov and Rubinstein, 1994b). The absence of the capsule reduces the virulence of the organism, yet the bacillus can still produce the toxin proteins PA, EF, and LF. Thus, the formation of active edema toxin and lethal toxin is possible.

Protective antigen vaccines. The U.K. and U.S. vaccines for humans are alum-precipitated cell-free filtrates of Bacillus anthracis. In the case of the U.S. vaccine, this precipitate is adsorbed onto aluminum hydroxide. The aluminum hydroxide adjuvant is included in the vaccine preparation to boost the immune response to the PA. Aluminum hydroxide is used in many vaccines and is thought to stimulate humoral rather than cell-mediated immunity (Ivins et al., 1998). The culture filtrates are processed to maximize the content of PA. The cell-free filtrate is primarily PA but also contains EF, LF, and other contaminants from culture (Ivins et al., 1998; Miller et al., 1998). PA vaccine for humans also elicited antibody production to EF and LF in rats and guinea pigs (Ivins et al., 1986; Turnbull et al., 1986; Ivins, 1988), suggesting that the contamination is sufficient to elicit a biological response.

The primary goal of anthrax vaccination is to produce neutralizing antibodies to PA. Subsequent exposure to anthrax infection would then eliminate the pathogenic potential of B. anthracis by eliciting the production of antibodies that neutralize PA. Without PA, EF and LF are incapable of acting as virulence factors. Barnard and Friedlander (1999) vaccinated guinea pigs with several different live recombinant Bacillus anthracis strains (pX01−, pX02−) that each produced a different amount of PA without producing the capsule, EF, or LF. The protective effect of these strains correlated with the production of PA and with the anti-PA antibody titer elicited in vivo. Studies by Turnbull and colleagues (1988) and Ivins (1988) in guinea pigs have provided evidence that PA is an essential component of the vaccine and that protection against anthrax in guinea pigs is possible in the absence of any detectable antibody to LF or EF. However, some studies have suggested that the protective effect of anthrax vaccine does not necessarily correlate with the antibody titer to PA in vivo. Studies by Little and Knudson (1986) indicated that a high titer to PA did not necessarily reflect the level of expected protection from infection. Studies by Turnbull and colleagues (1988) suggested that it is also important for the PA antigen to be presented to the immune system in such a way as to stimulate more than just a humoral immune response. Challenge tests with aerosol anthrax spores have shown that the Sterne live spore vaccine was more efficacious than PA-based vaccines (Ivins, 1988), suggesting that cellular immunity as well as humoral immunity is important for protection against anthrax infection.

Animal Studies

As noted earlier in this chapter, adverse health outcomes in animals after injection may result from the toxic effects of the injected substances or from stimulation of the immune system. Injection of the live spore vaccine can cause infection. Injection of protective antigen vaccines can result in adverse effects associated with administration of the exotoxins.

Live attenuated spore vaccines. Studies in veterinary use. Many of the studies using the live spore vaccine have involved large-scale vaccination of animals of economic importance. Avirulent, nonencapsulated strains of B. anthracis, including the Sterne live spore vaccine, have been used for vaccination. In general, these studies have relied on anecdotal reports from farmers and veterinarians to measure the incidence of reactions of livestock (primarily horses, cows, calves, sheep, lambs, and goats) to the vaccination. In some cases, data on the number of animals vaccinated and the number of deaths from anthrax have been collected by survey or from veterinarian reports.

In most cases, the primary focus has been on evaluating the efficacy of the vaccine, rather than on monitoring adverse effects; therefore, many studies of veterinary use of the live spore vaccine have not commented on adverse effects associated with vaccination (Sterne et al., 1942; Kaufmann et al., 1973; Salmon and Ferrier, 1992).

The primary health outcomes in animal studies are edema at the site of injection, a febrile response, or death. The extent of the edema ranged from no reaction, to mild irritation, to lameness in some animals. Edema is due to elaboration of the edema toxin or to an allergic response to a previously administered vaccine. An early study by Sterne (1939) used a retrospective questionnaire to solicit complaints regarding the effectiveness and adverse effects of vaccinating sheep and cattle with live spore vaccines. With a limited response to the questionnaire, the majority of reports for cattle indicated that no reactions occurred. However, some cattle experienced lameness and transient decreases in milk yield. Some animals showed severe swelling at the site of injection and one death occurred. Kolksov and Mikhailov (1959) described either insignificant or mild reactions in the majority of 650,000 animals that were vaccinated. Some horses and cattle had swelling at the injection site measuring 12–40 cm2 and lasting 3 to 4 days, along with a slight temperature rise. Of the 650,000 animals (including cattle, horses, oxen, sheep, and goats) 20 animals were reported as dying from unspecified causes. In another study of the Sterne live spore vaccine, it was noted that three of the 34,000 cattle, horses, mules, hogs, and sheep receiving the vaccine experienced instances of excessive swelling (Lindley, 1963). Increased temperature of the animals, lasting for days, has been observed in other studies (Kolksov and Mikhailov, 1959; Kolosov and Borisovich, 1968; Kolesov et al., 1968). Tanner and colleagues (1978) reported a febrile period lasting less than 24 hours in 12 of 49 vaccinated cows but no change in daily food consumption. In addition, decreased activity or decreased milk production has been noted and is presumed to be due to the presence of inflammation. Studies of live spore vaccine in veterinary use are primarily descriptive, so the actual incidence of adverse reactions to the vaccine is not known. The committee did not find any long-term studies (greater than a year) that monitored adverse effects from vaccination with the live spore vaccine.

Studies in laboratory animals. Studies with the live spore vaccine in laboratory animals have been conducted under better controlled conditions than those in veterinary practice. Many of these studies make no mention of adverse effects in guinea pigs, hamsters, rabbits, or mice (Klein et al., 1962; Jaiswal and Mittal, 1979; Ezzell and Abshire, 1988; Turnbull et al., 1988; Stepanov et al., 1996). Small laboratory animals such as rabbits, guinea pigs, and mice are more susceptible than larger animals to dying from administration of the live spore vaccine (Welkos, 1987; Welkos and Friedlander, 1988; Ivins et al., 1990). High doses of the live spore vaccine killed one-third of the guinea pigs studied by Turnbull and colleagues (1986, 1988) and up to 60 percent of the guinea pigs in a study by Klein and colleagues (1962).

A thorough study by Gusman and Migulina (1967) histologically examined rabbits and guinea pigs immunized with live anthrax spore vaccines by subcutaneous injection. They monitored tissue from internal organs as well as from the site of administration of the vaccine for up to 210 days after vaccination. Edema occurred at the site of the injection, sometimes with hemorrhage and abscess formation. Dilation of the blood vessels and infiltration of the site with segmented white blood cells also occurred. Edema and inflammation lasted for 14 days, followed by the formation of granulation tissue. Over a 2-month period, researchers noted changes in the lymphoid organs, consistent with a response to an antigen. In addition, histological changes were evident in the liver and heart muscle but resolved within 2 months.

Vaccination with live spore vaccine may also lead to complications of otherwise symptomless infections or to death from anthrax when the animal is subject to trauma. Kolesov and Gutiman (1968) and Stefanova (1968) noted that rabbits injected with live spore vaccine would sometimes die. They confirmed by autopsy and bacteriological examination that the rabbits died from pasteurellosis infection, not from anthrax. Thus, vaccination with the live spore anthrax vaccine may activate an underlying infection in a rabbit that was in satisfactory health prior to vaccination. In a similar manner, death from anthrax may occur more readily in an animal whose health is compromised. Stefanova (1968) found that rabbits subjected to the trauma of an ear biopsy after vaccination with the live spore vaccine were more likely to die than animals not subject to the trauma.

Guinea pigs vaccinated with the live spore vaccine have delayed-type hypersensitivity reactions 1 year after vaccination (Shlyakhov, 1970; Shlyakhov and Rubinstein, 1994b), indicating stimulation of the cellular immune response. These studies have employed anthraxin, an incompletely defined antigen used for skin testing. A positive delayed-type hypersensitivity reaction to anthraxin was associated with hyperemia of 64 mm2 and a twofold thickening of the skin.

Protective antigen-based vaccines. Protective antigen vaccines have had little use in veterinary practice but have been tested in laboratory animals either with or without adjuvant. The primary goal of these studies has been to determine the efficacy of injections of PA, with or without adjuvant, in protecting against infection with Bacillus anthracis. In addition, the studies have attempted to correlate the presence of antibodies with the degree of protection afforded by the vaccination.

Most studies in laboratory animals with the protective antigen vaccine have not mentioned adverse effects associated with vaccination. Many studies conducted in guinea pigs employed different vaccination regimens using culture filtrates of PA with and without alum adjuvant. Reports of these studies did not mention adverse effects (DeArmon et al., 1961; Klein et al., 1961; Puziss and Wright, 1963; Gulrajani et al., 1968; Little and Knudson, 1986; Ezzell and Abshire, 1988; Ivins, 1988; Ivins et al., 1986, 1989, 1990, 1993, 1994, 1995; McBride et al., 1998). In addition, reports of studies with similar vaccinations in mice and rabbits did not mention adverse events after vaccination (Wright et al., 1954; DeArmon et al., 1961; Puziss and Wright, 1963; Gulrajani et al., 1968; Ivins et al., 1990). The lack of data in these instances can indicate that no adverse effects occurred, that adverse events were not monitored, or that adverse events were not sufficiently severe to warrant termination of the experiment. The primary purpose of most of the studies was to evaluate the effectiveness of the vaccine against Bacillus anthracis infection. In general, most of the studies monitored animals for 1 to 2 months. A few studies extended to 1 or 2 years.

In a study by Wright and coworkers (1954), 25 rabbits received five 0.5-ml intracutaneous injections of anthrax vaccine on alternate days. The rabbits were sacrificed 23 days later. Complete autopsies, including gross and microscopic examination of all organs, revealed no adverse effects. Limited studies have also been conducted in nonhuman primates. A study in rhesus monkeys using the licensed anthrax vaccine revealed no remarkable local or systemic reactions (Ivins et al., 1998). Darlow and colleagues (1956) vaccinated 30 rhesus monkeys with the alum precipitate of the PA antigen and found no evidence of toxicity in the 10 animals that were monitored for 2 years. In this study, one control and one vaccinated animal developed a transitory illness and recovered within 3 days. The authors attributed the illness to a transitory infection, unrelated to the anthrax study. Three of the immunized animals monitored for the 2 years were reported to have died from other causes during the experimental period. Darlow also found that 50 ml of the vaccine preparation injected intravenously into rabbits resulted in no deaths and no apparent adverse effects. Wright and coworkers (1954) injected five monkeys with the PA antigen and did not mention any adverse effects. A booster injection given 3 months later did not cause significant local reactions or lesions observable on autopsy 3 weeks later.

Recombinant protective antigen-based vaccines. No adverse effects were reported after vaccination of mice or guinea pigs with either Bacillus subtilis expressing recombinant PA (Welkos, 1987; Welkos and Friedlander, 1988; Miller et al., 1998) or DNA encoding for PA (Gu et al., 1999). Using recombinant techniques, Singh and colleagues (1998) generated a noncleavable PA mutant that bound to the receptor with an affinity equal to that of native PA but failed to bind LF or EF. The authors did not comment on adverse effects when they used the mutant PA to vaccinate guinea pigs (Singh et al., 1998).

Protective antigen-based vaccines with miscellaneous adjuvants. In an attempt to increase the effectiveness of PA vaccines, investigators have employed different adjuvant preparations in combination with PA. Ivins and coworkers (1992) immunized mice with PA and a bacterial cell wall adjuvant preparation. This combination occasionally resulted in a small, nonnecrotic granuloma. A later study by Ivins and colleagues (1995) used many combinations of adjuvants with PA. The authors reported adverse effects only when they used purified PA combined with all of the following: squalene, Tween, Pluronic block copolymer L121, and threonyl muramyl dipeptide. Five of the twenty animals immunized with this combination died within 1 to 5 days. The cause of death was unknown, but significant vascular congestion in multiple organs suggested that the animals suffered from shock and cardiovascular collapse. Other adjuvants (including other combinations that included squalene) provided adequate protection and did not elicit adverse reactions.

Conclusions on Animal Studies

Few meaningful conclusions for humans can be drawn from animal studies of the anthrax vaccine. Many of the animal studies have used the anthrax live spore vaccine and therefore, have limited applicability for evaluating the toxicity of protective antigen vaccines. Additionally, animal studies using the PA vaccine typically have reported only on short-term local reactions to injection of the vaccine. Further, most of the studies do not indicate whether the authors monitored for adverse consequences of vaccination.

Human Studies

The committee used only the peer-reviewed literature to form its conclusions on the weight of the evidence for associations of the anthrax vaccine with adverse health effects. Only a few published peer-reviewed studies have examined potential adverse effects of the anthrax vaccine when administered to humans. In considering the need for future research, the committee evaluated other studies in addition to peer-reviewed publications.

Live Attenuated Spore Vaccine Studies

The committee examined information on the human studies and extensive field trials conducted in the Soviet republics from the 1940s to the 1970s (described in Shlyakhov and Rubinstein, 1994a). These Soviet studies used the live spore anthrax vaccine, which differs substantially from the protective antigen anthrax vaccines used in the United States and the United Kingdom, in terms of composition, reactogenicity, and potential residual virulence. Moreover, the Soviet studies performed neither passive nor active long-term monitoring. For these reasons, the committee did not include the live spore vaccine studies in its analysis.

The committee notes a recent literature review on anthrax vaccine studies (Demicheli et al., 1998) conducted according to Cochrane Collaboration guidelines for systematic reviews of health care interventions. Only the Brachman study (described below) met the Cochrane criteria for prospective randomized or quasi-randomized studies of a protective antigen vaccine.5

Healthy-Volunteer Studies

During the development of the anthrax vaccine, several early studies examined adverse reactions in humans but did not provide detailed information on the nature of the monitoring for adverse effects. These studies used early versions of the culture filtrate (protective antigen) vaccine. Wright and colleagues (1954) described the reactions of 660 persons at Camp Detrick who received a total of 1,936 injections. They found that 0.7 percent of vaccinated subjects reported systemic reactions—typically consisting of mild muscle aches, headaches, and mild-to-moderate malaise lasting 1 to 2 days. Significant local reactions—typically swelling (5–10 cm in diameter) and local pruritus (itching)— occurred in 2.4 percent of the subjects. The incidence of local reactions increased with the number of injections.

In another study at Fort Detrick (Puziss and Wright, 1963), 0.5-ml injections of protective antigen led to similar results. The study reported low rates of erythema, edema, or pruritus at the site of injection (no details were provided) and no systemic reactions.

Darlow and colleagues in Great Britain (1956) reported on the administration of 1,057 injections of the anthrax vaccine to 373 individuals (369 persons received two or more injections) over a period of 4 years. Most of the reactions were mild and brief (local tenderness and swelling). There was an increase in the number of persons experiencing pain after the second dose, and local reactions increased with successive booster injections. The study reported that three people had brief and mild fever.

Brachman Study

Brachman and colleagues (1962) conducted the only randomized clinical trial of vaccination with a protective antigen anthrax vaccine. Although the vaccine used in this study was similar to the current vaccine (used to immunize Gulf War troops and currently available in the United States) in that it was a PA vaccine, the manufacturing process has since changed and a different strain of anthrax bacillus is now used (GAO, 1999c).

The clinical trial was conducted among 1,249 eligible workers6 at four goat hair processing mills in which some raw materials were contaminated by anthrax bacilli. Both cutaneous and airborne anthrax were endemic; approximately one case of anthrax occurred per 100 employees per year in these mills. After the initial series of three injections, the study had to be terminated at the largest mill, which employed nearly half of the subjects, because of an outbreak of inhalation anthrax that required the vaccination of all employees. At the remaining mills, 480 participants completed the series of injections (230 of whom were randomized to receive active vaccinations and 250 to receive placebo injections) and 81 participants did not complete the series.7 The study subjects did not know whether they had received the active vaccine or placebo; the article does not state whether the investigators were also blinded to the allocation.

The report of the study does not always clearly state whether the results in the three mills apply only to the 480 subjects who completed the vaccination series or also included results from the 81 subjects who did not complete the series. Neither does it state whether the results apply only to the 480 subjects in the three mills who completed the series or whether the results include the subjects from the largest mill who had been randomized, received the initial injections, and were partially evaluated prior to the decision to withdraw the mill's employees from the study.

Participants were examined 24 and 48 hours following each vaccination to assess both local and systemic reactions to the vaccine. There were no reports of subsequent active or passive surveillance for possible adverse effects beyond 48 hours after each vaccination (there was further monitoring for the vaccine's efficacy).

Of the 230 actively vaccinated subjects who completed the inoculations, one individual (0.4 percent) developed anthrax. Of the 250 individuals receiving placebo, 12 (4.8 percent) developed anthrax. The great majority of cases of anthrax were of the cutaneous type; there were not enough cases of inhalation anthrax to determine if vaccination was effective against this, the most lethal form of anthrax.

