Anthrax Vaccines

Clinical Description

There are three primary forms of anthrax: cutaneous, inhalational, and gastrointestinal. Secondary meningitis can occur with all three forms but is most often observed with inhalational anthrax. Rarely, meningitis has been reported without a primary site identified. In the United States, approximately 95% of reported cases of clinical anthrax have been cutaneous and 5% inhalational.

Bacteriology

B. anthracis , the causative agent of anthrax, is a large, Gram-positive, spore-forming, nonmotile bacillus (1.0 to 1.5 × 3 to 10 mm). The organism grows readily on sheep blood agar aerobically and is nonhemolytic under these conditions. The colonies are large, rough, and gray-white in color, with irregular, tapered, curving outgrowths that cause the typical “medusa head” appearance. A loop drawn up through a colony makes the disturbed part of the colony stand upright like whipped egg white. In the presence of high concentrations of carbon dioxide, the organisms form antiphagocytic capsules and two exotoxins, and colonies are noted to be smooth and mucoid. In tissue, the bacteria are encapsulated and appear singly or in chains of two or three bacilli. Bacterial identification is confirmed by the production of toxin, lysis by a specific γ bacteriophage, the presence of a capsule and cell wall polysaccharide confirmed by fluorescent antibody testing, and confirmed virulence when injected in mice and guinea pigs. Polymerase chain reaction (PCR) tests for toxin and capsule genes are also confirmatory. Genetic analyses of different isolates reveal that B. anthracis is one of the most monomorphic, homogeneous bacterial species known. ^,

The spores are quite resistant to environmental extremes and may survive for decades in soil (see “Epidemiology” below). Recent evidence also suggests that B. anthracis infected with lysogenic bacteriophage can survive in soil and colonize earthworm intestines for prolonged periods in the vegetative state and may serve as a source of infectious spores. Viable spores were reported to persist for weeks to months within the lungs of rhesus monkeys after inhalation, while receiving antibiotic treatment. After the cessation of antibiotic treatment, residual spores remain viable and can cause fatal disease.

Pathogenesis

The major pathogenic virulence determinants of B. anthracis are the capsule and two protein exotoxins, lethal toxin and edema toxin. More recently, additional factors have been shown to contribute to virulence, although to a lesser degree than the capsule and toxins. These other factors include a capsule depolymerase responsible for attachment of the capsule to the cell wall peptidoglycan, a manganese adenosine triphosphate (ATP)-binding cassette transporter, siderophore biosynthesis genes, nitric oxide synthase, a caseinolytic protease component (ClpX), a surface protein that may be involved in cell adhesion (BslA), and a high temperature requirement A gene involved in the bacterial response to stress.

The importance of the capsule was appreciated early in the 20th century when Bail demonstrated that organisms without a capsule were avirulent. However, extensive studies by Sterne and others in the 1930s showed that nonencapsulated strains could induce immunity to anthrax. The strains developed by Sterne have proved remarkably effective as live vaccines for domesticated animals and are used worldwide.

As is true for many bacterial virulence factors, the genes encoding the anthrax capsule are carried on an extrachromosomal 96-kb plasmid (pX02). ^, Anthrax strains lacking the capsule plasmid fail to produce capsule and are attenuated. The capsule is a homopolymer of poly- d -glutamic acid and enhances virulence by making the organism resistant to phagocytosis and to lysis by cationic proteins. ^, Capsule released from the surface may also interfere with resistance, maturation of antigen presenting cells resulting in reduced chemotaxis to chemokines, and encapsulation inhibits human dendritic cell responses.

A role for toxins in anthrax pathogenesis was suspected from the earliest studies by Koch, but it was not firmly established until 1954, when Smith and Keppie demonstrated that sterile plasma from experimentally infected guinea pigs was lethal when injected into other animals. Evans and Shoesmith showed that B. anthracis culture filtrates produced edema after injection into the skin of rabbits. Additional work in the 1950s and 1960s evaluated the impact of toxins in disease and immunity. ^, Since the mid-1980s, many advances have been made in the understanding of the molecular biology of the toxins and their effects on multiple cell types in vivo and in vitro. Nevertheless, their precise role in pathogenesis remains undefined. Anthrax disease has been characterized by some as being due to a large bacterium that produces a feeble toxin. Although it is clear that anthrax is an invasive disease and that anthrax lethal toxin, when given intravenously, is relatively impotent compared with other bacterial toxins, the lethal and edema toxins are essential for virulence and thought to be important in the establishment of disease by impairing host defenses.

