Plague vaccines

SECTION TWO: Licensed vaccines

Plague vaccines E. Diane Williamson Petra C. F. Oyston

During the past two millennia, the bacterium Yersinia pestis has been responsible for social and economic devastation on a scale unmatched by other infectious diseases or armed conflicts. The first reliable reference is to the Justinian plague (AD 542–750), which originated in central Africa and spread throughout the Mediterranean Basin. The second pandemic, the Black Death, which started on the Eurasian border in the mid-14th century, may have caused 25 million deaths in Europe (25% to 30% of the population), persisted on the continental land mass for several centuries, and culminated in the Great Plague of London in 1665. The third pandemic started in China in the mid-19th century, spread east and west, reaching North America in the late 19th century, and caused 10 million deaths in India alone. Credible estimates indicate that almost 200 million deaths could be attributed to plague,1 which swept across Europe in these three major epidemics.2 The disease occurred in both bubonic and pneumonic (“black death”) forms. The bubonic form spreads as a result of transmission of the bacterium from rodents to humans via the bites of infected fleas (usually the rat flea, Xenopsylla cheopsis2). The close contact of humans with infected rats undoubtedly contributed to the spread of the disease by this route. In some instances, bacteremic spread of plague bacilli to the lungs leads to the development of the secondary pneumonic form of the disease; subsequent person-to-person transmission by respirable droplets can result in rapid epidemic spread of primary pneumonic plague.3 It is the pneumonic form of the disease that is most feared and that is associated with a mortality rate approaching 100% when untreated.4,5 For reasons that are not fully understood, epidemics of urban plague have dramatically waned, but data from the 1990s reported by the World Health Organization (WHO) indicate that plague was still a significant public health problem, especially in Africa, Asia, and South America (Figure 24–1).6,7 In 2005, an outbreak of pneumonic plague at a diamond mine in northern Congo caused 114 confirmed infections among the miners, 54 of whom died.8 The outbreak was limited only by the dispersal of miners fleeing in panic from the area.8 Between 2005 and 2009, more than 12,500 cases of plague were reported to the WHO, with a fatality rate of 6.7%, from 16 countries.9 The majority of these cases (> 97%) were reported from eight African countries, with less than 150 cases each in Asia and the Americas, resulting in 23 and 6 deaths on these continents, respectively. During 2009, the United States reported 27 plague cases resulting in five deaths, and an outbreak in the Qinghai Province of China, associated with the marmot hunting season, resulted in 12 cases of pneumonic plague with three deaths.9 Thus, even in the 21st century, plague still occurs episodically

24

in parts of the world where environmental conditions permit. In plague-endemic areas of the world, vaccines may be useful in preventing plague in people at high risk, as well as in those involved in research and diagnosis of the disease.

Clinical description Bubonic plague Bubonic plague is the form of disease typically transmitted to man by an infected flea that fed previously on an infected rodent. In some circumstances, infection can occur via open wounds that are exposed to infected material through handling and other direct contact.4 Within 2 to 6 days of infection, the patient develops a fever, headache, and chills.10 Occasionally, lesions develop at the site of inoculation. The classic feature of bubonic plague is the development of swollen and tender lymph nodes called buboes, from the Greek bubon, meaning groin. The buboes are often located in the inguinal and femoral lymph nodes, which drain the original site of infection on a lower extremity.4 Bacteremia is common in patients with bubonic plague, typically resulting in blood culture counts ranging from fewer than 10 and up to 4 × 107 colony-forming units (CFU) per milliliter.4 High levels of bacteremia are often associated with gastrointestinal symptoms such as vomiting, nausea, abdominal pain, and diarrhea.4 Intervention early in the course of disease, with an antibiotic such as streptomycin, gentamicin, tetracycline, chloramphenicol, or a fluoroquinolone, usually leads to rapid recovery.11

Septicemic plague Primary septicemic plague is a clinical diagnosis in a patient who has acute toxic illness, large numbers of Y. pestis in the bloodstream, and no identifiable anatomic site of infection, such as a peripheral bubo.10 Clinically, the disease appears similar to other Gram-negative septicemias, with elevated temperature, chills, headache, malaise, and gastrointestinal disturbances. In the absence of aggressive treatment, life-threatening complications of the systemic inflammatory response syndrome occur, such as disseminated intravascular coagulation and bleeding, adult respiratory distress syndrome, shock, and organ failure. Because of the absence of localizing signs, the diagnosis of primary plague sepsis may be delayed, which may result in a high case-fatality rate. The overall case-fatality rate for persons with plague sepsis is in the range of 20% to 40%.

494

SECTION TWO • Licensed vaccines

World distribution of plague, 1998 Countries reported plague, 1970–1998 Regions where plague occurs in animals Figure 24–1  Occurrence of plague in the modern world. The map depicts the probable foci of plague in red, with countries reporting plague (1998) in ochre. (Adapted from World Distribution of Plague [www.cdc.gov/ncidod/dvbid/plague]. Atlanta, GA: Centers for Disease Control and Prevention, Division of Vectorborne Diseases, 1998.)

Pneumonic plague Some colonization of pulmonary tissues occurs in virtually all untreated fatal cases of plague, with the potential to develop into a secondary pneumonic plague. However, most of these patients do not develop a transmissible plague pneumonia.10 However, when there is colonization of the alveolar spaces after a respiratory exposure, a suppurative pneumonia develops, and during the terminal stages of disease there is coughing and the production of a highly infectious, watery, and bloody sputum. Pneumonic plague is the most widely feared form of the disease because it is proposed that Y. pestis can be spread from person to person as respiratory droplets formed during coughing.12 A number of experiments with animals (guinea pigs, lemurs, and nonhuman primates) have demonstrated the potential for the cross-infection of control animals from infected animals showing the symptoms of pneumonic disease.13-17 More significantly, good evidence supporting the potential for the airborne spread of infection in humans has been derived from an analysis of an outbreak of pneumonic plague in Madagascar.18 In this outbreak, four cases of pneumonic plague were attributed to contact with one patient who had developed secondary pneumonic plague. These four infected persons apparently then transmitted the disease to 11 others, one of whom transmitted the disease to two further persons.18 In total, 18 persons contracted pneumonic plague, and eight died. The available evidence indicates that the inhalation of airborne droplets containing Y. pestis by susceptible persons leads to the development of pneumonic plague in 1 to 3 days.10 The rapidity with which the infection spreads between persons, along with the relatively short incubation period, makes control of the disease difficult, and antibiotic therapy may be ineffective after pulmonary symptoms have developed.4,10,11 Primary pneumonic plague is now rare; in the United States, the few cases that have been reported in recent years have been

in ­veterinarians and owners of domestic cats who have contracted pneumonic plague from their pets4,5 or in persons who have been exposed to infected wild animals. Nevertheless, the potential for pneumonic plague to spread quickly in human populations is evident—for example, the 2005 outbreak in the Northern Congo diamond mine.8