The typical reaction was described as a ring of erythema (1–2 cm in diameter) at the injection site, with local tenderness that lasted 24–48 hours. Some individuals (the authors did not report the number) reported more extensive edema, erythema (>5 cm in diameter), pruritus, induration, and/or small painless nodules at the injection site (lasting up to several weeks). Twenty-one persons had moderate local edema that lasted up to 48 hours. Three individuals had edema extending from the deltoid to the mid-forearm (in one case, to the wrist) that dissipated within 5 days. Systemic reactions occurred in two individuals (0.9 percent of the actively immunized subjects) who experienced “malaise” lasting 24 hours following vaccination. The study notes that three individuals who received the placebo (0.1 percent alum) had mild reactions.

Other Studies and Information

Several other studies, discussed below, had information on the safety of the anthrax vaccine, but these studies have not been published in the peer-reviewed literature and were not considered in the committee's conclusions regarding the strength of the evidence for associations with adverse health outcomes. Publication of these studies would substantially increase the body of information needed to form conclusions regarding health effects of the anthrax vaccine. Most of the studies are currently described only in secondary sources (e.g., reviews, congressional testimony, General Accounting Office [GAO] reports). Additionally, a recently published article in the CDC Morbidity and Mortality Weekly Report (CDC, 2000) provided summary results of several of the studies. A few of the studies have only recently been completed or are ongoing.

Investigational new drug data. In the early 1970s, CDC submitted data on the safety of the anthrax vaccine in support of an application to license the vaccine. The committee did not have access to primary data but examined the information provided in secondary sources (Friedlander et al., 1999; GAO, 1999b,c,d). At the end of its study, after the committee had completed its work, the committee received the IND information that had been requested earlier through a FOIA (Freedom of Information Act) request. Another Institute of Medicine committee that is currently studying the safety and efficacy of the anthrax vaccine will be able to examine this information.

The IND data included information on the reactions of approximately 7,000 individuals who had received approximately 16,000 doses of the vaccine (four lots manufactured by MDPH). With active monitoring (there was no description of the monitoring methods), mild local reactions (≤3 cm) occurred in 3–20 percent of all doses, moderate local reactions (>3 to <12 cm) in 1–3 percent of all doses, and severe reactions (≥12 cm) in less than 1 percent of doses. Four individuals reported transient systemic reactions consisting of fever, chills, nausea, and body aches (Friedlander et al., 1999).

VAERS reports. As described earlier in this chapter, the Vaccine Adverse Event Reporting System is a passive surveillance system consisting of reports filed by health care providers, individuals receiving vaccinations, family members, or others. As noted earlier, VAERS data are useful as a sentinel for adverse events but are limited in their usefulness for assessing the rate of adverse events since underreporting is likely and the information may be incomplete or duplicative, or may not always have been confirmed by medical personnel (IOM, 1994).

The committee reviewed summaries of VAERS data but did not review the individual VAERS forms. From its inception in 1990 through July 1, 1999, there have been 215 VAERS reports regarding anthrax vaccination (Ellenberg, 1999). The majority of the reports describe local or systemic symptoms including injection site edema, injection site hypersensitivity, rash, headache, and fever. Twenty-two of the VAERS reports are considered serious events8 and were described as occurring (or being diagnosed) from 45 minutes to 4½ months after receiving the vaccination. Reports of serious events include five patients hospitalized with severe injection site reactions, one individual with a widespread allergic reaction, one individual with a case of aseptic meningitis 9 days after vaccination, two individuals who experienced Guillain-Barré syndrome, one individual diagnosed with bipolar disorder 3 weeks after receiving the vaccine, one individual with an onset of multi-focal inflammatory demyelinating disease, and one individual who experienced the onset of lupus (Ellenberg, 1999). In recent congressional testimony FDA stated, “None of these events, except for the injection site reactions, can be attributed to the vaccine with a high level of confidence, nor can contribution of the vaccine to the event reported be entirely ruled out…. [T]he reports on anthrax vaccine received thus far do not raise any specific concerns about the safety of the vaccine” (Ellenberg, 1999). An external review panel, the Anthrax Vaccine Expert Committee, has recently been established by the Department of Health and Human Services at the request of the Department of Defense (DoD) to review each VAERS report received regarding anthrax vaccination.

Special Immunization Program Safety Study. Since 1973, 1,590 workers at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) have received 10,451 doses of the anthrax vaccine (Claypool, 1999; Friedlander et al., 1999). Visits to the occupational health clinic were used as a method of collecting and passively monitoring information on adverse reactions. Four percent of doses resulted in a local reaction (redness, induration, itching, or edema) at the site of injection. Systemic reactions (headache, fever, chills, malaise, muscle and joint aches) occurred with 0.5 percent of doses. Individuals received annual physical exams. Diseases and unexplained symptoms occurred at a rate that would be expected in a comparable group (Claypool, 1999).

Fort Bragg Booster Study. Pittman and colleagues (1997) studied the frequency of possible vaccine-related adverse outcomes during an open-label study of DoD personnel who received anthrax and/or botulinum toxoid vaccines during Operation Desert Shield/Desert Storm in 1990–1991. The objectives of the study were to assess the persistence of antibodies to the vaccines, determine the serological response 30 days after receiving a booster dose, and evaluate the reactogenicity of the vaccines. All of the 486 subjects were male volunteers who had documented records of receiving one or more doses of the anthrax or botulinum vaccine during the Gulf War. Subjects received booster shots (in different arms) of either the anthrax or the botulinum toxoid vaccine, or both, depending on what they had received in 1990–1991. The report states that there was daily monitoring for systemic and local reactions but does not state the total duration of follow-up.

Approximately 20–25 percent of subjects complained of erythema, induration, or swelling at the site of the anthrax vaccine injection; about 3 percent described the reaction as severe. Fever (defined as an oral temperature of 100.5°F or greater) occurred in 2.8 percent of subjects. Systemic symptoms within the first 7 days after receiving the vaccine(s) occurred in 44 percent of study subjects. Symptoms included muscle aches (30 percent), headache (16.5 percent), feeling ill (16 percent), and rash (16 percent). Other symptoms included loss of appetite, difficulty breathing, joint aches, and nausea. The study reports that most of the symptoms were mild; however, 20 volunteers had severe symptoms (the authors did not define “severity”).

Canadian Armed Forces Study. A study monitored 547 individuals in the Canadian Armed Forces who received the anthrax vaccine in 1998 (Claypool, 1999; Friedlander et al., 1999). Mild local reactions occurred with 10.1 percent of doses, moderate local reactions occurred with 0.5 percent of doses, and there were no reports of severe local reactions. Systemic reactions occurred with 1.5 percent of doses (five individuals had fever, two had heartburn, one experienced a “transient nerve disorder”) (Claypool, 1999). Reactions were transient except for one individual who reported persistent nodules. The type of monitoring for adverse health effects was not described.

USAMRIID Reduced Dose and Route Change Study. A pilot study at USAMRIID compared the safety of three doses of anthrax vaccine delivered by subcutaneous injection at 0, 2, and 4 weeks (the current primary schedule and route) with two doses given subcutaneously, and with two intramuscular deltoid injections (Claypool, 1999); 173 people were studied. The incidence of systemic effects did not differ between the three groups: headache (14 percent), malaise (9 percent), loss of appetite (3 percent), nausea or vomiting (3 percent), muscle ache (3 percent), itchiness (3 percent), and low-grade fever (3 percent). Local reactions (e.g., redness and swelling at the injection site, subcutaneous nodules) occurred more frequently with subcutaneous than with intramuscular injections (5–7 percent). Male vaccine recipients reported local reactions less frequently after subcutaneous injections (5–32 percent) than female vaccine recipients (39–66 percent). The report did not describe the type of monitoring for adverse health effects.

Tripler Army Medical Center Survey. A self-administered questionnaire was used to collect data on 603 health care personnel who received the anthrax vaccine at Tripler Army Medical Center beginning in September 1998 (CDC, 2000). As reported in congressional testimony, the survey found a high incidence of local transient reactions (70 percent with subcutaneous nodules and 65 percent with muscle soreness) (Claypool, 1999). Muscle aches, the most frequently reported systemic complaint, were reported in 15 percent of vaccine recipients. Three VAERS reports were submitted on the individuals in this study, and one individual lost more than a day of work; there were no hospitalizations. Gender differences in the number of reactions have been noted in this study. A higher proportion of women reported outpatient visits (e.g., after the second dose, 2.0 percent of males and 13.8 percent of females reported making outpatient visits) and local reactions (e.g., after the second dose, 20.4 percent of males and 46.9 percent of females reported moderate to severe redness) (GAO, 1999d).

Additional studies. The U.S. Air Force is completing a study comparing visual acuity in 354 vaccinated aircrew members with 363 aircrew personnel who were not vaccinated against anthrax. Preliminary analysis reported in congressional testimony indicates that changes in visual acuity occurred in 12 percent of vaccinated and 16 percent of unvaccinated crew members during the course of a year (Claypool, 1999).

Service members stationed in Korea completed a mandatory questionnaire when they reported for anthrax vaccination. Questions included the service member's reaction to the previous dose of the anthrax vaccine. Data from 6,879 questionnaires noted gender differences in the reported rate of transient adverse reactions, with higher rates in women. After the first or second dose of the vaccine, 82 (1.9 percent) of 4,348 men and women reported limited effects on their work performance, 21 (0.5 percent) went to the clinic for evaluation, and 1 required hospitalization for an injection site reaction (CDC, 2000).

Conclusions on Human Studies

There is a paucity of published peer-reviewed literature on the safety of the anthrax vaccine. The committee located only one randomized peer-reviewed study of the type of anthrax vaccine used in the United States (Brachman et al., 1962). However, the formulation of the vaccine used in that study differs somewhat from the vaccine given to Gulf War veterans (and currently in use). The Brachman study (and other early experimental studies) found transient local and systemic effects (primarily erythema, edema, induration) of the anthrax vaccine. There was no long-term monitoring for adverse outcomes. The committee did not compare the incidence of transient effects with other vaccines.

Studies of the anthrax vaccine have not used active surveillance to systematically evaluate long-term health outcomes. This situation is unfortunately typical for all but a few vaccines. The committee strongly encourages active monitoring to evaluate the long-term safety of the anthrax vaccine.

To date, published studies have reported no significant adverse effects of the vaccine, but the literature is limited to a few short-term studies. Reviewing the large body of results that have not yet been published would enable more definitive conclusions about the vaccine's safety. The committee strongly urges investigators conducting studies on the safety of the anthrax vaccine to submit their results to peer-reviewed scientific journals for publication.

The committee's findings are best regarded as an early step in the complex process of understanding the safety of the anthrax vaccine, which began with the vaccine's licensure in 1970 and the 1985 FDA advisory panel finding that categorized the vaccine as safe and effective. Active long-term monitoring of large populations will provide further information for documenting the relative safety of the anthrax vaccine.

The committee concludes that there is sufficient evidence of an association between anthrax vaccination and transient acute local and systemic effects (e.g., redness, swelling, fever) typically associated with vaccination.

The committee concludes that there is inadequate/insufficient evidence to determine whether an association does or does not exist between anthrax vaccination and long-term adverse health effects.

The latter finding means that the evidence reviewed by the committee is of insufficient quality, consistency, or statistical power to permit a conclusion regarding the presence or absence of an association between the vaccine and a health outcome in humans.

BOTULINUM TOXOID

Botulinum toxins, known primarily for causing cases of foodborne botulism,9 are produced by the anaerobic bacterium Clostridium botulinum. The organism itself is not thought to play a role in the poisoning syndrome (Middlebrook et al., 1994). However, different strains of the bacillus produce seven distinct botulinum toxins (A–G). These toxins are among the most toxic compounds per body weight of agent, with an LD50 of 0.001 μg/kg in mice (for comparison, the LD50 of sarin in laboratory mice is 100 μg/kg) (USAMRIID, 1996). It is interesting to note that botulinum A toxin is successfully used to relax muscular spasms for a number of therapeutic purposes.10 The doses used are so minute that they do not produce toxic reactions, nor are they immunogenic.

Work on modifying the botulinum toxin to the nontoxic form of a toxoid began in 1924.11 Experimental work on a toxoid for use in humans was first reported in the 1930s by the Russian scientist Velikanov (Anderson and Lewis, 1981; Middlebrook and Brown, 1995). A bivalent toxoid (for serotypes A and B) was developed in the United States in the 1940s. Further research led to a pentaPublic Health (now BioPort Corporation) produced the pentavalent toxoid. valent toxoid (serotypes A–E) first produced in large lots by Parke, Davis, and Company in 1958 under contract to the U.S. Army (Anderson and Lewis, 1981). CDC submitted an Investigational New Drug application for the pentavalent toxoid in 1965 (IND 161; Rettig, 1999). In the 1970s, the Michigan Department of

Currently, the toxoid is in IND status. As described below, the toxoid has been administered to volunteers for testing purposes and to occupationally at-risk workers. Additionally, it is estimated that 8,000 U.S. troops received the toxoid during the Gulf War. Under an FDA Interim Rule (U.S. DHHS, 1990), the FDA commissioner was given the authority to waive IND requirements (e.g., informed consent) in specific military exigencies (Rettig, 1999).

The schedule for the pentavalent toxoid calls for subcutaneous injections at 0, 2, and 12 weeks, followed by annual boosters. Contraindications for administering the vaccine include alum, formaldehyde, or thimerosal sensitivities or hypersensitivity after receiving a previous dose (USAMRIID, 1996). Recent advances in molecular cloning techniques and new knowledge about the molecular mechanisms of action of the toxins have opened up avenues for new botulinum vaccine development (Middlebrook, 1995).

Toxicology

Mechanism of Action

Knowledge of the pathogenic mechanisms of Clostridium botulinum can provide insight into the potential adverse effects associated with administration of the botulinum toxoid vaccine. The signs and symptoms of botulism are due to the action of neurotoxins that are synthesized during cell growth of C. botulinum and released prior to or after lysis of the bacteria.

There are seven immunologically distinct types of botulinum neurotoxins: types A through G. Human botulism is caused principally by types A, B, E, and F toxins, and animal botulism is principally caused by types C and D (Sellin, 1984). In horses, the Clostridium botulinum that colonizes the intestinal tract produces type B toxin, causing the symptoms of shaker foal syndrome.

The mechanism of action of botulinum neurotoxins has recently been reviewed (Simpson, 1989; Brin, 1997). The seven neurotoxins are all metalloenzymes that cleave various components of the proteins involved in the release of the neurotransmitter, acetylcholine. Botulinum toxins share a number of structural features with tetanus toxins, even though the clinical symptoms of poisoning are quite different. The toxin molecule has three functional domains. The carboxy terminal portion of the molecule mediates binding of the toxin to the presynaptic nerve terminal at the neuromuscular junction. The central third of the molecule acts to internalize the neurotoxin inside the nerve ending. After internalization and disulfide cleavage, the light chain or amino terminal section then translocates the toxin to the cytosol where it inhibits the binding of synaptosomal vesicles to the axon terminal membrane, thus inhibiting the release of acetylcholine. Inhibiting the release of acetylcholine significantly affects nerve transmission between motor nerves and the voluntary muscles, causing paralysis and loss of respiration, a process that occurs at doses lower than required to affect the autonomic nervous system. All ganglionic synapses require acetylcholine and thus are disrupted. In addition, the postganglionic parasympathetic nervous system requires acetylcholine. The clinical effect of the toxin is thought to be due primarily to its effects on the peripheral nervous system because the botulinum neurotoxin does not cross the blood–brain barrier (Simpson, 1993). Extreme cases of poisoning with botulinum toxin result in total paralysis, with the patient incapable of moving or breathing.

To produce the toxoid, toxins are partially purified from culture supernatants and exposed for prolonged periods to formaldehyde. After exposure to formaldehyde they are tested for toxicity in mice and guinea pigs to ensure that the neurotoxin was inactivated.

Animal Studies

As noted earlier in this chapter, adverse health outcomes can result either from toxic effects of the injected toxoid preparation or from stimulation of the immune system. Toxic effects of the vaccination itself could be due to traces of formaldehyde or preservative in the toxoid preparation, contaminant proteins in the toxoid preparation, toxin that has not been inactivated by formaldehyde, and/or the adjuvant.

Studies in veterinary use. Botulinum toxoids type C and D. Animal botulism is caused principally by the type C and D neurotoxins. In veterinary use, vaccination of mink and ferrets (Pranter, 1976; Shen et al., 1981), chickens (Dohms et al., 1982; Kurazono et al., 1985), pheasants (Kurazono et al., 1985), and cattle (Tammemagi and Grant, 1967) has been reported using either monovalent type C toxoid or bivalent types C and D. These studies did not mention adverse effects from vaccination. In the study using type C toxoid in chicks (Dohms et al., 1982), survivors of the toxin challenge showed no lesions at the site of vaccination. In addition, Davidson (1976) reviewed the use of types C and D toxoids for veterinary purposes and did not mention adverse effects due to vaccination. The primary purpose of most of the studies was to evaluate the effectiveness of the vaccine against challenge with the botulinum neurotoxin, not to evaluate adverse effects. Most of the studies monitored animals for 1–2 months.