The anthrax lethal and edema toxins, like many bacterial toxins (e.g., diphtheria, tetanus, botulinum), have a binding domain by which they bind to target cell receptors and an active domain that is responsible for the biochemical and usually enzymatic activity of the toxin. However, the anthrax toxins are unusual in that the binding and active domains exist in two distinct proteins, and the two active domain proteins share the same binding protein. This binding protein is called protective antigen (PA). PA combined with a second protein called lethal factor (LF) constitutes the anthrax lethal toxin, which is lethal when injected into experimental animals. ^, The same PA combined with a third protein, edema factor (EF), constitutes the edema toxin, which causes edema when injected into experimental animals ^, and can also be lethal for some animals. The edema toxin is responsible for the massive edema that may be present in cases of anthrax, whether in the skin or mediastinum. The 89-kDa EF is a calmodulin-dependent adenylate cyclase that raises intracellular cyclic adenosine monophosphate (AMP) levels. The 85-kDa LF is a zinc metalloprotease that inactivates mitogen-activated protein kinase and also inhibits the phosphoinositide-3-kinase pathway, thus interfering with multiple host cell signaling pathways. ^, Toxicity for macrophages appears to be mediated through activation of caspase-1, requires a specific inflammasome protein, and results in lysosomal permeabilization. Consistent with this model, each of the individual proteins alone lacks biologic activity.

The crystal structures of PA, LF, and EF have all been determined. The current model, based on cell culture studies, proposes that PA first binds to a host cell toxin receptor ( Fig. 12.1 ). Two of these receptors have now been identified, ^, tumor endothelial marker 8 and capillary morphogenesis gene 2. Both receptors have an extracellular van Willebrand factor A domain involved in binding of PA and additional cell-surface proteins may modulate their activity. ^, PA then is cleaved by a cell-surface protease, releasing a 20-kDa aminoterminal fragment. The cell-bound 63-kDa carboxyterminal fragment oligomerizes to a heptamer or octamer and creates a second binding domain to which either or both of the active proteins (LF or EF) binds. The complex then enters the cell through endocytosis with LF and EF passing through a PA channel induced by the low pH of the endosome, recently visualized by cryoelectron microscopy, ^, to exert their intracellular toxic effects. PA may also be cleaved by proteases in the circulation to form oligomers with LF and EF, which then bind to cell receptors. An alternative pathway has recently been described whereby LF within extracellular vesicles or exosomes can enter and intoxicate cells independently of the PA receptor.

The genes for toxin production are carried on a second 182-kb plasmid (pX01). Strains deleted of the plasmid coding for the toxin genes remain encapsulated, but are attenuated. ^, Of historical significance, it appears that the veterinary vaccine strains produced by Pasteur through passage at high temperature lacked the plasmid for the toxin genes, explaining their attenuation. It is thought that vaccine preparations consisted of a mixture of organisms, most of which lacked the toxin plasmid together with small numbers of fully virulent organisms that retained the toxin plasmid and were responsible for the vaccine’s efficacy. Further work has shown that deleting the PA gene alone eliminates the organism’s virulence, thus confirming the central role of PA in the activity of the two toxins and their role in virulence.

Early studies showed that crude toxin preparations or combinations of edema and lethal toxins inhibited neutrophil killing, chemotaxis, or phagocytosis, and that edema toxin inhibited neutrophil phagocytosis and priming of the respiratory burst of neutrophils. The toxins have effects early in infection on inhibiting the host innate immune responses and later during infection have systemic effects on the cardiovascular and other systems when toxin levels are greatly elevated but the relevance of studies with isolated toxins to the changes during infection remain to be confirmed. At low concentrations, lethal and edema toxins also appear to block the production of proinflammatory cytokines from macrophages, ^, directly inhibit macrophage chemotaxis, and interfere with the early protective inflammatory response. At high concentrations, lethal toxin is cytolytic for macrophages from some mouse strains. These mouse studies have shown that strains with macrophages and neutrophils resistant to lethal toxin, as seen in human cells, are more resistant to infection, with neutrophils more important for resistance than macrophages. However, the relevance of these studies in mice with attenuated unencapsulated strains to the mechanism of toxin pathogenesis of human disease remains to be determined. Lethal and edema toxins also have deleterious effects on a multitude of other cells, including lymphocytes and dendritic cells, endothelial cells, glucocorticoid receptor–positive cells, and cardiac myocytes, but the physiologically important cell target(s) in the infected host remains to be established. In terms of pathogenesis, the greater importance of lethal toxin versus edema toxin was demonstrated with a mouse model in which an anthrax strain producing the lethal toxin alone retained some virulence, whereas a strain producing only edema toxin was avirulent; the parent strain producing both toxins was the most virulent. More recent studies show that lethal toxin, but not edema toxin, is essential for virulence in nonhuman primates. In contrast, studies in rabbits show that strains producing either lethal or edema toxin are equally virulent but both toxins are necessary for full virulence. These results demonstrate the variability in pathogenesis in different animal models and the caution that must be used in extrapolating results in animals to humans.

In cutaneous anthrax, infection begins when a spore is introduced through the skin. At the local site, the spore germinates into the vegetative bacillus, which produces the antiphagocytic capsule. The edema and lethal toxins produced by the organism impair leukocyte function and contribute to the distinctive findings of tissue necrosis, edema, and relative lack of leukocytes in the local lesion. If not contained, the bacilli spread to the draining regional lymph nodes, allowing further production of toxins and induction of the typical hemorrhagic, edematous, and necrotic lymphadenitis. From the lymph nodes, the bacteria continue to multiply and enter the bloodstream, producing a systemic infection.