Bacteriology The etiologic agent of plague is Y. pestis, a Gram-negative bacterium that is a member of the family Enterobacteriaceae. The bacterium can grow at temperatures between 4° and 40° C and has nutritional requirements for l-isoleucine, l-valine, l-­methionine, l-phenylalanine, and glycine. The species has been subdivided into three biovars (orientalis, mediaevalis, and antigua) on the basis of the ability to convert nitrate to nitrite and to ferment glycerol; however, all three biovars show similar ­virulence in animal models.4 The genus Yersinia also includes Y. enterocolitica and Y. pseudotuberculosis species, both of which are pathogens of humans but rarely cause disease with a fatal outcome.1,11 It has been shown that Y. pestis evolved from Y. pseudotuberculosis (most probably serotype 1b) between 1,500 and 20,000 years ago.19 Recent attempts to reconstruct the genome sequence of the medieval Y. pestis from DNA isolated from the teeth of victims of the Black Death have not indicated major genetic differences between the medieval strain and a contemporary strain of Y. pestis (CO92), although reliance on CO92 as a reference could not be expected to indicate genomic regions that might have existed in the ancient strain and subsequently lost.20 An intermediate form, termed Pestoides, has been identified. Genome sequence analysis reveals that Pestoides strains do not contain the full complement of plasmids associated with Y. pestis, but that they do retain chromosomal loci found in

Plague vaccines

24

LPS (endotoxin)

pH6 antigen (aids establishment of infection)

Polysaccharide capsule incorporating aggregated F1 protein; capsule is antiphagocytic Yersinis outer proteins (Yop’s) with array of anti-host activities

Plasminogen activator; protease secreted early in infection which aids bacterial dissemination in the host

Secreted V protein is located with Yersinia secretory factor F (YscF) on bacterial cell surface; regulates Type Three secretion

Y. pseudotuberculosis but lost in Y. pestis.21,22 Pestoides isolates are virulent, particularly by aerosol routes,21,22 but can show attenuation by systemic routes.22 Evolution of Y. pestis has entailed the inactivation of a range of genes that are thought to be required for an enteric lifestyle, and acquisition of new virulence factors.23 The acquired virulence genes appear to be located primarily on two plasmids. Both Y. pestis and Y. pseudotuberculosis possess a 70-kilobase (kb) virulence plasmid called pYV that carries a type III secretion system operon.24,25 Y. pestis possesses two further unique plasmids, a 9.5-kb plasmid, pPCP1, and the 100- to 110-kb pFra plasmid.1 The pla gene on pPCP1 encodes a surface-bound protease (plasminogen activator), which has potent fibrinolytic activity (Figure 24–2).1 The pFra plasmid shows extensive sequence homology with plasmid pHCM2, possessed by some strains of Salmonella enterica serovar Typhi.26 However, some regions of pFra appear to be unique to Y. pestis, and one of these regions includes the caf operon responsible for synthesis of the protein capsule expressed by Y. pestis at 37° C.27

Pathogenesis Plague has a complex lifestyle, cycling between arthropod and mammalian hosts, and the primary mammalian hosts are rodents; humans are accidental hosts. As a result of this lifestyle, the organism has had to develop sophisticated strategies to ensure transmission from infected mammal to the flea, then from the flea to a new host. The flea ingests bloodborne bacteria from the infected rodent, and growth of the bacteria leads to blockage of the flea's foregut. The hemin storage system is thought to play an important role in the formation of this blockage.28 The blockage prevents digestion of the blood meal, and further ingestion of blood leads to regurgitation of bacteriacontaminated material.29 In achieving its transmission, the pathogen results in the death of both mammalian host and flea vector. High levels of septicemia in the infected mammal are required to allow the infection of a subsequent flea vector, and efficient transmission from the flea is required to infect the mammal.30 However, when humans and rodents are in close proximity, or when the rodent population is reduced as a result of the disease or of rodent control measures, humans and other warm-blooded mammals serve as alternative hosts. Y. pestis differs from the other human pathogenic Yersinia species in that it is an obligate parasite. The organism circulates in a “sylvatic” form in wild rodent populations, typically causing a fatal disease in mice and squirrels and a milder, subclinical infection in gerbilline and dipodids31 such as the Jerboas. Other reservoirs of

Figure 24–2 Cartoon depicting virulence factors expressed by Yersinia pestis.

infection include prairie dogs, rabbits, and members of the cat family, including the domestic cat.4 Thus, the infection of humans usually occurs as the result of a bite from an infected flea, and it has been suggested that as many as 24,000 bacteria are delivered into the host with a single bite.4 To date, only three factors have been positively confirmed as essential for transmission from flea to mammal: murine toxin, an extracellular polysaccharide, and a lipopolysaccharide (LPS) core modification locus. The murine toxin has phospholipase D activity, which is essential for survival in the flea midgut,32 whereas the exopolysaccharide and LPS modification are required for biofilm formation and blockage of the flea.28,33 However transcriptional analysis of Y. pestis in the flea gut identified a wide range of genes, such as the insecticide-like toxin yit and yip genes in Y.p.locus (y0181-0191) that were differentially regulated, expression of which meant that the bacteria regurgitated into a new host had increased resistance to innate immune effectors.34 With infection of a new host, the plague bacilli are vulnerable to phagocytosis by polymorphonuclear leucocytes or monocytes. However, bacteria from fleas showed significantly lower levels of phagocytosis than bacteria grown in vitro, indicating that their growth inside the flea contributes to their pathogenesis in a complex way.34 The insecticide-like toxins were a key component in resisting phagocytosis in the early stages of infection,34 before the antiphagocytic type III secretion system could be induced. There is controversial evidence that preferential phagocytosis into monocytes or macrophages may be facilitated by the chemokine receptor Ccr5,35-37 prevalent on these cells and on dendritic cells, but not on polymorphonuclear cells, such as neutrophils. A specific role for Ccr5 in plague pathogenesis remains uncertain, appearing possible from in vitro bacterial uptake studies but less likely from in vivo susceptibility studies in Ccr5 knockout mice, although Ccr5 is a coreceptor with CD4 facilitating entry of another infection—with the human immunodeficiency virus (HIV) —into host cells.35–37 The plague bacteria in polymorphonuclear leucocytes are destroyed, but those in monocytes survive and express various virulence determinants, thereby allowing growth and eventual release from the monocytes.38 One such virulence determinant is the pH6 antigen, a fibrillar adhesin induced by low pH conditions such as those encountered in the phagosome, which has a pH of 4.5.39 The expression of the pH6 antigen fimbriae enhances resistance to phagocytosis by macrophages.40 The F1 capsule might be expected to play a key role in avoiding further phagocytosis.41 However, mutants of Y. pestis that are unable to produce F1 antigen are still able to cause disease in the mouse, albeit in a less acute form.42 It appears, however, that possession of the locus encoding the F1 antigen increases transmissibility from the flea,43 although this is not observed after needle challenge in a laboratory setting.