Botulinum toxoid type B in veterinary practice and laboratory animals. Shaker foal syndrome is a neuromuscular condition affecting 2- to 8-week-old foals. Administering type B botulinum toxoid can mimic the symptoms of shaker foal syndrome. Swelling at the injection site has been reported in four horses administered two doses of type B botulinum toxoid (Thomas et al., 1988). These swellings were hard and approximately 75 cm2 in area. The report did not state if the swelling occurred with the first, second, or both doses of the toxoid.

Studies in laboratory animals. Studies with botulinum toxoids have been done in guinea pigs, rabbits, and mice with type E (Kondo et al., 1969), type A (Gendon, 1958), and pentavalent types A–E toxoid (Cardella, 1964); in mice with type F toxoid (Mikhailova, 1966); and in guinea pigs with types C and D toxoid (Mathews, 1976). In none of these studies did the authors mention adverse effects, which may indicate that no adverse effects occurred, that adverse events were not monitored, or that the adverse events were not sufficiently severe to warrant termination of the experiment.

Studies in guinea pigs suggest that skin-sensitizing anaphylactic antibodies may be produced in response to the administration of a combination of type B botulinum toxoid with the complex typhoid antigen (Yefremova, 1980). Such skin-sensitizing antibodies in the guinea pig are associated with immediate hypersensitivity reactions and respiratory allergy. Effects of administration of type B toxoid alone were not investigated in this study.

Recombinant DNA methods have been used to generate fragments of the botulinum neurotoxins, in hopes of developing a molecule without neurotoxicity but able to provoke an immune response and protect against botulinum neurotoxin activity. Such fragments of types A, B, and C botulinum neurotoxin have been generated and tested for toxicity and immunogenicity in mice (Middlebrook et al., 1994; Whalen et al., 1996; Kiyatkin et al., 1997; Bavari et al., 1998; Byrne et al., 1998; Smith, 1998). The monitoring period for adverse effects in these animal studies was no more than months. The authors of these studies did not mention adverse effects from vaccination with toxin fragments. Clayton and colleagues (1995) expressed a fragment of botulinum neurotoxin type A in Escherichia coli. Initial experiments immunized mice with a crude extract of E. coli expressing the gene of interest; several mice died following the second or third vaccination. The authors suspected endotoxin contamination. Subsequent experiments used purified neurotoxin fragments, and no toxicity occurred after repeated vaccination of these recombinant preparations.

Conclusions on Animal Studies

Animal studies using botulinum toxoid vaccines have reported minimal transient local reactions and swelling at the injection site. Only short-term outcomes were reported, and in most studies, the reports did not mention adverse effects.

Human Studies

Only a few published peer-reviewed studies have examined the potential adverse health effects of the botulinum toxoid vaccine when administered to humans. The committee based its conclusions about possible associations between the toxoid and health effects solely on the peer-reviewed literature and included other studies when assessing research needs.

Published Studies of Laboratory Workers

Early studies of the initial univalent botulinum toxoids in the 1940s reported a significant number of local and systemic reactions (Middlebrook and Brown, 1995). Toxoids were further purified in the 1950s, and a study published in 1962 (Fiock et al., 1962) reported on tests of bivalent toxoid preparations by Parke, Davis, and Company. This study of laboratory personnel at Fort Detrick focused on the efficacy of four different schedules of bivalent botulinum toxoid vaccination. Personnel, not previously immunized with botulinum toxoid, were assigned to four different vaccination schedules as they reported for initial vaccination (50 individuals followed a 0-, 2-, 4-, and 6-week schedule; 25 individuals followed a 0- and 8-week schedule; 50 individuals followed a 0-, 2-, and 10-week schedule; and 25 individuals followed a 0- and 10-week schedule). The study reports that after 800 injections, no systemic or severe local reactions had occurred. Some individuals (the number is not reported) had a small subcutaneous nodule that lasted 2–3 weeks. The report does not discuss the surveillance methods for monitoring adverse effects.

A subsequent study by Fiock and colleagues (1963) examined four different pentavalent (A–E) toxoid lots prepared by Parke, Davis, and Company. This study focused on the immunological response to pentavalent toxoids. In the first part of the experiment, 17 laboratory workers received 0.5-ml injections of a pentavalent toxoid on a 0-, 2-, and 10-week schedule. As a control group, additional personnel received a univalent toxoid on the same schedule (each univalent toxoid [A, B, C, D, or E] was given to five or six individuals). Four months later an additional 15 individuals received the same lot of the pentavalent toxoid on the same schedule. Satisfactory antitoxin titer levels were seen in these initial experiments. Groups of 30 individuals (the study does not indicate the number of groups or whether the individuals were new participants) were then immunized with one of the pentavalent toxoid lots on a 0-, 2-, and 12-week schedule, with a booster at 52 weeks. The only statement about adverse reactions made by the investigators in their report was that 400 individuals received the pentavalent toxoid with “no marked local or marked systemic reactions.” Three persons had either a moderate local or moderate systemic reaction (the authors provided no details), and the authors stated that the incidence of mild local reaction was somewhat greater for the pentavalent toxoids than the control group toxoids (Fiock et al., 1962).

Other Studies and Reports

The committee received reports on several other studies, discussed below, with information on the botulinum toxoid vaccine. These studies have not been published in the peer-reviewed literature and were not considered in the committee's conclusions regarding the strength of the evidence for associations between botulinum toxoid and adverse health outcomes.

USAMRIID studies. The Proceedings of the 1981 International Conference on the Biomedical Aspects of Botulism (Lewis, 1981) details further studies on the pentavalent toxoids. Investigators at USAMRIID hypothesized that injections with formulations of the pentavalent botulinum toxoid containing less formaldehyde preservative would result in reduced pain at the site of injection. They conducted a full-series double-blind study to evaluate two formulations of the pentavalent toxoid with varying formaldehyde content; 36 previously nonimmunized laboratory workers received injections at 0, 14, and 84 days (13 individuals received the low-formaldehyde lot, 11 received the higher-formaldehyde lot, and 12 received the control toxoid [with an intermediate level of formaldehyde]). Reactions were recorded immediately and then at 24, 48, and 72 hours in all volunteers and after 72 hours as needed. The study reported no discernible differences in immediate pain. The group receiving the low-formaldehyde lot reported a slightly higher percentage of moderate local reactions (17.9 percent compared to 15 percent in those receiving the high-formaldehyde lot and 3 percent of controls). Moderate reactions were defined as edema or induration >30 mm and < 210 mm in any one diameter. No severe reactions occurred for any of the lots.

Fort Bragg Booster Study. Pittman and colleagues (1997) conducted an open-label study of DoD personnel 2 years after they had received anthrax and/or botulinum vaccines during Operation Desert Shield/Desert Storm in 1990–1991. As described above in the discussion of the anthrax vaccine, the objectives of the study were to assess the persistence of antibodies to the vaccines, to determine the serological response 30 days after receiving a booster dose (subjects received boosters for either the anthrax or the botulinum vaccine or both, depending on what they had received in 1990–1991), and to evaluate the reactogenicity of the vaccines. Of the 459 volunteers who received booster injections of the anthrax and botulinum vaccines, 13 percent reported erythema, and 15 percent noted induration at the site of the botulinum toxoid injection. The report states that the local reactions measured 3–60 mm (no severe local reactions [>210 mm] occurred). Low-grade fever occurred in 2.8 percent of the recipients.

CDC reports. As noted above, the botulinum toxoid is an Investigational New Drug and has not yet received FDA approval. As the holder of the IND, CDC submits annual progress reports to the FDA. These annual reports provide information on the number of injections given and summarize the adverse effects noted on the CDC Response to Investigational New Drug forms.12 Additionally, the report provides summary statistics on injections since 1970. The committee examined five of the most recent progress reports (CDC, 1994b, 1995, 1996, 1997, 1998).

The CDC progress report for March 2, 1997, to March 1, 1998 (the most recent one reviewed by the committee) reported on 955 injections (728 primary and 227 booster injections) given to 422 individuals (CDC, 1998). Of the 955 total, 879 recipients (92 percent) noted no reaction or a mild reaction; 56 (5.9 percent) experienced a moderate reaction, 4 (0.4 percent) had a severe local reaction and/or systemic reaction,13 and no response was recorded for 16 injections (1.7 percent). The four severe reactions were a 21-cm area of erythema and swelling, induration of approximately 35 cm, a severe local reaction, and severe swelling below the elbow.

Summary tables in the CDC report record the number of reactions for the 16,676 injections of the botulinum toxoid administered between 1970 and 1997 (CDC, 1998). Of this total, local reactions were characterized as 15,207 (91.2 percent) none or mild; 1,208 (7.2 percent) moderate; 63 (0.4 percent) severe; and no response recorded for 198 reactions (1.2 percent). Another summary table indicated the number of systemic reactions from 1970 to 1997 (total of 747 systemic reactions): 348 reports of swelling, lumps, soreness, stiff back, or stiff neck; 87 reports of itching hives; 79 reports of general malaise; 69 reports of chills and fever; 63 reports of headache; 63 reports of nausea, diarrhea, vomiting, and GI problems; and 38 reports of blurred vision or dizziness. The report implies that all of these reactions were of limited duration.

Conclusions on Human Studies

Studies have noted transient local and systemic effects of the botulinum toxoid vaccine. However, studies of the botulinum toxoid vaccine have not used active surveillance to systematically evaluate long-term health outcomes. This situation is unfortunately typical for all but a few vaccines.

The committee concludes that there is sufficient evidence of an association between botulinum toxoid vaccination and transient acute local and systemic effects (e.g., redness, swelling, fever) typically associated with vaccination.

The committee concludes that there is inadequate/insufficient evidence to determine whether an association does or does not exist between botulinum toxoid vaccination and long-term adverse health effects.

The latter finding means that the evidence reviewed by the committee is of insufficient quality, consistency, or statistical power to permit a conclusion regarding the presence or absence of an association between the vaccine and a health outcome in humans.

MULTIPLE VACCINATIONS

Military personnel often receive several vaccinations as they prepare for service in an environment with many endemic diseases. People have been expressed concerns that multiple vaccinations prior to and during Gulf War service may have caused adverse health effects. In this section, the committee examines the issue of multiple vaccinations.

Vaccination programs in the U.S. military began in 1777 when Continental Army recruits received inoculation against smallpox (Takafuji and Russell, 1990). As vaccines were developed for other endemic diseases that would take a heavy toll on the health of the military population (e.g., diphtheria, yellow fever, influenza), vaccinations were made mandatory for U.S. troops.

Each of the military services determines the immunization requirements for its personnel. However, there is general consensus, as evidenced in the triservice regulation on immunizations (Table 7.1). Some vaccinations (e.g., measles–rubella, influenza, diphtheria–tetanus) are administered routinely to all military recruits; others are administered for deployment to specific geographic or high-risk areas (e.g., typhoid, plague, cholera); and other vaccinations are specific to the occupational setting (e.g., hepatitis B vaccination for health care workers; rabies vaccination for animal control officers). During the first 2 weeks of military training, recruits may receive as many as 17 different antigens (Takafuji and Russell, 1990). Other vaccines may be given later in the training cycle or prior to deployment. The Armed Forces Epidemiologic Board, a group of civilian medical consultants, has been advising DoD since World War II on establishing and implementing DoD's preventive medicine guidelines. These guidelines are similar to civilian guidelines developed by the CDC and the Advisory Committee on Immunization Practices (IOM, 1996).

TABLE 7.1. Vaccinations Prescribed for Military Personnel.

TABLE 7.1

Vaccinations Prescribed for Military Personnel.

Combining several vaccines into a single injection or administering multiple vaccinations at the same clinical visit decreases the number of injections required to achieve protection against multiple diseases and reduces the number of visits to health care providers, thereby resulting in wider protection of the population (Goldenthal et al., 1995; Rappuoli, 1996; Choo and Finn, 1999). However, researchers continue to investigate whether interactions may decrease the efficacy of the component vaccines (i.e., as manifested in lower seroconversion rates and lower antibody titers) or increase the frequency of adverse effects.

In the United States, multiple vaccinations are routinely given to children, to adults prior to travel to areas of risk, and to military personnel. A number of combination vaccines have been licensed in the United States including: diphtheria–tetanus–pertussis (DTP), measles–mumps–rubella (MMR), 23-valent pneumococcal polysaccharide, and 4-valent meningococcal polysaccharide (Parkman, 1995; Ellis, 1996; CDC, 1999a; Choo and Finn, 1999). Additionally, safety and immunogenicity reports have appeared in the literature for several pediatric combinations including: DTP–hepatitis B (HBV); DTP–IPV (inactivated poliovirus vaccine)–HIB (Haemophilus influenzae type B); and MMR– varicella (Choo and Finn, 1999). Evidence suggests that although some combination vaccines interfere with immunogenicity, others enhance it (Insel, 1995). Short-term follow-up trials have shown these combination vaccines to be safe (Choo and Finn, 1999).

Some sets of vaccines can be administered simultaneously (i.e., on the same day but not at the same anatomical site) including yellow fever and measles; MMR and trivalent oral polio vaccine (OPV); and DTP, OPV, and MMR (CDC, 1994a; Insel, 1995; Parkman, 1995). In general, this practice does not appear to affect immune response to individual vaccines, nor does it result in substantial adverse effects in short-term (2–6 months and occasionally 2 years) follow-up studies (Grabenstein, 1990; CDC, 1994a; Parkman, 1995). The frequency of local and systemic reactions is generally the same as in separate vaccine administrations; however some patients experience greater local tenderness (King and Hadler, 1994). CDC states that simultaneous administration of inactivated vaccines that commonly produce local or systemic reactions, (e.g., cholera, parenteral typhoid, plague) may accentuate the reactions. CDC advises injections on separate days for these vaccines (CDC, 1994a). A study of the safety of the simultaneous administration of several childhood vaccines found that the proportion of MSAEFI (Monitoring System for Adverse Events Following Immunization, the precursor of VAERS) reports that described a set of specific adverse effects remained constant compared to the frequency reported for separate administrations, with one exception: local reactions were more frequent with simultaneous vaccinations (Chen et al., 1995). The MSAEFI system was a passive surveillance system based on reports by people who had been vaccinated, family members, and other individuals.

Toxicology

The intended effect of vaccination is to protect individuals from infection by stimulating the immune response to a particular antigen. Vaccination with multiple antigens could possibly lead to polyclonal activation of the immune system. Webb (1997) has speculated that multiple vaccinations may shift the immune response to a Th2 profile that favors hypersensitivity responses. However, others have suggested that vaccinations may induce protective Th1 responses that should prevent the development of atopic disease (Barnes, 1999; Holt et al., 1999).

Animal Studies

Animal studies involving multiple vaccinations examine three categories of adverse effects: those specific to an individual vaccine (adverse health outcomes for anthrax and botulinum vaccines individually have been discussed earlier in this chapter), reduced protection to one antigen when the immune system is coping with multiple antigens simultaneously, and the development of adverse immunological reactions.

In examining animal studies on multiple vaccinations, the committee focused on studies in which at least one of the immunogens was the anthrax or botulinum vaccine. The committee did not examine the extensive literature on animal studies of vaccine combinations that have been in use for many years (use of the DTP vaccine began in the 1940s, and the MMR vaccine was approved in 1971; Plotkin and Mortimer, 1994).

Alteration in the protective effect of the vaccination. Multiple vaccinations may lead to an insufficient protective response to one or several of the antigens. Pilipenko and Miroshnichenko (1963) found that simultaneous injections of live vaccines for anthrax, plague, tularemia, and brucellosis in guinea pigs reduced the development of protective immunity to anthrax without adversely affecting the response to the other live vaccines. The only adverse effect of the vaccination itself was erythema and an infiltrate at the site of injection of the live spore anthrax vaccine, a response that resolved within 3 to 5 days. A similar study by Borodko and Samsonovich (1965) in guinea pigs reported a reduced immune response to all of the four antigens as determined by allergic skin responses and challenge with the infectious agent. The authors did not mention adverse effects from the vaccination.

In the following animal studies, the degree of protective immunity was unaffected by multiple vaccinations. In sheep, Safarov and Ibragimov (1968) investigated the combination of vaccination with live spore anthrax vaccine and the standard vaccine for braxy and infectious enterotoxemia. The studies did not mention any adverse effects from the vaccination itself. A study by Zuffa and colleagues (1972) combined vaccination with botulinum toxoid types C and D and vaccination for the viral Aujeszky's disease in mink; the authors did not discuss adverse effects. A similar study by Gorski and Motz (1984) found that vaccination with distemper virus at the same time as administration of botulinum type C toxoid did not affect immunity. The botulinum toxoid caused a small area of skin swelling that was evident for 2 weeks. Another study with mink and ferrets (Shen et al., 1981) examined combined vaccination with live distemper virus, inactivated mink virus enteritis, and type C botulinum toxoid vaccine. Good protection was afforded to all antigens, and the authors did not mention any adverse effects. In guinea pigs, a comparative study was done to assess the efficacy of a protective antigen anthrax vaccine, either alone or in combination with a pertussis vaccine (Turnbull, 1990). The authors did not mention adverse effects from administration of either vaccine. Studies by Ramyar and Baharsefat (1969) in sheep indicated that the administration of the anthrax live spore vaccine in combination with sheep pox virus with saponin resulted in local skin reactions. Adverse effects occurred at the same rate for animals vaccinated with both anthrax and pox virus compared to each vaccination alone.