In inhalational anthrax, spores ingested by alveolar phagocytes and lung dendritic cells are transported to the tracheobronchial and mediastinal lymph nodes, where they germinate. ^, Local production of toxins by extracellular bacilli leads to the massive hemorrhagic, edematous, and necrotic lymphadenitis and mediastinitis characteristic of this form of the disease. The bacilli then spread to the blood, causing septicemia. In approximately half the cases there may be hemorrhagic meningitis. PA and LF are detectable in the blood early and at high concentrations later, ^, with the lethal toxin occurring as a complex of PA and LF at late stages of disease. Capsule is also present at high levels. The site of action and the role of lethal and edema toxins in the mechanism of death from infection remain obscure. Death is a result of respiratory failure with overwhelming bacteremia that is often associated with meningitis and subarachnoid hemorrhage.

Diagnosis

A diagnosis of cutaneous anthrax should be considered after the appearance of a painless, pruritic papule that develops into a 1- to 2-cm vesicle, revealing a black eschar at the base of a shallow ulcer. Examination by Gram stain or culture of the vesicular fluid should confirm the diagnosis, but prior antibiotic therapy quickly renders the infected site culture-negative. Biopsy at the lesion edge, examined by Gram stain, immunohistology, and PCR, may be useful in people who have been treated with antibiotics. The latter two tests are available through the Laboratory Response Network (see below). In addition, there should be a history of exposure to materials contaminated with B. anthracis .

The diagnosis of inhalational anthrax is more difficult but should be suspected in cases with a history of exposure to an aerosol containing B. anthracis , followed by an initial phase during which symptoms are non-specific. Once the acute stage develops, a widened mediastinum seen on chest radiograph, often with pleural effusions, should suggest the diagnosis. In untreated cases, culture of blood and pleural effusions will readily establish the diagnosis, and more rapid detection methods, including PCR for the PA gene, immunoassay for capsule antigens, and mass spectrometry for LF or lethal toxin, and PA may all be useful. ^{, ,} These tests are available through the Laboratory Response Network and state health departments ( http://www.cdc.gov/anthrax/labs/labtestingfaq.html ). In cases previously treated with antibiotics, these assays for toxin and capsule in blood and pleural fluid, as well as immunohistologic examination of pleural fluid or transbronchial biopsy specimens, are of particular value ^{, , ,} and may be positive as long as 6–8 days after initiation of antibiotic therapy. ^, Because primary pneumonia is not a feature of inhalational anthrax, sputum examinations do not aid in the diagnosis. The radiographic differential diagnosis should include histoplasmosis, sarcoidosis, tuberculosis, and lymphoma. A computed tomography scan of the chest may be helpful to detect hemorrhagic mediastinal lymphadenopathy and edema, peribronchial thickening, and pleural effusions.

Gastrointestinal anthrax is difficult to diagnose because of its rarity and similarity to other more common severe gastrointestinal diseases. An epidemiologic history of ingesting contaminated meat, particularly in association with other similar outbreak cases, suggests the diagnosis. Microbiologic cultures are not helpful in confirming the diagnosis unless bacteremia is present. The diagnosis of oral–oropharyngeal anthrax can be made from the clinical and physical findings, but the role of bacteriologic cultures in confirming this diagnosis remains unclear.

Treatment and Prevention With Antibiotics

Mild cases of cutaneous anthrax may be treated effectively with oral penicillin, fluoroquinolone, doxycycline, or another antibiotic, depending on antimicrobial susceptibility. If rapid spread or prominent systemic symptoms are present, high-dose parenteral therapy as for inhalational anthrax (see below) should be given until there is a clinical response. Effective therapy reduces the edema and systemic symptoms but does not change the evolution of the skin lesion. In injectional anthrax parenteral antibiotic therapy as for inhalational anthrax (see below) should be initiated and surgical debridement and fasciotomy may be required.

Treatment of inhalational anthrax requires high-dose intravenous therapy (ciprofloxacin or another fluoroquinolone) with at least one or more antibiotics with good central nervous system penetration, for example, meropenem and linezolid, because of the high incidence of meningitis. ^{, , ,} This approach also applies to gastrointestinal anthrax. Regimens should be altered based on susceptibility testing available at the Laboratory Response Network and state health departments along with the clinical status. The successful treatment of six of the 11 inhalational cases in the 2001 bioweapon attacks suggests that, with rapid antibiotic treatment and modern supportive care, including aggressive management of respiratory distress and pleural effusions, mortality is similar to that of other causes of sepsis.

After exposure to an aerosol of anthrax, postexposure prophylaxis or presymptomatic treatment should include oral antibiotics for 60 days or more, depending on individual circumstances (e.g., extent of exposure, vaccination status; see later text). ^, Ciprofloxacin or doxycycline are recommended. Other oral antibiotics, including other fluoroquinolones, clindamycin, or amoxicillin or penicillin VK if the strain is susceptible, are alternatives. Levofloxacin is indicated as a second-line drug because of limited safety data beyond 30 days. Amoxicillin is recommended for children and pregnant or lactating women, depending on microbial resistance. ^, Pre-exposure or postexposure vaccination may enable shorter courses of antibiotics (see later text). ^, Postexposure vaccination alone would not be expected to protect quickly enough to be effective.