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SECTION TWO • Licensed vaccines

However, the dominant antiphagocytic effect is the result of induction of the type III secretion system carried on the large virulence plasmid. The type III system effectors were historically called Yersinia outer membrane proteins (Yops), and their secretion into host target cells results in phagocyte killing. The function of many of the Yops has been determined in this wellcharacterized secretion system, which serves as a paradigm for type III secretion systems.24 For example, the YopE protein is a cytotoxin that is transferred into host cells after bacterial contact;44 the YopH protein is a tyrosine phosphatase that has ­antiphagocytic-cell activity.44 The V antigen component of the type III system appears to play several roles in the pathogenesis of disease. First, it is shown to be present at the tip of the injectisome, which delivers Yops into the host cell.45 Additionally, V antigen secreted from Y. pestis is reported to exert a local immunomodulatory effect in the host, by downregulating the production of interferon γ and tumor necrosis factor α.46,47 Another major virulence factor required for dissemination from the site of infection is the Plasminogen activator (Pla). Pla is a protease located in the outer membrane, and it appears that its essential contribution in infection is that it has driven the selection of the rough phenotype; rare among Gram-negative pathogens, Y. pestis lacks an O antigen.48,49 However, the forced expression of O antigen blocks the activity of Pla and is thus attenuating by both dermal and respiratory routes of infection50,51 but not after intravenous inoculation.52 Pla interacts with the DEC-205 receptor on antigen-presenting cells to promote uptake, and it appears that dissemination occurs only after phagocytosis.51 The bacteria are disseminated from the site of primary infection into regional lymph nodes that drain these tissues. In the lymph node, further growth of the bacteria, accompanied by a massive inflammatory reaction, leads to lymphadenopathy and the formation of buboes. In the bubo, bacteria are predominantly extracellular, mainly because of the type III secretion system, which is highly expressed in the lymph node.43 An ability to proliferate in the bubo requires efficient iron acquisition systems. Y. pestis possesses 10 iron acquisition systems, which are apparently actively utilized in the bubo.43 Eventually, the bacteria are disseminated by the lymphatic system, gain access to the bloodstream, and colonize pulmonary tissues, which may lead to development of the pneumonic form of the disease. Untreated, pneumonic plague is almost invariably fatal, inducing an overwhelming septicemia that triggers septic shock in the host. However, the precise mechanisms that lead to death of the host have not been identified, although the systemic induction of nitric oxide synthase may be the terminal event, as in other Gram-negative septicemias.53

suitable for the evaluation of plague vaccines,60 and this species was approved by the US Public Health Service for the testing of plague vaccines. Disease arising from the delivery of Y. pestis by the subcutaneous route (median lethal dose [MLD], 1 to 2 CFU61) is considered to mimic bubonic plague, whereas the exposure of mice to the bacteria via aerosols inhaled through the nose results in the pneumonic form of the disease (MLD, most recently estimated to be in the range 601-2951 cfu). The efficacy of the original Cutter Killed Whole Cell (KWC) vaccine was determined by measuring the ability of sera passively transferred from immunized mice, guinea pigs, monkeys, or humans to protect naïve mice against Y. pestis, with the derivation of a mouse protection index (MPI).63 A potency test based on active immunization and organism challenge in the mouse model is being developed to test the efficacy of a next generation subunit plague vaccine for clinical lot release.

Diagnosis of plague The clinical diagnosis of plague is supported by laboratory tests. Bacteriologic diagnosis of plague is usually made on the basis of the analysis of aspirates taken from a bubo, or from blood or sputum samples. The most straightforward analysis involves Gram- or Wyson-staining of air-dried smears on microscope slides.64,65 After staining, Y. pestis cells appear as small Gramnegative rods with a characteristic bipolar staining (safety pin appearance). Culture on Congo red agar, ideally at 26° to 28° C, results in so-called bulls-eye colonies, which have a redpigmented central region and paler margins.65 However, culture methods are generally considered to be too slow (typically taking 48 hours) in the context of the rapid progression of the disease (and especially pneumonic plague), so a presumptive diagnosis should be made before such tests are carried out. A variety of confirmatory tests for Y. pestis have been proposed, including fluorescent antibody to the capsular F1 antigen, passive hemagglutination, polymerase chain reaction, and enzyme-linked immunosorbent assay (ELISA).61,66–68 These tests can be used with samples such as bubo aspirates for the rapid diagnosis of disease.66 An exciting development is the demonstration that an immunogold-chromatography dipstick can be used for the direct analysis of bubo aspirates, serum, or urine samples.69 Conceivably, such tests might be used outside the laboratory and at the patient's bedside for the rapid diagnosis of plague.

Treatment and prevention with antimicrobials Models of disease and protection Y. pestis causes disease in a wide variety of laboratory animals,4 and animal models of the disease in humans have been developed using not only mice but also the brown Norway rat,54 guinea pigs, and nonhuman primates.55 Additionally, nonlaboratory species such as the black-footed ferret56 and the prairie dog57 have been actively immunized against plague. However, most preliminary experimental work has been carried out with the mouse model of disease, which has been accepted as a meaningful indicator of the likely responses to infection and protection in humans. However, the disease in the mouse model may not faithfully mimic disease in humans because of the susceptibility of mice to the murine exotoxin.58 Although this ­limitation might be resolved by using the guinea pig model of disease, the protracted nature of the disease in this species59 suggests that the mouse model is a better indicator of the infection that occurs in humans. In a comparative study with mice and guinea pigs, it was concluded that mice were more

The successful treatment of plague depends on the prompt commencement of therapy during its early stages. In cases of bubonic plague treated with antibiotics, the fatality rate is generally less than 5%. In contrast, the successful treatment of septicemic or pneumonic plague is less certain because of the rapidity with which the disease develops and the difficulties of making an early diagnosis. A number of antibiotics including streptomycin, tetracycline, and ciprofloxacin are approved by the US Food and Drug Administration for the treatment of plague.64,70 For the treatment of plague meningitis, intravenously administered chloramphenicol is the antibiotic of choice because of its ability to cross the blood–brain barrier.64,70 For all antibiotics, a 10-day course is recommended. In mice infected with Y. pestis by the airborne route, successful treatment of disease with ciprofloxacin depended on starting antibiotic dosing within 24 hours of exposure to the pathogen.70 Improvements in the condition of patients suffering from bubonic plague are seen within 2 to 3 days, but buboes may

Plague vaccines

remain for several weeks after successful treatment. A strain of Y. pestis showing plasmid-mediated resistance to tetracycline, streptomycin, chloramphenicol, and sulfonamides was isolated from a case of bubonic plague in Madagascar.71 The failure of a patient to respond to treatment with these antibiotics should alert the clinician to the possibility of an antibiotic-resistant strain of Y. pestis. Family members and other close contacts of persons suffering from plague should be maintained under surveillance for at least 7 days after their last possible exposure to Y. pestis. It may be appropriate to use tetracycline or doxycycline prophylactically for 7 days in these people, especially if the primary case is pneumonic plague. The prophylactic use of antibiotics is recommended even for those who were previously vaccinated with the KWC vaccine and who might have been exposed to airborne Y. pestis,5 because of the limited ability of this type of vaccine to provide protection against pneumonic disease.

Epidemiology Endemic foci of animal plague occur mainly in semiarid regions of the world (see Figure 24–1). The main foci have been identified in the southwestern United States, the former Soviet Union, South America, South Africa, and Asia. Not surprisingly, these are the regions of the world that also report the highest incidence of human plague. Each year there are several thousand reported cases of the disease worldwide72 with a fatality rate between 5% and 15%,6,7 and plague has been classified as a reemerging disease by the WHO. In the United States, an average of 18 cases are reported each year, with a death rate of 1 in 7. There was an apparent increase in the incidence of disease during the 1990s, although it is possible that this increase was the result of more efficient diagnosis and reporting of cases. However, recent statistics on the global incidence of plague serve as a reminder that the disease can reemerge suddenly in endemic regions.9 A significant outbreak of plague occurred at Surat in India in 1994. Serologic testing of persons during this outbreak showed that there were 876 presumptive cases of plague and 54 fatalities.4,73 However in the past few decades, the major incidence of plague activity in the world has shifted from Asia to Africa,9 where plague is now endemic in many countries.74 An outbreak of plague cases in northeastern Tanzania was reported in 1980,75 and by 2004, more than 7,000 cases, predominantly of bubonic plague, had occurred. A report on this focus of plague in northeastern Tanzania from 1986 to 2002 indicated that the risk of infection for children 10 to 14 years old was twice that for adults, and that there was a higher incidence in women than men.74 This femaleto-male bias was also reported for outbreaks in Kenya and Mozambique,76 although in Madagascar the bias reversed, with greater incidence reported in male adults.77 Whether these are true sex effects on susceptibility to plague or more the result of social, occupational, or environmental risk factors is unclear. In Tanzania, plague outbreaks appear to be associated with the wet season, whereas in Madagascar the occurrence of plague cases correlates with the cool dry season.74 In the United States, a new trend in plague epidemiology appears to be related to the residential encroachment on former rural areas that contain enzootic foci,4 putting humans at risk for exposure to flea bites or into close contact with infected wild rodents or other animals.