Adverse immunological reactions. Repeated exposure to the same antigen (Hyperimmunization). Studies in experimental animals have shown that repeated or large-dose injection with a single antigen may result in adverse health effects in animals. These hyperimmunization studies have been conducted only in animals, and it is not known whether the much smaller doses of antigen normally administered to humans as vaccines would result in similar adverse health effects.

Many studies, including those of Germuth (1953), have demonstrated that intravenous injection of a single high-dose antigen (0.5 g of bovine serum albumin) will stimulate the immune system, antibody production, and the development of a hypersensitivity serum sickness-like response in rabbits, characterized by glomerulonephritis and arteritis. Repeated injection of rabbits with egg albumin (20–40 mg intravenously divided into approximately 20 vaccinations over 2 months) led to the development of antibodies to antibodies (Aho and Wager, 1961). In addition, extensive injections of rabbits with casein (0.5 g injected subcutaneously twice a week for up to 15 months or more) led to the development of amyloidosis (Giles and Calkins, 1958). However, amyloidosis can also occur in the absence of extensive vaccination (Anderson, 1971). Other studies have demonstrated that injection of Balb/c mice with mineral oil can lead to the development of myeloma (Azar, 1968; Potter, 1971). For unknown reasons, the development of myeloma in response to mineral oil is unique to the Balb/c mouse strain, strongly suggesting a role for the genetic composition of this mouse strain in this unusual response.

Exposure to multiple antigens. A multiple vaccination study by Classen (1996) examined adverse immunological reactions. Classen found that vaccination, whether with one antigen or many, may predispose certain laboratory animals to the development of autoimmune conditions. Classen used strains of rats and mice that spontaneously develop diabetes. These animals are models of human insulin-dependent diabetes mellitus, an autoimmune disease. NOD/ MrkTacfBR mice were used as well as diabetic-prone and diabetic-resistant BB/Wor rats. Classen also used a group of MRL/lpr mice that develop an autoimmune disease that closely resembles human systemic lupus erythematosus. Animals were immunized with a protective antigen-based anthrax vaccine, alone or with various combinations of the diphtheria–tetanus (DT) vaccine; combined diphtheria, tetanus, and whole-cell pertussis (DTP) vaccine; combined diphtheria, tetanus, and acellular pertussis (DTPa) vaccine; or plague vaccine. Researchers noted that the development of autoimmune disease in the groups of animals could be accelerated or inhibited depending on the timing of the vaccination. These experiments suggest that immune stimulation by vaccination may alter the development of autoimmune disorders. As expected, such alterations may depend on the genetic susceptibility of the animal to autoimmune disease.

Conclusions on Animal Studies

Studies in animals found no difference in the degree of protective immunity with multiple vaccinations. Studies have suggested that a single high dose of an antigen or repeated stimulation with the same antigen may result in the production of autoantibodies or malignancies. However, the antigen loads in these studies far exceeded those used in human vaccine schedules with repeated vaccination. One study (Classen, 1996) did suggest that vaccination, whether single or multiple, could influence the development of autoimmune disorders in genetically susceptible animals. Thus, although it is plausible that an exaggerated immune stimulation from vaccination could lead to long-term effects of autoimmune-type disease with the attendant multiorgan system pathology, no long-term animal studies supporting this hypothesis have been reported.

Human Studies

Studies of three populations are particularly relevant to examining the effects of intensive administration of a large number of vaccinations. The first population is a group of Finnish army recruits who received many routine vaccines during the first weeks of service. The second group of studies followed laboratory workers at Fort Detrick who received an intensive vaccination program for occupational reasons. Further, the committee examined several studies of Gulf War veterans.

Finnish Army Studies

A series of studies of Finnish military recruits who received many vaccinations investigated whether intensive vaccination would stimulate the immune system to develop autoantibodies (Aho et al., 1962, 1967). The autoantibodies measured in the studies were rheumatoid factor (RF) and immunoconglutinin (IC). All servicemen were healthy during the observation period. Blood samples were drawn before vaccination and on two instances afterwards. Table 7.2 shows sample sizes and summarizes the vaccination and blood sample schedules. Study subjects appear to have participated in a single study.

TABLE 7.2. Vaccination and Testing Schedules.

TABLE 7.2

Vaccination and Testing Schedules.

Of the total of 626 individuals tested, sera from 13 individuals showed positive RF responses before vaccinations and remained so throughout (with no clear change in titer) (Aho et al., 1962, 1967). Among those that were negative before vaccinations, there were 6 transient RF reactions (testing positive in the second sample but negative in the third), 5 that were positive in both the second and the third samples, and 12 that were positive only in the final blood sample. The IC responses were studied in 189 of the servicemen (Aho et al., 1967). The IC titers increased rapidly and then declined. None of the 22 individuals with significant (greater than fourfold) immunoconglutinin titers had RF reactions. Immunoconglutinin responses occurred within 2 weeks, whereas RF responses appeared within 3–4 weeks. Study results suggest that transient RF and IC reactions may be related to the immune response to intensive vaccination. However, the number of subjects with positive results was small, and no control group was used to test for spontaneous fluctuations in RF response.

Fort Detrick Studies

A group of skilled laborers and laboratory workers employed at Fort Detrick, Maryland, was followed for up to 25 years to investigate the potential subclinical effects of intensive vaccination. These individuals had received numerous vaccinations because of their potential occupational exposure to a variety of virulent microorganisms. They were selected from the 700 employees because of their long and more intensive vaccination history. The study group underwent clinical and laboratory examinations in 1956, 1962, and 1971 (Peeler et al., 1958, 1965; White et al., 1974). No members of the study group suffered unexplained clinical symptoms attributable to the vaccination program that required them to take sick leave.

1956 examination (Peeler et al., 1958). At the first examination in 1956, the ages of the 99 Caucasian male subjects ranged from 28 to 65 (with a mean of 40.1 years). All had been vaccinated against botulism, tularemia, Rocky Mountain spotted fever, Q fever, plague, typhus, psittacosis, and eastern, western, and Venezuelan equine encephalitis (referred to as the basic occupational vaccinations); 95 were also vaccinated against smallpox, 37 against brucellosis, 28 against anthrax, and 25 against diphtheria. The initial series of injections was followed by boosters every 6 to 12 months. The duration of the vaccination schedule varied between 8 and 13 years (with a mean of 10.4 years). In addition, these subjects underwent frequent skin tests with a number of antigens (average of 20 skin tests). On average, 2.8 reactions to the vaccinations were recorded per subject (with a range of 0 to 16).

These individuals were evaluated by a review of outpatient and hospital records, complete medical history (n = 93), physical examination at the time of the study (n = 93), and serum electrophoresis and an extensive array of laboratory tests (n = 89). The sera of 44 individuals (18–50 years) who had not undergone multiple vaccinations served as the control group for the electrophoretic studies (controls were not done for the other tests). The controls were not matched on age, gender, or occupational exposure.

The incidence of past illness in the study group was comparable to that of the general population for this age group. Unexplained clinical and laboratory findings at this exam included leukocytosis (n = 20), lymphocytosis (n = 40), and abnormalities in liver function tests (n = 53).

In the serum electrophoretic studies, the mean total serum nitrogen and albumin were significantly higher in the intensively vaccinated group. Also, there was poor separation of α2- and β-globulin fractions in 23 of the subjects but normal separation in the control group. There appeared to be no relationship between the occurrence of this abnormal pattern and the subject's age, duration of immunization, number of skin tests, or administered antigen amount.

1962 examination (Peeler et al., 1965). In 1962, 76 members of the original study group were available for follow-up. The duration of immunization ranged from 12 to 18 years (with a mean of 15.3 years). In addition to the basic occupational vaccinations, all had been immunized against smallpox, 72 against anthrax, 70 against yellow fever, 66 against Rift Valley fever, 63 against tetanus, 54 against influenza, 37 against poliomyelitis, 34 against brucellosis, 20 against diphtheria, 1 against cholera, and 1 against typhoid–paratyphoid. The number of skin tests varied from 9 to 44 per subject (with an average of 30).

All study subjects were evaluated by a complete medical history, outpatient records, physical examination, serum electrophoresis (also performed in 1958), and extensive laboratory tests. Sera of 64 individuals were also examined for γ-globulin level and were screened for rheumatoid factor. Seven of the subjects also underwent a gingival biopsy. Three men who had persistent proteinuria underwent a percutaneous renal punch biopsy for detection of amyloid; the results were normal. Sera were drawn from 102 healthy blood donors in the same age group to serve as a control group for the electrophoretic and hexosamine studies.

Again, none of the nonoccupational illnesses occurred at a higher rate than in the general population, and there were no clinical illnesses that could be attributed to intense vaccination. Clinical and laboratory findings that are unexplained by previous illnesses included leukocytosis (n = 11), lymphocytosis (n = 24, with 6 subjects having lymphocytosis at both examination times), and some abnormalities in tests of renal or liver function.

Serum electrophoresis results showed poor separation of α2- and β-globulin factors (n = 19). The mean serum hexosamine level was significantly elevated in the intensively vaccinated group compared to the control group in both 1958 and 1962. Of the 64 sera exhibiting abnormalities on laboratory tests, 10 had γ-globulin levels above normal, 12 showed agglutination of anti-D coated cells, and 22 had positive latex tests for rheumatoid factors (3 subjects were positive on all tests). The amount of antigens received did not explain these positive results. In the seven individuals who did not receive vaccinations in the 2 years preceding this study, anti-γ-globulin factors could not be detected. The causes of the four deaths since 1956 (three acute coronary occlusions and one colon carcinoma) were deemed unrelated to the vaccination program. In the three postmortem examinations performed, there was no evidence of amyloid deposition or other abnormalities in sections of liver, spleen, and kidney.

1971 examination (White et al., 1974). In 1971, 77 members of the original study group were re-examined, ranging at that time from 43 to 79 years of age with a mean age of 55 years. In addition to the basic occupational vaccinations, at least 60 were vaccinated against Rift Valley fever, influenza, vaccinia, yellow fever, brucellosis, and anthrax; 41 against poliomyelitis; and 37 against diphtheria. The individuals had received an average of 55 skin tests (range = 6–93). The vaccination program was discontinued 10 months before this study.

As in the previous studies, each subject underwent a complete medical history, physical examination, electrocardiogram, and extensive laboratory tests. The control group consisted of 26 age-matched, long-term, civilian male employees of Fort Detrick who had never received special vaccinations and were not exposed to laboratory infections. The authors concluded that there were no clinical sequelae attributable to intense long-term immunization. Only one laboratory abnormality, elevated serum hexosamine, occurred that had also been described in the previous studies on the intensively immunized group. During the 1971 exam, there were slight but statistically significant differences in serum albumin, α2-globulin, and β-globulin levels compared to the control group. By 1971, 11 of the original group had died; this mortality rate agrees with the rate estimated from actuarial data. No amyloid deposition was found in the one biopsy and four postmortem examinations performed.

Summary and conclusions on the Fort Detrick studies. There were no clinical sequelae (e.g., neoplasms, amyloidosis, autoimmune diseases) attributable to intense long-term immunization in this well-studied cohort. None of the subjects suffered unexplained clinical symptoms requiring them to take sick leave that could be attributed to the vaccination program. There was some evidence of a chronic inflammatory response, as characterized by certain laboratory test abnormalities: elevated levels of hexosamine (an acute-phase reactant), slightly abnormal white cell counts, slightly abnormal liver function test results, and polyclonal elevations in levels of gamma globulins. However, these changes cannot necessarily be attributed to the vaccinations, since the workers studied were occupationally exposed to a number of virulent microbes.

This series of longitudinal clinical studies had several shortcomings. There was no comparison cohort and no attempt to make the employees in the study representative of the broader population of workers. Further, the outcomes may be due in part to the healthy-worker effect since the subjects were selected for the intensity and length of their vaccination history, and individuals who either left employment or discontinued the vaccination program were not considered. Thus, the studies may have inadvertently focused on the most resilient individuals. However, the Fort Detrick study is valuable because careful monitoring did not disclose any evidence of serious unexplained illness in a cohort that received a series of intense vaccination protocols over many years.

Gulf War Veterans

Several studies of Gulf War veterans have looked for associations between health outcomes and exposure to a variety of agents, including vaccinations. The methodology and general results of these studies are described in Chapter 2.

U.K. Gulf War Veterans. Unwin and colleagues (1999) reported the results of a large cross-sectional postal survey on a random sample of U.K. Gulf War, Gulf War era, and Bosnia conflict veterans. The Gulf War and Bosnia troops were vaccinated against hepatitis A and B, yellow fever, typhoid, poliomyelitis, cholera, and tetanus (routine vaccinations), as well as against biological warfare agents (plague, anthrax administered simultaneously with the pertussis vaccine). Of the Gulf War cohort (n = 3,284; response rate = 70.4 percent), 31.8 percent reported that they had their vaccination records. The study found no difference between veterans with and without vaccine records regarding age, education, health outcomes, or rank, except that those with records were more likely to be reservists. A substantial fraction, 61.9 percent, of Gulf War veterans reported symptoms of the multisymptom syndrome (characterized by fatigue, mood or cognition, or musculoskeletal symptoms) using the CDC criteria (Fukuda et al., 1998). The study found that having received any routine vaccination was significantly associated with the multisymptom syndrome among all Gulf War veterans (odds ratio [OR] = 1.2, 95% confidence interval [CI] 1.1–1.4); however, when the analysis was restricted just to veterans who had formal records of their vaccinations, the association was not significant (OR = 1.0, 95% CI 0.7–1.3). There was also a significant association between reporting biological warfare vaccination and the multisymptom outcome in the Gulf War cohort (OR = 1.5, 95% CI 1.3–1.7). Data were available in the Bosnia cohort only for anthrax vaccination, where no significant association was seen (OR = 1.5, 95% CI 0.7–2.9). An association was seen for Gulf War veterans receiving the anthrax vaccine (OR = 1.5, 95% CI 1.3–1.7). Servicemen who recalled experiencing adverse effects immediately after vaccination were more likely to have current symptoms (Gulf War cohort: OR = 2.8, 95% CI 2.4–3.3; Bosnia cohort: OR = 2.2, 95% CI 1.6–3.1). Controlling for these perceived adverse effects immediately after vaccination weakened the association in the entire Gulf War cohort between vaccination and the long-term multisymptom outcome, except for tetanus vaccination (OR = 1.2, 95% CI 1.0–1.4). Controlling for immediate short-term adverse effects after vaccination in the statistical analysis allows an examination of a possible direct association between vaccination and long-term effects. However, this statistical procedure would tend to underestimate the association between vaccination and long-term effects if short-term adverse effects were really correlated with long-term adverse effects; such a correlation is biologically plausible.

The total number of vaccinations received bore a weak but significant relationship to the occurrence of the multisymptom outcome among all Gulf War veterans, even when the cohort was subdivided into groups according to whether the subject possessed his vaccination records or not. The association still existed after controlling for the receipt of biological warfare vaccines and for experiencing side effects after vaccination (although the addition of this independent variable weakened the association). Among Bosnia veterans, no association between the number of vaccinations and the occurrence of the multisymptom outcome was observed. Although recall bias may be the reason for the significant results for individual vaccines, this is unlikely to be the case when the overall number of vaccinations is considered. Thus, the U.K. Gulf War study provides some limited evidence of an association between multiple vaccinations and long-term multisymptom outcomes. The respondents were more likely to be older, to still be in service, and to have attended the Ministry of Defence's assessment program for Gulf War veterans with symptoms. This study was conducted through questionnaire and relied primarily on self-reports.

A recently released study (Hotopf et al., 2000) reported on a further analysis of the United Kingdom data. This study focused on the subcohort of U.K. Gulf War veterans who reported that they had copies of their vaccine records (n = 923; 28 percent of responders in the original survey). The analysis examined the vaccines received, the timing of vaccinations, and six health outcome measures (the CDC-defined multisymptom outcome, psychological distress, posttraumatic stress reaction, fatigue, health perception, and physical functioning). The questionnaire also queried whether the servicemen recalled being exposed to pesticides or had experienced traumatic events during the Gulf War. The authors noted that the scheduling of vaccinations prior to and during the Gulf War was such that routine vaccinations were more likely to be administered before deployment and biological warfare vaccines were more likely to be administered during deployment. All regression analyses controlled for the possible confounding effects of rank, age, branch of service, and education.

The study found that the number of vaccines received prior to deployment was associated with one of the health outcomes (posttraumatic stress reaction). This relationship did not occur when accounting for the number of reported stressors. The number of vaccinations received during deployment was associated with five of the health outcomes (all but posttraumatic stress reaction). Once all vaccinations were taken into account in the analysis, the only two vaccines that showed an association with the CDC multisymptom syndrome were tetanus (adjusted OR = 2.7, 95% CI 1.0–7.2) and cholera (adjusted OR = 2.9, 95% CI 1.0–7.9). However, few records included these vaccinations (3.8 and 3.1 percent, respectively). In an analysis not controlling for all vaccines received, anthrax (OR = 1.4, 95% CI 1.0–1.8) and pertussis vaccination (OR = 1.4, 95% CI 1.0–1.9) were also significant. When the authors further analyzed the data to see if confounders (e.g., number of vaccines received simultaneously, number of years in the military, length of deployment to the Gulf, whether side effects of vaccinations were reported) could account for the association, the association held true.