Potential use as an agent of bioterrorism Recently, plague vaccines have attracted considerable attention because of the potential for Y. pestis to be used as an agent of biological warfare, or bioterrorism.64 It is likely that

24

Y. pestis used in this way would be disseminated by the airborne route and would cause pneumonic plague. A report by the WHO in 197078 indicated that, in a worst-case scenario, the airborne release of 50 kg of Y. pestis over a city of 5 million would result in 150,000 cases of plague. Of these, 36,000 would be expected to die. The released bacteria would remain viable for 1 hour. Disease that resulted from the deliberate release of Y. pestis in this way would appear within 1 to 6 days of exposure, and most probably within 2 to 4 days.64 The initial symptoms would resemble other severe respiratory illnesses,64 which might make early diagnosis and appropriate treatment difficult. The indicators of an attack with Y. pestis would include the clustering of cases, the death of rodents in the area, the incidence of disease in persons without known risk factors, and disease in areas where plague is not known to be endemic.64 The unpredictable nature of such attacks and the potential for epidemic spread of disease from the index case might impose requirements for vaccines and immunoprophylactics that rapidly provide protection against disease (see “Postexposure prophylaxis”, later).

Passive immunization Early studies showed that serum from human volunteers immunized with purified F1 antigen or with a killed whole cell (KWC) vaccine could be used in the passive protection of mice against a parenteral challenge with 100 MLD of Y. pestis.79 More recently, there have been several reports that the passive immunization of mice with antiserum or monoclonal antibodies against the F1 and V antigens provides protection against parenteral challenge with Y. pestis,80,83 demonstrating the potential of postexposure immunoprophylaxis. However, there is currently no licensed immunoprophylaxis or immunotherapy for plague.

Active immunization Killed whole-cell vaccines Killed Y. pestis organisms have been used as a vaccine since 1897, when Waldemar Haffkine inoculated himself with an experimental vaccine. A KWC vaccine for human use was first produced in the United States in 1946 (the so-called Army Vaccine). Improvements to this vaccine led to the plague vaccine USP (for United States Pharmacopeia), which was produced from the virulent 195/P strain of Y. pestis.84 During the 1990s, there were several commercial suppliers of KWC vaccines against plague. Plague vaccine USP, which contains formaldehyde-killed bacteria, was formerly manufactured by Cutter Laboratories but from 1994 was manufactured by Greer Laboratories, Inc. Production of this vaccine was discontinued in 1999. An alternative KWC vaccine was manufactured by Commonwealth Serum Laboratories (CSL Australia)85 until November 2005, and this was licensed for clinical use in Australia. This vaccine contained heatkilled bacteria (Y. pestis strain 195/P) that were resuspended in saline containing 0.5% weight per volume of phenol to a concentration of 3 × 109 organisms per milliliter. The vaccine was given subcutaneously, and the initial course in adults was two 0.5-mL doses of vaccine at an interval of 1 to 4 weeks, with modified regimens for children (Table 24–1). A 6-monthly booster dose of this vaccine was recommended. There is now no licensed plague vaccine for general-use prophylaxis, although the recombinant subunit vaccine candidates (see “Subunit vaccines”, later) have investigational new drug (IND) status.

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SECTION TWO • Licensed vaccines Table 24–1 Schedules and Dosages of the Commonwealth Serum Laboratories Killed Whole-cell Vaccine Volume administered Age of recipient

First dose

Second dose*

Third dose*

Booster dose

6 mo to 2 yr

0.1 mL

0.1 mL

0.1 mL

0.1 mL†

3 to 6 yr

0.2 mL

0.2 mL

0.2 mL

0.2 mL†

7 to 11 yr

0.3 mL

0.3 mL

0.3 mL

0.3 mL†

> 12 yr

0.5 mL

0.5 mL

—

0.3 mL‡

*One to 4 weeks after previous dose. † At 6-month intervals after dose 3. ‡ At 6-month intervals after dose 2. Dose can be reduced to 0.1 mL intradermally for persons who had reactions to previous doses of the vaccine.

Live attenuated vaccines The first live attenuated Y. pestis strain used as an experimental vaccine was produced in 1895 by Yersin, but it was never tested in humans for fear of reversion to virulence. Subsequently, the EV76 strain was developed from the EV strain, which had been isolated in Madagascar from a human bubonic plague victim in 1926. Attenuation was identified after 6 years of in vitro passage.86 The EV76 strain and its derivatives have been used to vaccinate humans, especially in the former Soviet Union and the French colonies. However the genetic lesion that results in attenuation of EV76 has not been defined, although the strain is known to be a pigmentation (pgm) mutant, which prevents the bacterium from assimilating hemin4 and may also result in a change in surface properties of the bacterium.27 Other live vaccine strains included Tjiwidej, developed in the 1930 s;87 the attenuation of Tjiwidej appeared to result from loss of V antigen expression.88 This strain was used in limited field trials in Madagascar and Java.86 At present, however, no live vaccine is commercially available or licensed for use in humans because of risks of reversion and the severity of disease caused by these strains.86 Inoculation with the EV strain resulted in reactions ranging from local swelling and erythema of up to 15 cm diameter, to a rise in temperature accompanied by headache, weakness, and malaise that could be severe enough to require hospitalization in some cases. Although immunization with these strains could protect against both bubonic and pneumonic infections, the risks associated with them may outweigh the benefits.

Subunit vaccines Vaccines comprising purified recombinant proteins are now in advanced development and represent the next generation of plague vaccines. One such vaccine, comprising recombinant F1 and V proteins in an alhydrogel-adjuvanted liquid formulation, has successfully undergone phase 1 clinical trials. The F1 and V proteins, naturally produced by the parent organism as virulence factors, have been cloned and expressed under good manufacturing practice (GMP) conditions in harmless Escherichia coli89–92 and purified prior to coadsorption to alhydrogel. The resulting vaccine has been demonstrated to be safe and immunogenic in a total of 145 clinical trial volunteers to date. A variation of this vaccine, in which the C-terminus of F1 is genetically fused to the N-terminus of V to produce a recombinant fusion protein (rF1/V), is currently the presentation of choice and is in advanced development.93 Major advantages of the subunit formulation are the reduced immunization regimen required, the rapid induction of protective immunity, and the reduced reactogenicity, compared with KWC vaccine formulations.

Results of vaccination Immune responses The immune response to KWC vaccine formulations was mainly directed against the F1 antigen, as the content of V antigen in these vaccines is low or undetectable.94 There is good experimental evidence that the titer of F1 antibody, determined by passive hemagglutination, correlates with protection against plague in animal models.95 Ninety percent of animals with antiF1 passive hemagglutination titers of 128 survived challenge with 1 × 103 to 5 × 105 CFU of Y. pestis, whereas the proportions of animals with titers of 32 to 64 or 16 that survived were 46% and 6%, respectively. Passive immunization studies with sera obtained from immunized animals63 or humans96 also suggest that anti-F1 passive hemagglutination titers of 1:128 or greater were protective. However, the accepted test for demonstrating the efficacy of vaccines involves passive immunization of mice with sera and the demonstration of an acceptable mouse protection index (MPI). More recent trials with the Greer Laboratories' vaccine suggest that 55% to 58% of persons develop an antibody response with an acceptable MPI after two vaccinations;5 however, even after multiple (an average of five) vaccinations, 8% of persons fail to develop any antibody response. The rationale for combining the F1 and V antigens in the next generation vaccine is to induce an immune response to antagonize the key virulence mechanisms orchestrated by these antigens during the natural infection. Evidence from animal models94 and from clinical trials97 indicates that both antigens are immunogenic and that they are additive in their effect. The lead vaccine candidate is a recombinant genetic fusion of the F1 and V antigens (rF1/V) adsorbed to alhydrogel,93 although much work has also been published on an alternative formulation, in which the recombinant F1 and V antigens are simply combined and adsorbed to alhydrogel (rF1+rV)94 (see “Mechanism of protective immunity”, later).

Clinical immunogenicity The human immune response to the rF1/V vaccine has been assessed in more than 400 healthy volunteers and has been shown to be safe and immunogenic.98 Assay methods have been developed to assess the titer and functionality of antibodies raised to the rF1/V fusion, including ELISA and ­ opsonophagocytosis, with a proposal to carry out passive transfer of immune serum into naïve animals prior to challenge.99 Similarly, the rF1+rV vaccine has been assessed during phase 1 safety and immunogenicity trials in 32 healthy volunteers initially,97 and subsequently in a further 139 (unpublished data). The data from the first trial have been published and showed that all the subjects produced specific IgG in serum after the priming dose, which peaked in value after the booster dose (day 21), with the exception of one individual in the lowest doselevel group, who responded to rF1 only.97 The assessment of functional antibody has been aided by the identification of a mouse monoclonal antibody (mAb) to the V antigen, directed against a protective B-cell epitope in V antigen and which provides solid protection against live organism challenge by passive transfer into the mouse.97 Human antibody (as well as a reference macaque antibody) was found to compete with this mAb for binding to rV in vitro and also to protect naïve mice by passive transfer.97 This suggests that recognition of this protective B-cell epitope in the V antigen is conserved between mouse, macaque, and man. Total IgG to rV in human volunteers, and the titer of IgG competing for binding to rV, correlated significantly at days 21 and 28 (P < .001).97 The passive transfer of protective immunity in immune human sera into mice also correlated s­ignificantly with the IgG titers to both

Plague vaccines

Log IgG (rF1 + rV)

4.8

3.8

2.8 5

10

20

40

Dose level of rF1 + rV in mcg Figure 24–3 Correlation between vaccine dosage level and response in phase 1 clinical trial of rF1+rV vaccine. Volunteers were administered a priming immunization and a booster dose on day 21. The IgG response was measured by combining areas under the curve for log10 IgG responses to rF1 and to rV, measured in the period between days 21 (prior to the booster dose) through day 70 of the immunization schedule. (From Williamson ED, Flick-Smith HC, LeButt CS,

et al. Human immune response to a plague vaccine comprising recombinant F1 and rV antigens. Infect Immun 73:3598–3608, 2005.)

rF1 and rV at days 21 (P < .001) and 28 (P < .03), as determined by regression analysis (Figure 24–3).97 Thus, potential serologic immune correlates of protection have been identified that can be used as surrogate markers of protection in future clinical trials of the rF1/V vaccine, for which conventional phase 3 efficacy trials will not be possible.99 Surrogate markers of protection that rely on the induction of cell-mediated immune mechanisms may also be required, based on the indications from animal models.55,100 However, assays of cellmediated immunity during large trials conducted with nonhospitalised subjects are more problematic, relying as they do on fresh whole blood samples. Flow cytometric analysis of such samples from subjects responding to vaccination showed no significant changes in markers of cellular activation.97

Integration of clinical and nonclinical data None of the plague vaccines that have been developed have been subjected to a randomized, clinically controlled field study in humans.101 Although controlled clinical studies are desirable, the sporadic and relatively low incidence of plague means that such studies would be difficult to conduct. Thus, evidence for the efficacy of vaccines must be based on comprehensive studies in more than one animal model, and on evidence that they can, for example, induce antibody in humans that can passively protect animals. Also, anecdotal data, such as that obtained from Vietnam, can be useful. From 1961 to 1971, many thousands of Vietnamese civilians developed plague (333 cases per106 person-years of exposure), but the incidence of disease in KWCvaccinated US troops based in Vietnam during this period was low.102 (The total of eight cases represents one case per 106

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person-years of exposure.)5,102 Although this difference in the incidence of disease might be attributed to different exposures of these populations to Y. pestis, it is worth noting that many military personnel developed murine typhus, which is also transmitted by Xenopsylla cheopis.5 Furthermore, serologic studies indicated that some immunized persons were exposed to Y. pestis and developed subclinical infections.102 The effectiveness of KWC vaccines against the pneumonic form of the disease is more questionable, however, and cases of pneumonic plague have been reported in vaccinated persons.78,103 Thus, for diseases such as plague, where the logistics of a clinical field trial are difficult and efficacy testing is neither practical nor feasible, the only recourse to predict efficacy in man is to compare the immunologic readouts from the clinic with those gained from nonclinical studies, particularly where the latter can be related to a measurement of protective efficacy, to derive immune correlates of protection (Figure 24–4). Based on these data, surrogate markers can be sought from clinical studies, which would indicate that the vaccinee has developed protective immunity.104 Next, the nonclinical evidence that indicates the efficacy of the subunit vaccines in relevant animal models is reviewed.

Protective efficacy in nonclinical studies In the murine model of disease, immunization with two doses of a KWC vaccine provided solid protection against a subcutaneous challenge with 5,000 MLD of Y. pestis and partial protection (60% survival) against a subcutaneous challenge with 50,000 MLD of Y. pestis.61 In contrast, when challenged with approximately 18 MLD of Y. pestis by the airborne route, none of the mice that had been immunized with two doses of the vaccine survived.105 Although these data alone do not necessarily indicate an inability of the vaccine to protect against pneumonic plague in humans, when viewed alongside the reports previously cited, the overall evidence is that a KWC vaccine is better able to protect against bubonic plague than against pneumonic plague. All of these studies, however, have been carried out using F1+ strains of Y. pestis. However, since the main protective component of KWC vaccines appeared to be the F1 antigen, the efficacy of this vaccine type to provide protection against F1− strains of Y. pestis, which are rarely encountered in natural infections, has not been fully tested. Immunization of mice with the EV76 live attenuated vaccine strain does induce protection against both subcutaneous and inhalation challenges with virulent strains of Y. pestis,61 and in this respect the performance of this vaccine is better than the performance of a KWC vaccine (Table 24–2). The enhanced protection offered by the live vaccine over a KWC vaccine, might be the result of three factors. First, the KWC vaccine does not contain detectable levels of V antigen,94 and no ex vivo lymphoproliferative memory response for, or serum IgG to, the V antigen could be detected in KWC vaccinees.106 Second, it has been suggested that the chemical or heat inactivation of the bacterium

Immune correlates

Predict protected status

Immunological readouts

Protective efficacy

Figure 24–4 Integration of nonclinical and clinical studies with the identification of immune correlates of protection.