This study is consistent with the hypothesis that receiving multiple immunizations within a narrow window of time, during a period of presumed stress (deployment to a theater of war), could be associated with the development of multiple symptoms and impaired functional status. One theory for which no direct evidence has been obtained in humans is that such a combination of events could cause alterations in the immune system, in particular a shift in the Th1 to Th2 response (Rook and Zumla, 1997). However, this study was limited by its cross-sectional nature and the fact that it relied on vaccine records that had been retained by only 28 percent of the survey respondents. Other possible confounding factors could be considered, including the timing of the vaccines (servicemen who received multiple vaccines during deployment tended to have been sent to the Gulf earlier, and medical personnel tended to report more vaccines during that period). Despite its limitations, this study was quite large and carefully evaluated the possibility of both confounding and response bias—concluding that neither explained its result.

CDC study. As part of the CDC study (see Chapter 2), Fukuda and colleagues (1998) performed clinical evaluations on a group of Gulf War veterans (n = 158), all of whom volunteered for the evaluation and were a subset of the index unit of Pennsylvania Air Force National Guard (45 percent of this unit had been deployed to the Persian Gulf). Participants in the clinical study were classified as cases (sufferers of the chronic multisymptom condition, as described in Chapter 2) or noncases, based on responses to the questionnaire. As part of an extensive laboratory evaluation, the authors tested serum samples for antibodies to type A botulinum toxin and the anthrax protective antigen or lethal factor (to screen for exposure to the vaccines or toxins). The study found that 10 of the 158 individuals had antibodies to the botulinum toxin and 14 to the anthrax protective antigen; however there were no differences in reactivity rates between cases and noncases.

The clinical evaluation part of this study examined a small sample of veterans from one unit deployed to the Gulf War theater and did not rely on a random sample. The issues of bias and lack of power must be considered in interpreting the results of this study.

Conclusions on Human Studies

Certain multiple vaccination regimens can lead to suboptimal antibody responses, but there is little evidence, largely because of a lack of active monitoring, of other adverse clinical or laboratory consequences beyond the transient local and systemic effects seen frequently with any vaccination.

No long-term, identifiable clinical sequelae attributable to intense long-term immunization occurred in the Fort Detrick cohort. There was some evidence of a chronic inflammatory response, but these changes cannot necessarily be attributed to the vaccinations, since the workers studied were occupationally exposed to a number of virulent microbes. This series of longitudinal clinical studies also had several shortcomings. However, the Fort Detrick study is valuable because careful monitoring did not disclose any evidence of serious unexplained illness in a cohort that received a series of intense vaccination protocols over many years.

The U.K. Gulf War studies provide some limited evidence of an association between multiple vaccinations and long-term multisymptom outcomes, particularly for vaccinations given during deployment (Unwin et al., 1999; Hotopf et al., 2000). There are some limitations and confounding factors in these studies, and further research is needed.

The committee concludes that there is inadequate/insufficient evidence to determine whether an association does or does not exist between multiple vaccinations and long-term adverse health effects.

This finding means that the evidence reviewed by the committee is of insufficient quality, consistency, or statistical power to permit a conclusion regarding the presence or absence of an association between multiple vaccinations and health outcomes in humans.

SQUALENE

The committee was asked to examine the literature on potential health effects of squalene since it has been raised as an issue of concern to Gulf War veterans. A recent GAO (1999a) report found that at the time of the Gulf War, DoD had concerns about having a sufficient quantity of the anthrax vaccine and sufficient time to fully immunize the troops (GAO, 1999a). However, DoD has stated that it decided against the use of novel adjuvant formulations (e.g., formulations with squalene) because of lengthy FDA relicensure requirements (GAO, 1999a). The following section provides a brief overview of the animal and human studies that have been conducted and concludes with the committee's thoughts on directions for future research. The committee was not asked to draw conclusions on the strength of the evidence for an association between exposure to squalene and adverse health effects.

Squalene14 is a polyunsaturated terpene hydrocarbon that is widely distributed in nature. It is found in human sebum (a skin surface lipid) and is a precursor in the synthesis of human cholesterol (Final Report, 1982; Kelly, 1999). Its name stems from its abundance in shark (Squalus spp.) liver oil,15 from which it was first isolated (Liu et al., 1976). Squalene also is found in other fish oils, olive oil (0.7 percent), wheat germ oil, rice bran oil, and many other foods.

For more than 25 years, squalene has been used commercially as an emollient for topical application of more than 300 cosmetic formulations, including suntan preparations, bath oil, eye makeup, hair preparations, lipstick, baby powder, and skin care preparations. Cosmetic concentrations range from less than 0.1 to more than 50 percent. Squalene also is available as a dietary supplement; as a constituent of certain pharmaceuticals, including suppositories; and as a carrier of lipid-soluble drugs (Final Report, 1982; Kelly, 1999). As described below, squalene is being investigated for a number of potential medical purposes.

Dietary Intake, Absorption, Distribution, and Metabolism

In the 1970s, the average dietary intake of squalene in the United States was calculated at 24 mg per day (given a daily dietary intake of 2,000 calories) (Liu et al., 1976). Olive oil is particularly rich in squalene. Among Asians, shark liver oil supplements containing squalene are popular over-the-counter folk remedies (Asnis et al., 1993). The average total squalene exposure of humans from all routes of administration does not appear to have been studied. In case studies, excessive ingestion of squalene from dietary supplements has led to lipoid pneumonia (Asnis et al., 1993).

Squalene is absorbed through several routes of administration, depending on the species (Final Report, 1982). In mice, squalene penetrates slowly and poorly through the skin at a rate of 0.12 nmol/cm2 per minute (Final Report, 1982). Subcutaneous administration in rabbits leads to increases of stored squalene in liver, muscle, and skin (Final Report, 1982). Virtually all squalene administered orally to rats (96–100 percent) is unabsorbed (Albro and Thomas, 1970).

In humans, about 60 percent of dietary squalene is absorbed through the gastrointestinal tract, with the remainder excreted in feces (Strandberg et al., 1990). A significant fraction of absorbed squalene is converted into cholesterol. Squalene is distributed throughout human tissues, with greatest concentrations in skin and fat (Kelly, 1999). Squalene in human serum comes from endogenous cholesterol synthesis and from diet (Strandberg et al., 1990; Kelly, 1999). Peak serum levels are attained 9–12 hours after ingestion (Gylling and Miettinen, 1994).

Animal Studies

There are few published studies of squalene toxicity in animals or humans (Kelly, 1999). Kamimura and colleagues (1989) examined subacute toxicity in dogs after a single oral squalene dose of 1,200 mg/kg. Over the next 3 months, accumulation was noted in several tissues, especially liver, but there were no signs of toxicity based on testing of serum and liver function. In contrast to humans, who absorb 60 percent of ingested squalene, this study reported that dogs absorb a relatively small percentage and excrete most in feces (83 percent). Thus, the relevance of this study to humans is unclear.

Squalene's toxicity is considered low, with an oral LD50 (median lethal dose) in mice at greater than 50 ml/kg (Final Report, 1982). No toxic responses were noted after subcutaneous and intramuscular injections of 0.5 ml per 20g mouse (25 ml/kg) of squalane (C30H62), a saturated and more stable version of squalene.

In a neuropathology study, squalene was administered subcutaneously to 10 rats (and 5 control rats) at a dose of 20 g/kg body weight for 4 consecutive days (Gajkowska et al., 1999). The rats' peripheral and central nervous systems were examined via electron microscopy 7 or 30 days from initiation of the experiment. After 7 days, disturbances in the myelin sheath were observed; these disturbances were more pronounced at 30 days. There was some swelling of Schwann cells in the peripheral nervous system. Fibroblasts were activated and showed signs of increased collagen production. In the central nervous system, squalene triggered swelling of astrocytes in white matter and in the hippocampus, especially near blood vessels. Lipid droplets accumulated in myelin in both the central and the peripheral nervous systems.

The pertinence of this neuropathology study is difficult to gauge because the dose was extremely high and the report provided minimal information about the study's methodology (especially handling of controls). Additionally, it is not uncommon to detect occasional astrocytic or neuronal swelling or mitochondrial swelling in electron micrographs of normal tissue. Further, damage appeared to be localized, not global, targeting, for example, a single myelin fiber or axon.

Squalene has attracted the interest of arthritis researchers because of its ability to activate the immune system nonspecifically. It was one of the constituents used in the 1970s to create the first animal models of multiple sclerosis, known as experimental allergic encephalomyelitis (EAE) (Beck et al., 1976). Squalene is one of several adjuvants (such as incomplete Freund's adjuvant) found to induce arthritis in susceptible rat strains and has been used in the generation of animal models of arthritis (Whitehouse et al., 1974; Lorentzen, 1999). The effect is so pronounced that researchers have coined the term “squalene-induced arthritis.” After a single intraarticular injection of 50 μL squalene into Lewis (Yoshino, 1996) and Dark Agouti rats (Yoshino and Yoshino, 1994), animals experienced moderate joint inflammation by day 6, followed by more severe chronic arthritis by day 21. The inflammation was marked by joint swelling and infiltration of CD5+ and αβ+ T cells. Similarly, intradermal injection of 200 μL squalene into Dark Agouti rats produced arthritis (Lorentzen, 1999). Although the mechanisms are not fully understood, the inflammation is blocked by agents that suppress T cells (Yoshino, 1996; Sverdrup et al., 1998). Animal studies do not report whether injection of squalene produces antisqualene antibodies.

In summary, there is limited published information about squalene toxicity. The human relevance of what has been published is unclear because of species differences in absorption. Squalene has been found to produce arthritis and neuropathology under select conditions in animals; the relevance to humans of these toxicity findings is uncertain.

Use of Squalene as a Vaccine Adjuvant

Squalene is currently being studied for a number of medical purposes including treatment of hypercholesterolemia (Chan et al., 1996); as an antidote to reduce the toxicity of accidentally ingested drugs (Kelly, 1999); and as an adjunctive therapy in cancer treatment to potentiate the cytotoxic activity of some chemotherapeutic agents (Kelly, 1999). The area of research that is of particular relevance to this chapter is the use of squalene as a vaccine adjuvant or as a component of a vaccine adjuvant.

The dose of an adjuvant is typically small (in the microgram range), and the route of administration is usually intramuscular. Squalene has been tested primarily as one component of the vaccine adjuvant MF59. MF59 is an oil-in-water microemulsion, consisting of squalene, polysorbate 80 (Tween 80, polyoxethylene sorbitan monooleate), and sorbitan trioleate (Graham et al., 1996). FDA has not yet approved any experimental vaccines with squalene-containing adjuvants.

The safety and efficacy of MF59 has been tested in a number of animal species with recombinant and natural antigens. Both short-term (approximately 2 weeks) and long-term (8 months) studies have been conducted and have detected some minor and transient changes in clinical laboratory parameters and histopathology (Ott et al., 1995). As described earlier in this chapter, a study by Ivins and colleagues (1995) on numerous combinations of adjuvants with the purified anthrax protective antigen found adverse effects from one of the adjuvant combinations containing squalene; other adjuvants containing squalene did not elicit adverse reactions. Tests of an HIV candidate vaccine with MF59 found no embryotoxic or teratogenic effects in dogs or rabbits (Ott et al., 1995).

Clinical studies of MF59 and other squalene-containing adjuvants have been conducted with candidate malaria, HSV (herpes simplex virus), HIV (human immunodeficiency virus), and influenza vaccines (Ott et al., 1995; GAO, 1999a). Study populations for the clinical trials have included adults, elderly, and children and infants (Ott et al., 1995).

HIV Vaccine Trials

Keefer and colleagues (1996) investigated the safety and immunogenicity of a candidate HIV-1 vaccine in combination with MF59, with or without an additional immune modulator, MTP–PE (muramyl tripeptide linked covalently with dipalmitoyl phosphatidylethanolamine). Vaccination with the candidate vaccine Env 2-3 in MTP–PE/MF59 was associated with significant adverse effects; severe, though short-lived, systemic and/or local reactions occurred in 15 of 30 vaccinees. In contrast, Env 2-3 in MF59 without MTP–PE was relatively well tolerated; severe local and/or systemic reactions occurred in only 2 of 18 subjects. There were no severe reactions in the eight subjects that received MF59 alone.

Graham and colleagues (1996) evaluated the safety and immunogenicity of another candidate vaccine for HIV, the recombinant glycoprotein 120, formulated with MF59 with or without MTP–PE. Vaccines that contained MTP–PE caused a greater number of moderate or severe local and systemic reactions (of 16 participants, 4 had local reactions and 13 had systemic reactions) than did vaccine formulated with MF59 alone. Of 16 vaccinees, 7 had local reactions and 0 had systemic reactions.

The National Institute of Allergy and Infectious Diseases (NIAID)-sponsored AIDS Vaccine Evaluation Group examined safety data from 1,398 HIV-negative, healthy volunteers who were enrolled in 25 multicenter, randomized double-blind studies evaluating 11 HIV candidate vaccines (Keefer et al., 1996). The study examined the adverse effects of a number of adjuvants, including MF59 and MF59 formulated with the biological response modifier MTP–PE. MTP–PE was associated with moderate to severe local reactions as well as with self-limited severe systemic reactions that resolved within 2–3 days. The same vaccines in the MF59 emulsion alone were well tolerated (Keefer et al., 1996).

Influenza Vaccine Trials

The safety and efficacy of MF59 have been evaluated in pilot studies (Keitel et al., 1993) and clinical trials (Martin, 1997; Menegon et al., 1999; Minutello et al., 1999). A study by Martin (1997) assembled data from eight randomized controlled clinical trials over four influenza seasons; 984 elderly volunteers (older than 65 years) received the adjuvanted vaccine, and 823 elderly volunteers received a conventional influenza vaccine. More than 20 percent of the volunteers who received the adjuvanted vaccine had local reactions. Myalgia was the only systemic effect to have been significantly more common in those receiving the vaccine with a squalene-containing adjuvant (3.9 percent) than those receiving the vaccine without the adjuvant (1.8 percent). All adverse events were recorded for 1 week after vaccination. Hospitalization and mortality were followed during the influenza season. The group receiving the adjuvanted vaccine had similar hospitalization rates and lower mortality than subjects receiving the conventional vaccine.

To date, clinical studies of the MF59 adjuvant that contains squalene have not shown any adverse health effects beyond transient acute effects.

Gulf War Issues

A recent study by Asa and colleagues (2000) reports on the development of an anti-squalene antibody assay to detect antibodies to squalene in the circulation. Blood samples from 144 Gulf War era veterans or military employees, 48 blood donors, 40 patients with systemic lupus erythematosus (SLE), 34 patients with silicone breast implants, and 30 patients with chronic fatigue syndrome (CFS) were studied for squalene antibodies. The study reports that a blinded test of serum samples found antibodies to squalene in more than 95 percent of 38 veterans deployed to the Gulf War who developed chronic illness symptoms; in all of the 6 veterans not deployed to the Gulf War who developed chronic illness symptoms; and in none of 12 veterans deployed to the Gulf War who were healthy. In an unblinded test, the study reported antibody reactivity to squalene in 5 percent of blood donors, 10 percent of patients with SLE, 10 percent of patients with silicone breast implants, and 15 percent of patients with CFS.

This study has several shortcomings. The subjects were self-selected, rather than being chosen at random from a larger sample, which can introduce substantial selection bias and does not allow inferences to the broader population of Gulf War veterans. Sample sizes were small, and the study may suffer from misclassification errors since the group of Gulf War veterans categorized as healthy (n = 12) was not devoid of individuals with serious symptoms (1 had fibromyalgia, 1 had thyroid disease, 3 had memory loss, and 4 had chronic fatigue). Further, the report provides inadequate evidence that the assay is able to accurately detect antibodies to squalene. Many of the methods used in the study are not described; as a result it is not possible to fully assess the study's methodology or to reproduce the assay. The study did not attempt to demonstrate that the substance giving the positive response in the assay was found in the immunoglobulin G (IgG) fraction of serum where antibodies are found. Further, the authors did not show that the assay was specific to squalene. To prove the specificity of the assay, the investigators would have had to show inhibition, in a dose–response manner, with squalene and no inhibition with other substances, as is seen in most reports of new enzyme-linked immunosorbent assays (ELISAs). The committee does not regard this study as providing evidence that the investigators have successfully measured antibodies to squalene.

Future Research Directions Regarding Squalene

As squalene continues to be investigated for a number of clinical uses, ongoing toxicity studies will provide the additional information that is needed about its toxicity, both in animals and in humans. It will be important to examine the relevance of animal studies because of species differences in the absorption of squalene and the susceptibility of certain strains of animals to squalene's effects. In considering future research directions, the committee focused on squalene's potential use as a vaccine adjuvant. Research questions that remain to be addressed include the following:

  • What types of immune responses does exogenous squalene evoke?
  • Does the immune response differ with the route of administration or entry (i.e., oral, cutaneous, intramuscular)?
  • How does the response vary according to the dose of squalene?
  • Is the presence of antibodies to squalene abnormal, and if so, what is their functional significance?
  • Could antibodies to squalene represent the consequences of, rather than the cause of, a pathological process?