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SECTION TWO • Licensed vaccines Table 24–2 Protection Afforded by Killed Whole-cell Vaccine, EV76 Vaccine, or F1- and V-antigen Subunit Experimental Vaccine Against Challenge with Yersinia pestis Strain GB Protection (% survival) against challenge (MLD) Vaccine

Subcutaneous route

Inhalational route

Control

0 (10 )*

0 (102 to 103)†

Killed whole-cell vaccine

60 (2 × 106)*

80 (19)‡

EV76

100 (2 × 109)*

100 (150)§

F1 + V

100 (2 × 10 )*

100 (102 to 104)†,‡

1

9

*Tested in BALB/c strain mice. (Data from Williamson ED, Eley SM, Griffin K, et al. A new improved sub-unit vaccine for plague: the basis of protection. FEMS Immunol Med Microbiol 12:223–230, 1995.) † Tested in BALB/c, CBA, C57Bl6, and CB6F1 strain mice. (Data from Jones SM, Day F, Stagg AJ, et al. Protection conferred by a fully recombinant sub-unit vaccine against Yersinia pestis in male and female mice of four inbred strains. Vaccine 19:358–366, 2001.) ‡ Tested in Porton strain outbred mice. (Data from Williamson ED, Eley SM, Stagg AJ, et al. A sub-unit vaccine elicits IgG in serum, spleen cell cultures and bronchial washings and protects immunized animals against pneumonic plague. Vaccine 15:1079–1084, 1997.) § Tested in Porton strain outbred mice by intranasal route. (Data from Russell P, Eley SM, Hibbs SE, et al. A comparison of plague vaccine, USP and EV76 vaccine induced protection against Yersinia pestis in a murine model. Vaccine 13:1551–1556, 1995.) EV76, attenuated live vaccine strain of Y. pestis; F1 + V, subunit vaccine containing the F1 and V antigens of Y. pestis; MLD, minimum lethal dose.

results in structural changes in other surface components such as the F1 antigen.91 Third, local responses to vaccination with EV76 in mice suggest that a limited local infection is initiated, and that the duration of exposure to the antigenic complement is therefore prolonged. The immune response induced by the live attenuated vaccine may be more authentic than that induced by KWC preparations. A significant amount of work has been carried out in the mouse,107 guinea pig,59 and macaque108-113 models, from which immune correlates have been proposed. Although a titer of neutralizing antibody provides protection against exposure, it is clear that the development of cell-mediated immunity is also critical to full protection and eventual clearance of the bacterial challenge from the host. Intraperitoneal or intramuscular immunization with native F1 or rF1 antigens adjuvanted with alum induced an immune response that protected mice against a subcutaneous challenge with as many as 105 CFU of virulent Y. pestis.94,114 Although the F1 antigen also induced protection against inhalation challenge with 100 MLD of an F1+ strain of Y. pestis,114 there was concern that a vaccine based solely on the F1 antigen would not provide protection against naturally occurring but virulent F1− strains of Y. pestis (although only one such naturally occurring strain has been reported). Intraperitoneal immunization of mice with the V antigen adjuvanted with alum induced protection against a subcutaneous challenge with 4 × 106 CFU of Y. pestis.89 A significant advantage over the F1 antigen is the ability of the V antigen to induce protection against virulent F1− strains of Y. pestis; protection against 1,000 or more MLD of either virulent F1+ or F1− strains of Y. pestis given by the inhalation route has been reported.115 The protection afforded individually by the F1 and V antigens was defeated by very high subcutaneous challenge of the immunized mouse with, for example, levels of 109 CFU of Y. pestis.94 However, protection against this challenge was achieved after intraperitoneal immunization in multiple doses with a mixture of the F1 and V antigens.94 The vaccine also protected mice against 100 MLD of Y. pestis given by the inhalation route (see Table 24–2), suggesting that the vaccine would provide protection against pneumonic plague in humans.105 The advantages of such a combined subunit vaccine lie not only with the enhanced level of protection afforded against disease but also with the ability of the vaccine to confer protection against both F1+ and F1− strains of Y. pestis. A systematic study using mice of four different haplotypes and both sexes showed that all responded to immunization with the rF1 and rV antigens with high antibody titers.116 The response to either antigen is affected by the conformation of

the protein,91,117 and an optimal ratio of rF1 to rV was found when rF1 was in a twofold molar excess to rV.118 However, mice can also mount an antibody response to these antigens when the C-terminus of the F1 antigen is genetically fused to the N-terminus of the V antigen.93 The rF1+rV vaccine has also been used successfully to immunize guinea pigs and nonhuman primates. Antibody titer to rF1+rV in guinea pigs also correlated with protection and passive transfer of specific antibody (IgG) from the guinea pig into naïve mice protected the latter against ­challenge.59 Cynomolgus macaques immunized with rF1 and rV antigens produced high titers of specific antibody that competed with the V-specific and protective mAb.7.3 for binding to V antigen and also passively protected naïve mice against challenge,108 and the antibody was used as a positive reference serum in the passive transfer of human serum.97 A more recent study showed that the immunization of cynomolgus macaques with rF1+rV or rF1/V in alhydrogel fully protects them against pneumonic plague after inhalation exposure to Y. pestis.109,111,112

Mechanism of protective immunity The mechanism by which the rF1+rV vaccine induces protective immunity has been the subject of considerable investigation during the past few years. The F1 capsule is thought to inhibit phagocytosis of the bacterium by preventing complement-­mediated opsonization.40,119 Therefore, it is possible that antibody induced to the F1 antigen opsonizes the bacterium and promotes antibody-dependent cellular cytotoxicity. Some vaccinated animals do appear to harbor the pathogen even in a mutated F1− form.114 The KWC vaccines are less effective than purified F1 antigen in inducing high titers of antibody to F1.94,114 Antibody against the V antigen may also be of overriding importance in protection against plague, and immunization with V antigen has been shown to restore the ability of the host to produce tumor necrosis factor (TNF)-α and interferon (IFN)-γ in response to infection.47 Therefore, immunization with the V antigen would enable the host to mount a normal inflammatory response and thereby enable host phagocytes to clear bacteria opsonized by antibody to the F1 antigen. Additionally, because V antigen forms part of the injectisome of the type III system45 and is exposed on the bacterial surface,120 it is extracellular for enough time to be targeted by antibody raised in the host by vaccination, or passively transferred into the host.81-83 It has been shown that antibody to V antigen can inhibit the translocation of Yops by Y. pestis into the host cell.120 It was found that this inhibitory effect depended on

Plague vaccines

the binding of antibody to the bacterial cell surface, and it was attributed to the direct promotion of phagocytosis of Y. pestis by polymorphonuclear leucocytes.121 Although protection against plague in the mouse model has been demonstrated to correlate with the specific IgG titer to the subunits (specifically of the IgG1 subclass in the mouse, in response to this alhydrogeladjuvanted vaccine),118 there is no doubt that the mechanism of protection after immunization with the rF1+rV proteins also involves T-cell memory, and this has been demonstrated in the mouse model.106,118,122 In the mouse, the T-cell response to alhydrogel -adsorbed F1+V is biased toward T-helper cell type 2 (Th2), and this response is highly protective. However, recent work has illustrated that although delivery of the F1+V proteins formulated in the Ribi adjuvant system to IL4T mice (genetic knock-outs for the interleukin [IL]-4 receptor) induced predominantly a Th1 response and the passive transfer of their antiserum into B-cell-deficient knock-out mice, with no intrinsic antibody, protected the latter fully against live-organism challenge.123 Furthermore, a targeted gene deletion in the Stat4 signaling pathway (preventing the ligation of the Stat4 receptor with IL-12 signaling), and abrogating a Th1 response, resulted in reduced protection, whereas mice with a targeted gene deletion in the Stat 6 pathway and no Th2 activity were fully protected against challenge.124 In general terms, therefore, antibody is highly protective against this predominantly extracellular infection, but cell-mediated immunity125 and particularly a balanced Th1/Th2 activity124 underpin the specific antibody responses to rF1 and rV and provide an optimal strategy for protection. This is supported by recent data that show that mice immunized with the rF1/V vaccine and depleted of TNF-α and IFN-γ just prior to challenge had very poor survival compared with positive controls that were only immunized,100 indicating key roles for these Th1 cytokines in the development of protective immunity.126,127

Correlates of protection and surrogate markers of efficacy In summary, correlates of protection to the rF1/V vaccine reside in both antibody-mediated and cellular mechanisms, which do not act independently. Knowledge of mechanisms derived from nonclinical models of vaccination and infection can lead to the identification of potential surrogate markers of efficacy.104 Assays of surrogate markers of efficacy may include the in vitro inhibition of the cytotoxicity of a Y. pseudotuberculosis construct expressing Y. pestis V antigen,128 the passive transfer of human immune serum into a naïve animal model,85 and an assessment of cell-mediated immunity using ex vivo peripheral blood mononuclear cells and a secondary recall response in, for example, an ELIspot or similar format. Much work is ongoing to identify statistically valid immune correlates of protection for plague and to use these to establish surrogate markers of efficacy. With an increasing understanding of the molecular basis of pathogenicity and of the innate and adaptive immune response mechanisms required to counter Y. pestis, more progress will be made in this area, which will expedite the development of next-generation vaccines.

Indications for vaccine use Currently there is no licensed vaccine available for plague. However, the indications for the KWC vaccine formulations would also apply to a vaccine licensed in the future. Vaccination is recommended, if possible, for persons who are working with fully virulent strains of Y. pestis (eg, researchers and laboratory workers) or for persons in regular contact with the wild animal hosts of plague or their fleas, in areas endemic for the disease.

24

It may also be considered for use in personnel who are deployed to work in endemic areas (eg, emergency workers, military personnel, field workers, agricultural consultants), especially if they may be exposed to epizootic plague arising from some acute geologic disturbance.5 However, vaccination against plague is not a statutory requirement for travel to any country. The vaccination of populations in plague-endemic areas of the world is not routinely indicated, because the incidence of disease is relatively low and most cases of disease are of the bubonic form, which, once diagnosed, can be treated with antibiotics. The previously available KWC vaccines had limited use in controlling sporadic plague epidemics, because they required several months to induce protective immunity. However, the response to the new rF1/V vaccine should be more rapid, so that, once licensed, it would be feasible to use it in this context and in the indigenous population in areas where there is recurrent or intense plague activity that cannot be controlled by other measures. Additionally, a licensed subunit vaccine for plague would be of benefit as a deterrent to the use of Y. pestis as a potential bioweapon. Although antibiotics might also provide protection against disease, it is not clear if sufficient quantities could be stockpiled to allow the treatment of large populations for long periods of time. In addition, there is concern over the appropriateness of long courses of broad-spectrum antibiotics being used in large numbers of persons. It seems likely that antibiotics will have a clear use for the protection of relatively small numbers of persons, whereas vaccination is more appropriate for entire populations. It seems unlikely that routine mass vaccination of entire populations, in the absence of any clear indication of an impending attack, would be undertaken. Therefore, vaccines for protection of the civilian population might need to be used at short notice and will need to induce a protective response rapidly.

Postexposure prophylaxis The previously available KWC vaccines were not suitable for postexposure use, because several months were required to complete the primary vaccination schedule. Even use of the rF1/V vaccine, which induces relatively rapid immunity when administered after exposure, would be unlikely to act fast enough to protect an infected and previously unvaccinated person from disease. The use of antibiotics should be considered in the case of possible exposure to Y. pestis, even in vaccinees who had previously completed the full KWC vaccination schedule, as this vaccine type offered poor protection against pneumonic plague. There is a real prospect for the future development of passive immunotherapy for postexposure situations.

Precautions and contraindications The KWC vaccine formulations were contraindicated in persons who had a history of hypersensitivity to any of the vaccine components (eg, beef protein, soya, casein, sulphite, phenol, formaldehyde). The safety and efficacy of the KWC vaccine formulations in people younger than 18 years or in pregnant women was not known, as is the case also for the rF1/V vaccine.

Persistence of immunity Few studies have examined the duration of immunity to Y. pestis after immunization with KWC vaccines. However, it is generally considered that immunity was short lived so that booster immunizations were needed at 6-month intervals to ensure that a protective response was maintained. A single dose of the live attenuated vaccine derivatives of strain EV76 could protect

501

502

SECTION TWO • Licensed vaccines

against bubonic and pneumonic plague for up to a year (see review).129 Nonclinical data indicate that immunization with the combined rF1+rV vaccine induces durable immunity up to 9 months after a single immunization in the mouse model.106 Cynomolgus macaques immunized with rF1+rV were observed to have functional IgG titers able to compete with Mab7.3 for binding to the V antigen more than 1 year after immunization.108 The establishment of an immunization regimen and the need for a booster immunization will require similar monitoring of volunteers during clinical trials of the rF1-V vaccine, for the persistence of immunity.

Safety The KWC vaccines were known to be reactogenic in humans.130,131 The manufacturer of the Cutter vaccine reported that reactions such as malaise, headaches, local erythema and induration, or mild lymphadenopathy, occurred in approximately 10% of vaccinees; this frequency of side effects was also reported by other workers.86 Data provided by the manufacturer of the Greer vaccine suggest that more than 10% of vaccinees suffered side effects. Allergic reactions induced by immunization with KWC vaccines, evidenced mainly as urticaria, occurred infrequently.130 One study found that the frequency of side effects was much greater in persons who had previously been immunized with the live EV vaccine.86 The safety of the EV76 live attenuated vaccine has been questioned by several workers. In a review of the use of live attenuated vaccines such as EV76, Meyer and colleagues reported that in one study in the former Soviet Union, a febrile response was reported in 20% of vaccinees, accompanied by headache, weakness, and malaise.132 Erythema surrounding the site of vaccination was frequently reported and could reach dimensions of 15 cm in diameter. Some severe systemic reactions required hospitalization. Numerous unsuccessful attempts were made to reduce the incidence of side effects, such as administering the vaccine by different routes, including by scarification, by the inhalational route, and even intraocularly.132 Of equal concern is the finding that the EV76 strain is able to cause disease in some animal species. Russell and colleagues reported that immunization of mice with Y. pestis EV76 induced severe side effects and occasional (approximately 1%) fatalities,61 and the vaccine is reported to cause fatal infections in vervets.132 By comparison, the rF1+rV vaccine was shown to be safe and well tolerated in the first reported clinical phase 1 trial in 32 healthy volunteers.97 Four dosage levels were assessed, and no serious or severe adverse events attributable to the vaccine were reported (Fig 24-3). Further clinical trials of the rF1/V formulation vaccine are ongoing in larger numbers of subjects to investigate the safety and immunogenicity of the formulation, as well as to predict its efficacy through surrogate markers of protection.99