CONCLUSIONS

The committee felt it would be helpful to the reader to restate the conclusions from this chapter. The conclusions listed below are identical to those made at the end of the respective sections of this chapter.

Anthrax Vaccine

There is a paucity of published peer-reviewed literature on the safety of the anthrax vaccine. The committee located only one randomized peer-reviewed study of the type of anthrax vaccine used in the United States (Brachman et al., 1962). However, the formulation of the vaccine used in that study differs somewhat from the vaccine given to Gulf War veterans (and currently in use). The Brachman study (and other early experimental studies) found transient local and systemic effects (primarily erythema, edema, induration) of the anthrax vaccine. There was no long-term monitoring for adverse outcomes. The committee did not compare the incidence of transient effects with other vaccines.

Studies of the anthrax vaccine have not used active surveillance to systematically evaluate long-term health outcomes. This situation is unfortunately typical for all but a few vaccines. The committee strongly encourages active monitoring to evaluate the long-term safety of the anthrax vaccine.

To date, published studies have reported no significant adverse effects of the vaccine, but the literature is limited to a few short-term studies. Reviewing the large body of results that have not yet been published may enable more definitive conclusions about the vaccine's safety. The committee strongly urges the investigators conducting studies on the safety of the anthrax vaccine to submit their results to peer-reviewed scientific journals for publication.

The committee's findings are best regarded as an early step in the complex process of understanding the vaccine's safety, which began with the vaccine's licensure in 1970 and the 1985 FDA advisory panel finding that categorized the anthrax vaccine as safe and effective. Active long-term monitoring of large populations will provide further information for documenting the relative safety of the anthrax vaccine.

The committee concludes that there is sufficient evidence of an association between anthrax vaccination and transient acute local and systemic effects (e.g., redness, swelling, fever) typically associated with vaccination.

The committee concludes that there is inadequate/insufficient evidence to determine whether an association does or does not exist between anthrax vaccination and long-term adverse health effects.

The latter finding means that the evidence reviewed by the committee is of insufficient quality, consistency, or statistical power to permit a conclusion regarding the presence or absence of an association between the vaccine and a health outcome in humans.

Botulinum Toxoid

Studies have noted transient local and systemic effects of the botulinum toxoid vaccine. However, studies of the botulinum toxoid vaccine have not used active surveillance to systematically evaluate long-term health outcomes. This situation is unfortunately typical for all but a few vaccines.

The committee concludes that there is sufficient evidence of an association between botulinum toxoid vaccination and transient acute local and systemic effects (e.g., redness, swelling, fever) typically associated with vaccination.

The committee concludes that there is inadequate/insufficient evidence to determine whether an association does or does not exist between botulinum toxoid vaccination and long-term adverse health effects.

The latter finding means that the evidence reviewed by the committee is of insufficient quality, consistency, or statistical power to permit a conclusion regarding the presence or absence of an association between the vaccine and a health outcome in humans.

Multiple Vaccinations

Certain multiple vaccination regimens can lead to suboptimal antibody responses, but there is little evidence, largely because of a lack of active monitoring, of other adverse clinical or laboratory consequences beyond the transient local and systemic effects seen frequently with any vaccination.

No long-term identifiable clinical sequelae attributable to intense long-term immunization occurred in the Fort Detrick cohort. There was some evidence of a chronic inflammatory response, but these changes cannot necessarily be attributed to the vaccinations, since the workers studied were occupationally exposed to a number of virulent microbes. This series of longitudinal clinical studies also had several shortcomings. However, the studies are valuable because careful monitoring did not disclose any evidence of serious unexplained illness in a cohort that received a series of intense vaccination protocols over many years.

The U.K. Gulf War studies provide some limited evidence of an association between multiple vaccinations and long-term multisymptom outcomes, particularly for vaccinations given during deployment (Unwin et al., 1999; Hotopf et al., 2000). There are some limitations and confounding factors in these studies, and further research is needed.

The committee concludes that there is inadequate/insufficient evidence to determine whether an association does or does not exist between multiple vaccinations and long-term adverse health effects.

This finding means that the evidence reviewed by the committee is of insufficient quality, consistency, or statistical power to permit a conclusion regarding the presence or absence of an association between multiple vaccinations and health outcomes in humans.