Future developments With the discontinuation of production of a KWC vaccine formulation, the most likely replacement in the medium term is a formulation comprising a combination of the recombinant F1 and V antigens, which is expected to induce more rapid and comprehensive immunity. Indeed, this subunit vaccine may be the first non-live vaccine to protect against the most dangerous form of the disease, pneumonic plague. An injectable, alhydrogel-­ adsorbed formulation is currently being developed, and an alternative formulation of the recombinant fusion protein may have the potential for self-administration or for mucosal delivery. A considerable body of work has been done on the polymeric

­ icroencapsulation of the F1 and V antigens and the mucosal m delivery of microencapsulated formulations to achieve protective immunity.122,133 In addition to permitting more flexibility in route of administration, polymeric encapsulation enhances vaccine stability and removes the need for cold chain storage. Many refinements have been made to optimize microencapsulated formulations of rF1 and rV antigens; the antigens have been either mixed for co-encapsulation or they have been separately encapsulated and mixed after encapsulation. In each case, both the subunits retained immunogenicity134 and protective efficacy.117,135 Further refinements have included the substitution of polymers conferring particular properties of stability and hydrophobicity, and approaches to control particle size and loading.107 A microsphere formulation suitable for nasal or parenteral administration has been derived that is fully protective against an inhalational challenge with Y. pestis after two separate136 or even a single immunizing dose137 in the mouse model. Liposomal encapsulation of the plague subunit vaccine offers an alternative approach to mucosal delivery.133 Alternatively, a spray-dried powder formulation of the rF1/V formulation has been shown to be highly immunogenic by intradermal or intramuscular delivery in mice.138 The delivery of vaccines by advanced transdermal patch is a possible growth area139 and would provide an alternative noninvasive approach to vaccination with the rF1 and rV subunits.140 The most promising approach to an oral vaccine for plague that has met with success is the use of Salmonella enterica serovar Typhimurium expressing the caf operon, which induced a high level of protection against subcutaneous challenge with Y. pestis.141,142 Formulation of the rF1 and rV subunits for oral delivery achieved more modest protection against challenge.143

Additional subunits Much effort has been made to identify additional protective subunit proteins, which was unsuccessful until recently. Yersinia secretory protein F (YscF) along with V-antigen form the injectisome of the type III system, and immunization with YscF has been shown to provide some protection against plague in the mouse.144 Thus, it may be beneficial to add YscF to a future subunit vaccine formulation to maximize its protective efficacy. A recent study has shown that immunization with Y. pseudotuberculosis that lacks the Lcr plasmid can provide protection against plague, suggesting that protective antigens other than F1 antigen and components of the type III system exist.145

Live attenuated vaccines The finding that the live EV76 vaccine induces protection against plague, although its use is accompanied by unacceptable local and systemic side effects,132 has prompted some researchers to develop rationally attenuated strains of Y. pestis as a vaccine. Work toward this goal has been based on the finding that other Gram-negative pathogens can be attenuated by the introduction of mutations into genes essential for bacterial growth, virulence, or survival in the host, and that the nature of the attenuating mutation can influence the immunogenicity and reactogenicity of these vaccines. Strains of Y. pestis with mutations in the aroA,146 phoP,147,148 or htrA149 genes have been constructed and tested but show only a modest reduction in virulence in mice and would not be suitable as the basis for the development of a live attenuated vaccine for use in humans. However, mutation of the dam gene resulted in a 2,000-fold attenuation of Y. pestis, and mice that were exposed to sublethal dosages of the mutant were protected against a subsequent challenge with fully virulent Y. pestis.150 Even more impressively, a pcm mutant of Y. pestis, which is believed to be

Plague vaccines

defective in its response to environmental stresses, was more than 107-fold attenuated in mice.151 The immune response induced after immunization with the pcm mutant was superior to that induced by the EV76 strain, suggesting that this might be an efficacious and safe alternative to the EV76 vaccine. The elucidation of the Y. pestis genome sequence has identified a wide range of other genes that might be inactivated to yield a live attenuated vaccine.22 For example, genes encoding lipoprotein (lpp) and an acyltransferase involved in modification of LPS (lpxM) have been shown to be attenuating when mutated.129,152 For a live attenuated vaccine to be safe, multiple mutations in independent pathways would be required to reduce any risk of reversion to virulence. The effect of combining attenuating mutations is hard to predict, as the multiple mutations may

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overly attenuate, which would have an effect on the induction of adequate protective immune responses. An alternative strategy would be to use a closely related species with lower virulence, and to exploit cross-immunity. For example, an attenuated dam mutant of Y. pseudotuberculosis was able to induce a robust protective immune response against challenge with Y. pestis.145 A naturally arising attenuated strain lacking the high pathogenicity island, the Y. pseudotuberculosis superantigens, and type IV pili was similarly able to induce a protective response against plague.153 In addition to reducing risk as a result of using a less virulent strain, this approach has the advantage that the immunity was induced after oral dosing, indicating that a needle-free vaccine is achievable.

Access the complete reference list online at http://www.expertconsult.com 8.

World Health Organization. Plague, Democratic Republic of the Congo. Wkly Epidemiol Rec 2005;80:65. 19. Achtman M, Zurth K, Morelli G, et al. Yersinia pestis, the cause of plague, is a recently emerged clone of Yersinia pseudotuberculosis. Proc Natl Acad Sci U S A 1999;96:14043–8. 22. Eppinger M, Worsham PL, Nikolich MP, et al. Genome sequence of the deeprooted Yersinia pestis strain Angola reveals new insights into the evolution and pangenome of the plague bacterium. J Bacteriol 2010;192:1685–99. 45. Mueller CA, Broz P, Muller SA, et al. The V-antigen of Yersinia forms a distinct structure at the tip of injectisome needles. Science 2005;310:674–6. 54. Anderson DM, Ciletti NA, Lee-Lewis H, et al. Pneumonic plague pathogenesis and immunity in brown Norway rats. Am J Pathol 2009;174:910–21. 55. Williamson ED. Plague. Vaccine 2009;27(Suppl. 4):D56–60. 59. Jones SM, Griffin KF, Hodgson I, et al. Protective efficacy of a fully recombinant plague vaccine in the guinea pig. Vaccine 2003;21:3912–8. 93. Heath DG, Anderson GW, Mauro M, et al. Protection against experimental bubonic and pneumonic plague by a recombinant capsular F1-V antigen fusion protein vaccine. Vaccine 1998;16:1131–7.

108. Williamson ED, Flick-Smith HC, Waters EL, et al. Immunogenicity of the rF1+rV vaccine with the identification of potential immune correlates of protection. Microb Pathog 2007;42:12–22. 125. Parent MA, Wilhelm LB, Kummer LW, et al. Gamma interferon, tumor necrosis factor alpha, and nitric oxide synthase 2, key elements of cellular immunity, perform critical protective functions during humoral defense against lethal pulmonary Yersinia pestis infection. Infect Immun 2006;74:3381–6. 140. Eyles JE, Elvin SJ, Westwood A, et al. Immunisation against plague by transcutaneous and intradermal application of sub-unit antigens. Vaccine 2004;22:4365–73. 145. Taylor VL, Titball RW, Oyston PFC. Oral immunisation with a dam mutant of Yersinia pseudotuberculosis protects against plague. Microbiol 2005;151:1919–26. 153. Blisnick T, Ave P, Huerre M, et al. Oral vaccination against bubonic plague using a live avirulent Yersinia pseudotuberculosis. Infect Immun 2008;76:3808–16.

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