REFERENCES

  • Ahmed SA, Hissong BD, Verthelyi D, Donner K, Becker K, Karpuzoglu-Sahin E. 1999. Gender and risk of autoimmune diseases: Possible role of estrogenic compounds. Environ Health Perspect 107(Suppl 5):681–686. [PMC free article: PMC1566250] [PubMed: 10502531]
  • Aho K, Wager O. 1961. Production of anti-antibodies in rabbits. Ann Med Exper Fenn 39:79–87. [PubMed: 13681773]
  • Aho K, Konttinen A, Wager O. 1962. Transient appearance of the rheumatoid factor in connection with prophylactic vaccinations. Acta Pathologica et Microbiologica Scandinavica 56(4):478–479. [PubMed: 14011370]
  • Aho K, Somer T, Salo OP. 1967. Rheumatoid factor and immuno-conglutinin responses following various vaccinations. Proc Soc Exp Biol Med 124(1):229–233. [PubMed: 6017774]
  • Albert LJ, Inman RD. 1999. Molecular mimicry and autoimmunity. N Engl J Med 341:2068–2074. [PubMed: 10615080]
  • Albro PW, Thomas R. 1970. Absorption of aliphatic hydrocarbons by rats. Biochem Biophys Acta 291:437–446. [PubMed: 5497201]
  • Anderson JH Jr, Lewis GE Jr. 1981. Clinical evaluation of botulinum toxoids. In: Lewis GH Jr, editor. , ed. Biomedical Aspects of Botulism. New York: Academic Press.
  • Anderson RE. 1971. Disseminated amyloidosis in germfree mice. Am J Pathol 65:43–50. [PMC free article: PMC2047519] [PubMed: 5109976]
  • Asa PB, Cao Y, Garry RF. 2000. Antibodies to squalene in Gulf War syndrome. Exp Mol Pathol 68(1):55–64. [PubMed: 10640454]
  • Asnis DS, Saltzman HP, Melchert A. 1993. Shark oil pneumonia. An overlooked entity. Chest 103(3):976–977. [PubMed: 8449116]
  • Azar HA. 1968. Significance of adjuvant-induced plasmacytomas of mice in relation to the pathogenesis of human multiple myeloma. Ann Allergy 26(6):293–298. [PubMed: 4172770]
  • Barnard JP, Friedlander AM. 1999. Vaccination against anthrax with attenuated recombinant strains of Bacillus anthracis that produce protective antigen. Infect Immunol 67(2):562–567. [PMC free article: PMC96355] [PubMed: 9916059]
  • Barnes PJ. 1999. Therapeutic strategies for allergic diseases. Nature 402(6760 Suppl): B31–B38. [PubMed: 10586893]
  • Bavari S, Pless DD, Torres ER, Lebeda FJ, Olson MA. 1998. Identifying the principal protective antigenic determinants of type A botulinum neurotoxin. Vaccine 16(19): 1850–1856. [PubMed: 9795391]
  • Beck FW, Whitehouse MW, Pearson CM. 1976. Improvements for consistently inducing experimental allergic encephalomyelitis (EAE) in rats: I. Without using mycobacterium. II. Inoculating encephalitogen into the ear. Proc Soc Exp Biol Med 151(3): 615–622. [PubMed: 815915]
  • BioPort. 1999. Anthrax Vaccine Adsorbed, Package Insert. [Online]. Available: http://www​.bioportcorp​.com/Anthraxins.htm [accessed June 1999].
  • Borodko SL, Samsonovich LG. 1965. Duration of Immunity in Guinea Pigs Inoculated with Combined Live Vaccine Against Plague, Tularemia, Brucellosis and Anthrax. Available from the National Technical Information Service. AD-638 588/XAB.
  • Brachman PS, Gold H, Plotkin S, Fekety FR, Werrin M, Ingraham NR. 1962. Field evaluation of a human anthrax vaccine. Am J Public Health 52:632–645. [PMC free article: PMC1522900] [PubMed: 18017912]
  • Brin MF. 1997. Botulinum toxin: Chemistry, pharmacology, toxicity, and immunology. Muscle Nerve Suppl 6:S146–S168. [PubMed: 9826987]
  • Byrne MP, Smith TJ, Montgomery VA, Smith LA. 1998. Purification, potency, and efficacy of the botulinum neurotoxin type A binding domain from Pichia pastoris as a recombinant vaccine candidate. Infect Immun 66(10):4817–4822. [PMC free article: PMC108595] [PubMed: 9746584]
  • Cardella MA. 1964. Botulinum Toxoids. Frederick, MD. Available from the National Technical Information Service. AD-443 673/9/XAB.
  • Cardoso F, Jankovic J. 1995. Clinical use of botulinum neurotoxins. Curr Top Microbiol Immunol 195:123–141. [PubMed: 8542751]
  • CDC (Centers for Disease Control and Prevention). 1994. a. General recommendations on immunization. Recommendations of the Advisory Committee on Immunization Practices. MMWR 43(RR-1). [PubMed: 8145710]
  • CDC (Centers for Disease Control and Prevention). 1994. b. Progress Report #28 BB-IND 161: Pentavalent Botulinum Phosphate Adsorbed (March 2, 1993 to March 1, 1994). Atlanta, GA.
  • CDC (Centers for Disease Control and Prevention). 1995. Progress Report #29 BB-IND 161: Pentavalent Botulinum Phosphate Adsorbed (March 2, 1994 to March 1, 1995). Atlanta, GA.
  • CDC (Centers for Disease Control and Prevention). 1996. Progress Report #30 BB-IND 161: Pentavalent Botulinum Phosphate Adsorbed (March 2, 1995 to March 1, 1996). Atlanta, GA.
  • CDC (Centers for Disease Control and Prevention). 1997. Progress Report #31 BB-IND 161: Pentavalent Botulinum Phosphate Adsorbed (March 2, 1996 to March 1, 1997). Atlanta, GA.
  • CDC (Centers for Disease Control and Prevention). 1998. Progress Report #32 BB-IND 161: Pentavalent Botulinum Phosphate Adsorbed (March 2, 1997 to March 1, 1998). Atlanta, GA.
  • CDC (Centers for Disease Control and Prevention). 1999. a. Combination vaccines for childhood immunization. MMWR 48(RR05):1–15.
  • CDC (Centers for Disease Control and Prevention). 1999. b. Vaccine Adverse Event Reporting System (VAERS). [Online]. Available: http://www​.cdc.gov/nip/vaers.htm [accessed June 1999].
  • CDC (Centers for Disease Control and Prevention). 2000. Surveillance for adverse effects associated with anthrax vaccination—U.S. Department of Defense, 1998–2000. MMWR 49(16):341–345. [PubMed: 10817479]
  • Chan P, Tomlinson B, Lee CB, Lee YS. 1996. Effectiveness and safety of low-dose pravastatin and squalene, alone and in combination, in elderly patients with hypercholesterolemia. J Clin Pharmacol 36(5):422–427. [PubMed: 8739021]
  • Chen RT, Haber P, Mullen JR. 1995. Surveillance of the safety of simultaneous administration of vaccines. Ann NY Acad Sci 754:309–320. [PubMed: 7625667]
  • Choo S, Finn A. 1999. Pediatric combination vaccines. Current Opin Pediatr 11(1):14– 20. [PubMed: 10084078]
  • Christopher GW, Cieslak TJ, Pavlin JA, Eitzen EM Jr. 1997. Biological warfare. A historical perspective. JAMA 278(5):412–417. [PubMed: 9244333]
  • Classen JB. 1996. The timing of immunization affects the development of diabetes in rodents. Autoimmunity 24(3):137–145. [PubMed: 9020406]
  • Claypool GR. 1999. The Anthrax Vaccine Immunization Program . Statement at the July 21, 1999 hearing of the Subcommittee on National Security, Veterans Affairs, and International Relations, Committee on Government Reform, U.S. House of Representatives. U.S. Army Medical Corps, Deputy Assistant Secretary for Health Operations Policy. Washington, DC.
  • Clayton MA, Clayton JM, Brown DR, Middlebrook JL. 1995. Protective vaccination with a recombinant fragment of Clostridium botulinum neurotoxin serotype A expressed from a synthetic gene in Escherichia coli. Infect Immun 63(7):2738–2742. [PMC free article: PMC173366] [PubMed: 7790092]
  • Committee on Veterans' Affairs, U.S. Senate. 1998. Report of the Special Investigation Unit on Gulf War Illnesses. 105th Congress, 2nd session. Washington, DC: Government Printing Office. S.PRT 105-39.
  • Coombs RR, Gell PG. 1968. Classification of allergic reactions responsible for clinical hypersensitivity and disease. In: Gell PG, editor; , Coombs RR, editor. , eds. Clinical Aspects of Immunology. 2nd edition. Oxford: Blackwell.
  • Cooper GS, Miller FW, Pandey JP. 1999. The role of genetic factors in autoimmune disease: Implications for environmental research. Environ Health Perspect 107(Suppl 5):693– 700. [PMC free article: PMC1566257] [PubMed: 10502533]
  • Darlow HM, Belton FC, Camb BA. 1956. The use of anthrax antigen to immunise man and monkey. Lancet 2:476–479. [PubMed: 13368432]
  • Davidson I. 1976. An international survey of clostridial sera and vaccines. Dev Biol Stand 32:3–14. [PubMed: 187508]
  • DeArmon IA Jr, Klein F, Lincoln RE, Mahlandt BG, Fernelius AL. 1961. Immunological studies of anthrax. I. An index to determine quantitative immunization. J Immunol 87:233–239. [PubMed: 13720971]
  • Demicheli V, Rivetti D, Deeks JJ, Jefferson T, Pratt M. 1998. The effectiveness and safety of vaccines against human anthrax: A systematic review. Vaccine 16(9–10): 880–884. [PubMed: 9682332]
  • Dohms JE, Allen PH, Cloud SS. 1982. The immunization of broiler chickens against type C botulism. Avian Dis 26(2):340–345. [PubMed: 7049149]
  • Ellenberg SS. 1999. Statement at the July 21, 1999 Hearing of the Subcommittee on National Security, Veterans Affairs, and International Relations, Committee on Government Reform, U.S. House of Representatives. Rockville, MD: Food and Drug Administration.
  • Ellis RW. 1996. Challenges in the development of combination vaccines. In: Cohen S, editor; , Shafferman A, editor. , eds. Novel Strategies in Design and Production of Vaccines . New York: Plenum Press. Pp.127–132.
  • Ezzell JWJ, Abshire TG. 1988. Immunological analysis of cell-associated antigens of Bacillus anthracis. Infect Immun 56(2):349–356. [PMC free article: PMC259287] [PubMed: 3123387]
  • Fauci AS, editor; , Braunwald E, editor; , Isselbacher KJ, editor; , Wilson JD, editor; , Martin JB, editor; , Kasper DL, editor; , Hauser SL, editor; , Longo DL, editor. , eds. 1998. Harrison's Principles of Internal Medicine. 14th edition. New York: McGraw Hill.
  • FDA (Food and Drug Administration). 1985. Biological products: Bacterial vaccines and toxoids: Implementation of efficacy review. Proposed Rule. Federal Register 50(240):51002–51117. [PubMed: 14968793]
  • Final Report. 1982. Final report on the safety assessment of squalane and squalene. J Am Coll Toxicol 1(2):37–56.
  • Fiock MA, Devine LF, Gearinger NF, Duff JT, Wright GG, Kadull PJ. 1962. Studies on immunity to toxins of Clostridium botulinum. VIII. Immunological response of man to purified bivalent AB botulinum toxoid. J Immunol 88:277–283. [PubMed: 13893049]
  • Fiock MA, Cardella MA, Gearinger NF. 1963. Studies on immunity to toxins of Clostridium botulinum. X. Immunologic response of man to purified pentavalent ABCDE botulinum toxoid. J Immunol 90:697–702. [PubMed: 14054890]
  • Franz DR, Jahrling PB, Friedlander AM, McClain DJ, Hoover DL, Bryne WR, Pavlin JA, Christopher GW, Eitzen EM Jr. 1997. Clinical recognition and management of patients exposed to biological warfare agents. JAMA 278(5):399–411. [PubMed: 9244332]
  • Friedlander AM. 1986. Macrophages are sensitive to anthrax lethal toxin through an acid-dependent process. J Biol Chem 261(16):7123–7126. [PubMed: 3711080]
  • Friedlander AM. 1997. Anthrax. In: Zajtchuk R, editor; , Bellamy R, editor. , eds. Textbook of Military Medicine: Medical Aspects of Chemical and Biological Warfare . Washington, DC: Office of the Surgeon General, Department of the Army. Pp.467–478.
  • Friedlander AM, Pittman PR, Parker GW. 1999. Anthrax vaccine: Evidence for safety and efficacy against inhalational anthrax. JAMA 282(22):2104–2106. [PubMed: 10591317]
  • Fukuda K, Nisenbaum R, Stewart G, Thompson WW, Robin L, Washko RM, Noah DL, Barrett DH, Randall B, Herwaldt BL, Mawle AC, Reeves WC. 1998. Chronic multisymptom illness affecting Air Force veterans of the Gulf War. JAMA 280(11):981– 988. [PubMed: 9749480]
  • Gajkowska B, Smialek M, Ostrowski RP, Piotrowski P, Frontczak-Baniewicz M. 1999. The experimental squalene encephaloneuropathy in the rat. Exp Toxicol Pathol 51(1):75–80. [PubMed: 10048717]
  • GAO (U.S. General Accounting Office). 1999. a. Gulf War Illnesses: Questions About the Presence of Squalene Antibodies in Veterans Can Be Resolved. GAO/T-NSIAD-99-5. Washington, DC: GAO.
  • GAO (U.S. General Accounting Office). 1999. b. Medical Readiness: Issues Concerning the Anthrax Vaccine . Statement of Kwai-Cheung Chan, Director, Special Studies and Evaluations, National Security and International Affairs Division, before the Subcommittee on National Security, Veterans' Affairs, and International Relations, Committee on Government Reform, House of Representatives. GAO/T-NSIAD-99-226. Washington, DC: GAO.
  • GAO (U.S. General Accounting Office). 1999. c. Medical Readiness: Safety and Efficacy of the Anthrax Vaccine . Statement of Kwai-Cheung Chan, Director, Special Studies and Evaluations, National Security and International Affairs Division, before the Subcommittee on National Security, Veterans' Affairs, and International Relations, Committee on Government Reform, House of Representatives. GAO/T-NSIAD-99-148. Washington, DC: GAO.
  • GAO (U.S. General Accounting Office). 1999. d. Anthrax Vaccine: Safety and Efficacy Issues. GAO/NSAID-00-48. Washington, DC: GAO.
  • Gendon YZ. 1958. Changes in the Fractional Composition of the Serum Proteins of Mice and Guinea-Pigs on Immunization with Type A Clostridium Botulinum Toxoids. Available from the National Technical Information Service. AD-682 631/XAB.
  • Germuth FG. 1953. A comparative histologic and immunologic study in rabbits of induced hypersensitivity of the serum sickness type. J Exp Med 97:257–282. [PMC free article: PMC2136196] [PubMed: 13022878]
  • Giles RB Jr, Calkins E. 1958. The relationship of serum hexosamine, globulins, and antibodies to experimental amyloidosis. J Clin Invest 37:846–857. [PMC free article: PMC1062744] [PubMed: 13549599]
  • Goldenthal KL, Burns DL, McVittie LD, Lewis BP, Williams JC. 1995. Overview: Combined vaccines and simultaneous administration. Past, present, and future. Ann NY Acad Sci 754:xi–xv. [PubMed: 7625640]
  • Gorski J, Motz J. 1984. Safety and immunogenic value of the vaccines against botulism and distemper simultaneously administered to the mink. Bull Vet Inst Pulawy 27(1– 4):16–22.
  • Grabenstein JD. 1990. Drug interactions involving immunologic agents. Part 1. Vaccine-vaccine, vaccine-immunoglobulin, and vaccine-drug interactions. DICP 24(1):67– 81. [PubMed: 2405589]
  • Graham BS, Keefer MC, McElrath MJ, Gorse GJ, Schwartz DH, Weinhold K, Matthews TJ, Esterlitz JR, Sinangil F, Fast PE, NIAID AIDS Vaccine Evaluation Group. 1996. Safety and immunogenicity of a candidate HIV-1 vaccine in healthy adults: Recombinant glycoprotein (rgp) 120. A randomized, double-blind trial. Ann Intern Med 125(4):270–279. [PubMed: 8678389]
  • Gu M-L, Leppla SH, Klinman DM. 1999. Protection against anthrax toxin by vaccination with a DNA plasmid encoding anthrax protective antigen. Vaccine 17(4):340–344. [PubMed: 9987172]
  • Gulrajani TS, Misra RP, Verma JC, Ahuja ML. 1968. Effect of adjuvants on immunising efficacy of Bacillus anthracis protective antigen. Indian Vet J 45(6):465–476. [PubMed: 5685170]
  • Gusman BS, Migulina TV. 1967. Morphology of Immunogenesis During Experimental Anthrax Vaccination. Available from the National Technical Information Service. AD-675 373/XAB.
  • Gylling H, Miettinen TA. 1994. Postabsorptive metabolism of dietary squalene. Atherosclerosis 106(2):169–178. [PubMed: 8060377]
  • Hanna PC, Acosta D, Collier RJ. 1993. On the role of macrophages in anthrax. Proc Natl Acad Sci USA 90:10198–10201. [PMC free article: PMC47741] [PubMed: 8234277]
  • Holt PG, Macaubas C, Stumbles PA, Sly PD. 1999. The role of allergy in the development of asthma. Nature 402(6760 Suppl):B12–B17. [PubMed: 10586890]
  • Hotopf M, David A, Hull L, Ismail K, Unwin C, Wessely S. 2000. Role of vaccinations as risk factors for ill health in veterans of the Gulf war: Cross sectional study. BMJ 320:1363–1367. [PMC free article: PMC27378] [PubMed: 10818024]
  • Ibrahim KH, Brown G, Wright DH, Rotschafer JC. 1999. Bacillus anthracis: Medical issues of biologic warfare. Pharmacotherapy 19(6):690–701. [PubMed: 10391414]
  • Insel RA. 1995. Potential alterations in immunogenicity by combining or simultaneously administering vaccine components. Ann NY Acad Sci 754:35–47. [PubMed: 7625671]
  • IOM (Institute of Medicine). 1991. Adverse Effects of Pertussis and Rubella Vaccines. Washington, DC: National Academy Press.
  • IOM (Institute of Medicine). 1994. Adverse Events Associated with Childhood Vaccines: Evidence Bearing on Causality. Washington, DC: National Academy Press. [PubMed: 25144097]
  • IOM (Institute of Medicine). 1996. Interactions of Drugs, Biologics, and Chemicals in U.S. Military Forces. Washington, DC: National Academy Press.
  • IOM (Institute of Medicine). 1997. Vaccine Safety Forum: Summaries of Two Workshops. Washington, DC: National Academy Press.
  • IOM (Institute of Medicine). 2000. Safety of the Anthrax Vaccine: A Letter Report. Washington, DC: National Academy Press.
  • Ivins BE. 1988. The Search for a New-Generation Human Anthrax Vaccine. Ft. Detrick, MD: U.S. Army Medical Research Institute of Infectious Diseases. Available from the National Technical Information Service. AD-A190178.
  • Ivins BE, Ezzell JW Jr, Jemski J, Hedlund KW, Ristroph JD, Leppla SH. 1986. Immunization studies with attenuated strains of Bacillus anthracis. Infect Immun 52(2):454– 458. [PMC free article: PMC261020] [PubMed: 3084383]
  • Ivins BE, Welkos SL, Little SF, Knudson GB. 1989. Cloned Protective Activity and Progress in Development of Improved Anthrax Vaccines. Ft. Detrick, MD: U.S. Army Medical Research Institute of Infectious Diseases. Available from the National Technical Information Service. AD-A216 312/9.
  • Ivins BE, Welkos SL, Knudson GB, Little SF. 1990. Immunization against anthrax with aromatic compound-dependent (Aro-) mutants of Bacillus anthracis and with recombinant strains of Bacillus subtilis that produce anthrax protective antigen. Infect Immun 58(2):303–308. [PMC free article: PMC258455] [PubMed: 2105269]
  • Ivins BE, Welkos SL, Little SF, Crumrine MH, Nelson GO. 1992. Immunization against anthrax with Bacillus anthracis protective antigen combined with adjuvants. Infect Immun 60(2):662–668. [PMC free article: PMC257681] [PubMed: 1730501]
  • Ivins BE, Fellows PF, Pitt ML, Welkos SL. 1993. Experimental anthrax vaccines: Efficacy studies in guinea pigs. Abstracts of the General Meeting of the American Society for Microbiology 93(0):160.
  • Ivins BE, Fellows PF, Nelson GO. 1994. Efficacy of a standard human anthrax vaccine against Bacillus anthracis spore challenge in guinea-pigs. Vaccine 12(10):872–874. [PubMed: 7975827]
  • Ivins B, Fellows P, Pitt L, Estep J, Farchaus J, Friedlander A, Gibbs P. 1995. Experimental anthrax vaccines: Efficacy of adjuvants combined with protective antigen against an aerosol Bacillus anthracis spore challenge in guinea pigs. Vaccine 13(18): 1779–1784. [PubMed: 8701593]
  • Ivins BE, Pitt ML, Fellows PF, Farchaus JW, Benner GE, Waag DM, Little SF, Anderson GW Jr, Gibbs PH, Friedlander AM. 1998. Comparative efficacy of experimental anthrax vaccine candidates against inhalation anthrax in rhesus macaques. Vaccine 16(11–12):1141–1148. [PubMed: 9682372]
  • Jaiswal TN, Mittal KR. 1979. Potency testing of anthrax spore vaccine (living) in guinea pigs. Indian Vet J 56(3):199–201. [PubMed: 113339]
  • Kamimura H, Koga N, Oguri K, Yoshimura H, Inoue H, Sato K, Ohkubo M. 1989. [Studies on distribution, excretion and subacute toxicity of squalene in dogs]. Fukuoka Igaku Zasshi 80:269–280. [PubMed: 2744688]
  • Kaufmann AF, Fox MD, Kolb RC. 1973. Anthrax in Louisiana, 1971: An evaluation of the Sterne strain anthrax vaccine. J Am Vet Med Assoc 163(5):442–445. [PubMed: 4200690]
  • Keefer MC, Graham BS, McElrath MJ, Matthews TJ, Stablein DM, Corey L, Wright PF, Lawrence D, Fast PE, Weinhold K, Hsieh RH, Chernoff D, Dekker C, Dolin R. 1996. Safety and immunogenicity of Env 2-3, a human immunodeficiency virus type 1 candidate vaccine, in combination with a novel adjuvant, MTP–PE/MF59. AIDS Res Hum Retroviruses 12(8):683–693. [PubMed: 8744579]
  • Keitel W, Couch R, Bond N, Adair S, Van Nest G, Dekker C. 1993. Pilot evaluation of influenza virus vaccine (IVV) combined with adjuvant. Vaccine 11(9):909–913. [PubMed: 8212835]
  • Kelly GS. 1999. Squalene and its potential clinical uses. Alternative Med Rev 4(1):29–36. [PubMed: 9988781]
  • Keusch GT, Bart KJ. 1998. Immunization principles and vaccine use. In: Fauci AS, editor; , Braunwald E, editor; , Isselbacher KJ, editor; , Wilson JD, editor; , Martin JB, editor; , Kasper DL, editor; , Hauser SL, editor; , Longo DL, editor. , eds. Harrison's Principles of Internal Medicine. 14th edition. New York: McGraw Hill.
  • King GE, Hadler SC. 1994. Simultaneous administration of childhood vaccines: An important public health policy that is safe and efficacious. Pediatr Infect Dis J 13:394– 407. [PubMed: 8072822]
  • Kiyatkin N, Maksymowych AB, Simpson LL. 1997. Induction of an immune response by oral administration of recombinant botulinum toxin. Infect Immun 65(11):4586– 4591. [PMC free article: PMC175658] [PubMed: 9353037]
  • Klein F, Mahlandt BG, Lincoln RE, DeArmon IA Jr, Fernelius AL. 1961. Immunization as a factor affecting the course of septicemic anthrax. Science 133:1021–1022. [PubMed: 13756651]
  • Klein F, DeArmon IA Jr, Lincoln RE, Mahlandt BG, Fernelius AL. 1962. Immunological studies of anthrax. II. Levels of immunity against Bacillus anthracis obtained with protective antigen and live vaccine. J Immunol 88:15–19. [PubMed: 14456743]
  • Kolesov SG, Gutiman AA. 1968. The Study of the Complications Resulting from the Application of STI Vaccine . Available from the National Technical Information Service. AD-674 877/XAB.
  • Kolesov SG, Mikhailov NA, Borisovich YF. 1968. Aluminum Hydroxide Vaccine Against Anthrax. Translation of Veterinariya (USSR) 34(10):39–45 1957. Available from the National Technical Information Service. AD-672 433/XAB.
  • Kolksov SG, Mikhailov NA. 1959. Studies of the Immunogenic Properties of the Aluminum Hydroxide Vaccine Against Anthrax and Testing It At-Large in the Practice . Available from the National Technical Information Service. AD-640 528/XAB.
  • Kolosov SG, Borisovich YF. 1968. Obtainance of Anthrax Strains for the Purpose of Prophylactic Vaccination. Available from the National Technical Information Service. AD-674 926/XAB.
  • Kondo H, Kondo S, Murata R, Sakaguchi G. 1969. Antigenicity of Clostridium botulinum type-E formol toxoid. Jpn J Med Sci Biol 22(2):75–85. [PubMed: 4899485]
  • Kurazono H, Shimozawa K, Sakaguchi G, Takahashi M, Shimizu T, Kondo H. 1985. Botulism among penned pheasants and protection by vaccination with C1 toxoid. Res Vet Sci 38(1):104–108. [PubMed: 3883454]
  • Leppla SH. 1982. Anthrax toxin edema factor: A bacterial adenylate cyclase that increases cyclic AMP concentrations in eukaryotic cells. Proc Natl Acad Sci USA 79: 3162–3166. [PMC free article: PMC346374] [PubMed: 6285339]
  • Leppla S, Friedlander AM, Singh EM, Bhatnagar R. 1990. A model for anthrax toxic action at the cellular level. Salisbury Medical Bulletin 68(Special Supplement):41– 43.
  • Lewis GE Jr, editor. , ed. 1981. Biomedical Aspects of Botulism. New York: Academic Press.
  • Lindley WH. 1963. Anthrax vaccination. J Am Vet Med Assoc 142(6):621–623.
  • Little SF, Knudson GB. 1986. Comparative efficacy of Bacillus anthracis live spore vaccine and protective antigen vaccine against anthrax in the guinea pig. Infect Immun 52(2):509–512. [PMC free article: PMC261029] [PubMed: 3084385]
  • Liu GC, Ahrens EH Jr, Schreibman PH, Crouse JR. 1976. Measurement of squalene in human tissues and plasma: Validation and application. J Lipid Res 17(1):38–45. [PubMed: 1255019]
  • Lopez GP. 1998. Botulinum toxin. In: Wexler P, editor. , ed. Encyclopedia of Toxicology. San Diego: Academic Press. Pp.184–185.
  • Lorentzen JC. 1999. Identification of arthritogenic adjuvants of self and foreign origin. Scand J Immunol 49(1):45–50. [PubMed: 10023856]
  • Martin JT. 1997. Development of an adjuvant to enhance the immune response to influenza vaccine in the elderly. Biologicals 25(2):209–213. [PubMed: 9236054]
  • Mathews AG. 1976. Antitoxin responses to Clostridium botulinum vaccines types C and D in guinea pigs. Dev Biol Stand 32:193–201. [PubMed: 793915]
  • McBride BW, Mogg A, Telfer JL, Lever MS, Miller J, Turnbull PC, Baillie L. 1998. Protective efficacy of a recombinant protective antigen against Bacillus anthracis challenge and assessment of immunological markers. Vaccine 16(8):810–817. [PubMed: 9627938]
  • Menegon T, Baldo V, Bonello C, Dalla Costa D, Di Tommaso A, Trivello R. 1999. Influenza vaccines: Antibody responses to split virus and MF59-adjuvanted subunit virus in an adult population. Eur J Epidemiol 15(6):573–576. [PubMed: 10485352]
  • Middlebrook JL. 1995. Protection strategies against botulinum toxin. Adv Exp Med Biol 383:93–98. [PubMed: 8644518]
  • Middlebrook JL, Brown JE. 1995. Immunodiagnosis and immunotherapy of tetanus and botulinum neurotoxins. Curr Top Microbiol Immunol 195:89–122. [PubMed: 8542761]
  • Middlebrook JL, Lapenotiere H, Clayton M, Brown D. 1994. Development of a molecularly engineered vaccine for botulinum toxins. FEMS Symposium 73:531–532.
  • Mikhailova IM. 1966. Cl. Botulinum F. Report II, Biochemical Properties. A Study of Toxin- and Toxoid-Formation. Available from the National Technical Information Service. AD-652 659/XAB.
  • Miller FW. 1999. Genetics of environmentally associated rheumatic disease. In: Kaufman LD, editor; , Varga J, editor. , eds. Rheumatic Diseases and the Environment. New York: Oxford University Press. Pp.33–41.
  • Miller J, McBride BW, Manchee RJ, Moore P, Baillie LW. 1998. Production and purification of recombinant protective antigen and protective efficacy against Bacillus anthracis. Lett Appl Microbiol 26(1):56–60. [PubMed: 9489035]
  • Minutello M, Senatore F, Cecchinelli G, Bianchi M, Andreani T, Podda A, Crovari P. 1999. Safety and immunogenicity of an inactivated subunit influenza virus vaccine combined with MF59 adjuvant emulsion in elderly subjects, immunized for three consecutive influenza seasons. Vaccine 17(2):99–104. [PubMed: 9987141]
  • Nass N. 1999. Anthrax vaccine: Model of a response to the biological warfare threat. Infect Dis Clin North Am 13(1):187–208. [PubMed: 10198799]
  • OSAGWI (Office of the Special Assistant for Gulf War Illnesses). 1999. Military Medical Recordkeeping During and After the Gulf War: Interim Report. Washington, DC: U.S. Department of Defense.
  • Ott G, Barchfeld GL, Chernoff D, Radhakrishnan R, van Hoogevest P, Van Nest G. 1995. Design and evaluation of a safe and potent adjuvant for human vaccines. In: Powell MF, editor; , Newman MJ, editor; , Burdman JR, editor. , eds. Vaccine Design: The Subunit and Adjuvant Approach . New York: Plenum Press. Pp.277–296.
  • Parkman PD. 1995. Combined and simultaneously administered vaccines: A brief history. Ann NY Acad Sci 754:1–9. [PubMed: 7625642]
  • Peeler RN, Cluff LE, Trever RW. 1958. Hyper-immunization of man. Bulletin of the Johns Hopkins Hospital 103:183–198. [PubMed: 13584945]
  • Peeler RN, Kadull P, Cluff L. 1965. Intensive immunization of man: Evaluation of possible adverse consequences. Ann Intern Med 63(1):44–57. [PubMed: 14305968]
  • Pile JC, Malone JD, Eitzen EM, Friedlander AM. 1998. Anthrax as a potential biological warfare agent. Arch Intern Med 158(5):429–434. [PubMed: 9508220]
  • Pilipenko VG, Miroshnichenko MG. 1963. Compatibility of STI anthrax vaccine with plague, tularemia and brucellosis vaccines. Zh Mikrobiol Epidemiol Immunobiol 40(3):T551–T554. [PubMed: 13943593]
  • Pittman PR, Sjogren MH, Hack D, Franz D, Makuch RS, Arthur JS. 1997. Serologic Response to Anthrax and Botulinum Vaccines (Protocol NO. FY92-5, M109, Log No. A-5747) . Final Report to the U.S. FDA. Fort Detrick, MD: U.S. Army Medical Research Institute of Infectious Diseases.
  • Plotkin SA, Mortimer EA Jr. 1994. Vaccines. 2nd edition. Philadelphia: W.B. Saunders Company.
  • Potter M. 1971. Myeloma proteins (m-components) with antibody-like activity. N Engl J Med 284(15):831–838. [PubMed: 4101199]
  • Pranter W. 1976. Results of potency tests of a vaccine against Cl. botulinum type C by different methods. Dev Biol Stand 32:185–191. [PubMed: 793914]
  • Puziss M, Wright GC. 1963. Studies on immunity in anthrax. X. Gel adsorbed protective antigen for immunization of man. J Bacteriology 85:230–236. [PMC free article: PMC278112] [PubMed: 13972632]
  • Ramyar H, Baharsefat M. 1969. A new approach to active immunization of sheep by a combined sheep pox and anthrax vaccine. Zentralbl Veterinarmed [B] 16(7):588– 592. [PubMed: 4312593]
  • Rao T, Richardson B. 1999. Environmentally induced autoimmune diseases: Potential mechanisms. Environ Health Perspect 107(Suppl 5):737–742. [PMC free article: PMC1566247] [PubMed: 10502539]
  • Rappuoli R. 1996. European Commission COST/STD Initiative. New vaccines especially new combined vaccines. Vaccines 14(7):691–700. [PubMed: 8799981]
  • Rettig RA. 1999. Military Use of Drugs Not Yet Approved by the FDA for CW/BW Defense: Lessons from the Gulf War. Santa Monica, CA: RAND.
  • Rook GAW, Zumla A. 1997. Gulf War syndrome: Is it due to a systemic shift in cytokine balance towards a Th2 profile? Lancet 349:1831–1833. [PubMed: 9269228]
  • Russell PK. 1999. Vaccines in civilian defense against bioterrorism. Emerg Infect Dis 5(4):531–533. [PMC free article: PMC2627741] [PubMed: 10458959]
  • Safarov YB, Ibragimov NM. 1968. Results of Simultaneous Vaccination of Sheep Against Anthrax, Braxy and Infectious Enterotoxaemia. Available from the National Technical Information Service. AD-675 163/XAB.
  • Salmon DD, Ferrier GR. 1992. Post vaccination occurrence of anthrax in cattle. Vet Rec 130(7):140–141. [PubMed: 1557881]
  • Sellin LC. 1984. Botulism: An update. Mil Med 149(1):12–16. [PubMed: 6142432]
  • Shen DT, Gorham JR, Ryland LM, Strating A. 1981. Using jet injection to vaccinate mink and ferrets against canine distemper, mink virus enteritis, and botulism, type C. Vet Med Small Anim Clin 76(6):856–859. [PubMed: 6911916]
  • Shlyakhov EN. 1970. Anthrax. Biological and immunological principles of diagnosis and prevention. 4. The dynamics and intensity of skin tests with anthraxin in guinea pigs inoculated with a STI vaccine. J Hyg Epidemiol Microbiol Immunol 14(4):464–468. [PubMed: 4992904]
  • Shlyakhov EN, Rubinstein E. 1994. a. Human live anthrax vaccine in the former USSR. Vaccine 12(8):727–730. [PubMed: 8091851]
  • Shlyakhov E, Rubinstein E. 1994. b. Delayed hypersensitivity after anthrax vaccination. I. Study in guinea pigs vaccinated against anthrax. Medecine Tropicale 54(1):33–37. [PubMed: 8196523]
  • Simpson LL. 1989. Botulinum Neurotoxin and Tetanus Toxin. San Diego, CA: Academic Press.
  • Simpson LL. 1993. Botulinum toxin. In: Corn M, editor. , ed. Handbook of Hazardous Materials . New York: Academic Press. Pp.91–98.
  • Singh Y, Ivins BE, Leppla SH. 1998. Study of immunization against anthrax with the purified recombinant protective antigen of Bacillus anthracis. Infect Immun 66(7): 3447–3448. [PMC free article: PMC108368] [PubMed: 9632621]
  • Smith LA. 1998. Development of recombinant vaccines for botulinum neurotoxin. Toxicon 36(11):1539–1548. [PubMed: 9792170]
  • Stefanova EP. 1968. On the Nature of Immunity in Anthrax. The Effect of Trauma and the Significance of Nervous System in the Immunity in Anthrax. Communication III. Available from the National Technical Information Service. AD-833 624/0/XAB.
  • Stepanov AV, Marinin LI, Pomerantsev AP, Staritsin NA. 1996. Development of novel vaccines against anthrax in man. J Biotechnol 44(1–3):155–160. [PubMed: 8717399]
  • Sterne M. 1939. The use of anthrax vaccine prepared from avirulent (uncapsulated) variants of Bacillus anthracis . Onderstepoort Journal of Veterinary Science and Animal Industry 13(2):307–312.
  • Sterne M, Nicol J, Lambrechts MC. 1942. The effect of large scale active immunization against anthrax. J S African Vet Med Assoc 13:53–63.
  • Strandberg TE, Tilvis RS, Miettinen TA. 1990. Metabolic variables of cholesterol during squalene feeding in humans: Comparison with cholestyramine treatment. J Lipid Res 31(9):1637–1643. [PubMed: 2246614]
  • Sverdrup B, Klareskog L, Kleinau S. 1998. Common commercial cosmetic products induce arthritis in the DA rat. Environ Health Perspect 106(1):27–32. [PMC free article: PMC1532946] [PubMed: 9417771]
  • Takafuji ET, Russell PK. 1990. Military immunizations: Past, present, and future prospects. Infect Dis Clin North Am 4(1):143–158. [PubMed: 2407777]
  • Tammemagi L, Grant KM. 1967. Vaccination in the control of bovine botulism in Queensland. Aust Vet J 43(9):368–372. [PubMed: 6052464]
  • Tanner WB, Potter ME, Teclaw RF, et al. 1978. Public health aspects of anthrax vaccination of dairy cattle. J Am Vet Med Assoc 173(11):1465–1466.
  • Thomas RJ, Rosenthal DV, Rogers RJ. 1988. A Clostridium botulinum type B vaccine for prevention of shaker foal syndrome. Aust Vet J 65(3):78–80. [PubMed: 3041951]
  • Tsui JK. 1996. Botulinum toxin as a therapeutic agent. Pharmacol Ther 72(1):13–24. [PubMed: 8981568]
  • Turnbull PC, editor. , ed. 1990. Proceedings of the International Workshop on Anthrax, Winchester, England, April 11–13, 1989. Salisbury Medical Bulletin 68:(Special Supplement).
  • Turnbull PC. 1991. Anthrax vaccines: Past, present and future. Vaccine 9(8):533–539. [PubMed: 1771966]
  • Turnbull PC, Broster MG, Carman JA, Manchee RJ, Melling J. 1986. Development of antibodies to protective antigen and lethal factor components of anthrax toxin in humans and guinea pigs and their relevance to protective immunity. Infect Immun 52(2):356–363. [PMC free article: PMC261006] [PubMed: 3084381]
  • Turnbull PC, Leppla SH, Broster MG, Quinn CP, Melling J. 1988. Antibodies to anthrax toxin in humans and guinea pigs and their relevance to protective immunity. Med Microbiol Immunol (Berl) 177(5):293–303. [PubMed: 3139974]
  • U.K. Ministry of Defence. 2000. Implementation of the Immunisation Programme Against Biological Warfare Agents for UK Forces During the Gulf Conflict 1990/1991. [Online]. Available: http://www​.mod.uk/policy​/gulfwar/info/immunisation_ch1.htm [accessed January 2000].
  • Unwin C, Blatchley N, Coker W, Ferry S, Hotopf M, Hull L, Ismail K, Palmer I, David A, Wessely S. 1999. Health of UK servicemen who served in Persian Gulf War. Lancet 353(9148):169–178. [PubMed: 9923871]
  • USAMRIID (U.S. Army Medical Research Institute of Infectious Diseases). 1996. Medical Management of Biological Casualties: Handbook. 2nd edition. Fort Detrick, MD: USAMRIID.
  • U.S. Department of the Air Force. 1995. Air Force Joint Instruction 48-110. Aerospace Medicine: Immunizations and Chemoprophylaxis (Army Regulation 40-5622/ Bumedinst6230.15/CG COM DINST M6230.4E). November 1, 1995.
  • U.S. DHHS (Department of Health and Human Services). 1990. Informed consent for human drugs and biologics: Determination that informed consent is not feasible. Federal Register 55(246):52814. [PubMed: 11645686]
  • Webb M. 1997. Re: Multiple vaccination. J R Soc Health 117(6):401. [PubMed: 9519681]
  • Welkos SL. 1987. Protective Efficacy and Safety of Live Anthrax Vaccines for Mice. Fort Detrick, MD: U.S. Army Medical Research Institute of Infectious Diseases. Available from the National Technical Information Service. AD–A190 150/3.
  • Welkos SL, Friedlander AM. 1988. Comparative safety and efficacy against Bacillus anthracis of protective antigen and live vaccines in mice. Microbial Pathogenesis 5(2):127–139. [PubMed: 3148815]
  • Whalen RL, Dempsey DJ, Thompson LM, Bucknell K, Kunitomo R, Okazaki Y, Harasaki H. 1996. Microencapsulated vaccines to provide prolonged immunity with a single administration. ASAIO J 42(5):M649–M654. [PubMed: 8944961]
  • White CS, Adler WH, McGann VG. 1974. Repeated immunization: Possible adverse effects. Ann Intern Med 81(5):594–600. [PubMed: 4417964]
  • Whitehouse M, Orr K, Beck F, Pearson C. 1974. Freund's adjuvants: Relationship of arthritogenicity and adjuvanticity in rats to vehicle composition. Immunology 27: 311–330. [PMC free article: PMC1445566] [PubMed: 4214125]
  • Whitford HW. 1987. A Guide to the Diagnosis, Treatment, and Prevention of Anthrax. WHO/ZOON./87.163. Geneva: World Health Organization.
  • Wright GG, Green TW, Kanode RG Jr. 1954. Studies on immunity in anthrax. V. Immunizing activity of alum-precipitated protective antigen. J Immunol 73:387–391. [PubMed: 13212061]
  • Yefremova VN. 1980. Experimental study of skin-sensitizing antibodies after aerosol and subcutaneous immunization. J Hyg Epidemiol Microbiol Immunol 24(1):29–35. [PubMed: 7190588]
  • Yoshino S. 1996. Oral administration of type II collagen suppresses non-specifically induced chronic arthritis in rats. Biomed Pharmacother 50(1):24–28. [PubMed: 8672728]
  • Yoshino S, Yoshino J. 1994. Recruitment of pathogenic T cells to synovial tissues of rats injected intraarticularly with nonspecific agents. Cell Immunol 158(2):305–313. [PubMed: 7522973]
  • Zilinskas RA. 1997. Iraq's biological weapons: The past as future? JAMA 278:418–424. [PubMed: 9244334]
  • Zuffa A, Banda I, Konrad J. 1972. Combined vaccination of mink against Aujeszky's disease and botulism. Zentralbl Veterinarmed [B] 19(9):728–738. [PubMed: 4650800]

Footnotes

1

The committee used the definitions of the Advisory Committee on Immunization Practices (ACIP), which defines “vaccination” as the physical act of administering any vaccine or toxoid and “immunization” as a more inclusive term denoting the process of inducing or providing immunity artificially by administering an immunobiologic. The ACIP states that although the terms are often used interchangeably, they are not synonymous because the administration of an immunobiologic does not automatically equate with the development of adequate immunity (CDC, 1994a).

2

An adjuvant is a substance that is used to increase the immune response to specific vaccine components.

3

Anthrax occurs most commonly in herbivores who ingest anthrax spores from the soil. Naturally occurring cases of human anthrax are the result of contact with anthrax-infected animals or contaminated animal products. There are three clinical forms of human anthrax infection: inhalation, cutaneous, and gastrointestinal. Inhalation anthrax naturally occurs only rarely, but the mortality rate approaches 100 percent (Fauci et al., 1998). Since 1950, the incidence of the disease in animals and man has dropped markedly due in large part to the availability of the vaccine, the use of antibiotics, and the implementation of strict quarantine laws in many countries (Whitford, 1987).

4

During the Gulf War, British troops received the U.K. anthrax vaccine administered simultaneously with the pertussis vaccine in an adjacent site in the deltoid muscle (U.K. Ministry of Defence, 2000).

5

The one live spore vaccine study that met the Cochrane criteria was a field trial conducted by Burgasov and colleagues. In this study, 107,285 individuals received the live spore anthrax vaccination and 49,974 individuals served as controls. The review by Demicheli and colleagues (1998) does not report any information from the Burgasov study regarding adverse effects of the vaccine.

6

Employees who had a previous case of anthrax were not eligible for the study. Of the 1,249 employees eligible for participation, 340 refused to participate in the study.

7

The authors state that there was a gradual decline in participation in the study, partly because of changes in the nature of the textile business and partly because some of the employees withdrew from the program. Reasons for withdrawal were not stated.

8

Adverse events resulting in life-threatening illness, hospitalization, permanent disability, extended hospital stay, or death (Ellenberg, 1999).

9

Botulism is a paralytic disease with three primary clinical manifestations: foodborne, wound, and infant (Sellin, 1984; Fauci et al., 1998). Incidence of the disease is low, fewer than 100 cases of botulism are reported in the United States each year (Lopez, 1998). Ingestion of botulinum spores is the primary exposure pathway. A trivalent equine antitoxin is available from CDC for the treatment of foodborne botulism. A heptavalent antitoxin is currently in IND status (Franz et al., 1997).

10

The primary use has been to reduce muscle spasm by blocking the release of acetylcholine at the neuromuscular junction. This is used in the treatment of strabismus, hemifacial spasm, cervical dystonia, and other spastic disorders (Cardoso and Jankovic, 1995; Tsui, 1996). Transient local muscle weakness can occur; however, no hypersensitivity reactions have been reported (Tsui, 1996). Systemic effects such as fever, malaise, fatigue, and flulike symptoms have been observed but in one double-blind study occurred less frequently than when placebo was given (Tsui, 1996).

11

A toxoid is a modified bacterial toxin that is made nontoxic but has the capacity to stimulate the formation of antitoxin antibodies (Fauci et al., 1998).

12

The directions on the form state that it should be completed for each individual who receives the initial series and should be returned following the third injection. Additionally, a form should be completed and returned to CDC following each booster injection.

13

Mild is defined as erythema, edema or induration 30 mm or less in any one diameter. Moderate is defined as edema or induration measuring greater than 30 mm but less than 210 mm in any one diameter. Severe is defined as any reaction measuring more than 210 mm in any one diameter or any reaction accompanied by marked limitation of motion of the arm or marked axillary node tenderness.

14

Its chemical formula is 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene.

15

Squalene concentrations in shark liver oil range between 50 and 80 percent (Liu et al., 1976).

Copyright 2000 by the National Academy of Sciences. All rights reserved.
Bookshelf ID: NBK222854

Views

  • PubReader
  • Print View
  • Cite this Page
  • PDF version of this title (1.6M)

Related information

  • PMC
    PubMed Central citations
  • PubMed
    Links to PubMed

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...