Plague Vaccines

Clinical Description

Bubonic Plague. Bubonic plague is the form of disease typically transmitted to man through the skin by an infected flea, having previously fed on an infected rodent. In some circumstances, infection can occur via open wounds, exposed to infected material through handling and direct contact. Similarly, human ectoparasites may be a transmission vector. ^, Within 2–6 days of infection, the patient develops a fever, headache, and chills. 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 word bubon , meaning groin. The buboes are often located within the inguinal and femoral lymph nodes, which drain the original site of infection on the lower extremities. Bacteremia is common in patients, typically resulting in organisms in the blood ranging from fewer than 10 to up to 4 × 10 ⁷ colony-forming units [cfu] per mL. High levels of bacteremia are often associated with gastrointestinal symptoms such as vomiting, nausea, abdominal pain, and diarrhea. Early treatment with antibiotics such as streptomycin, gentamicin, tetracycline, chloramphenicol, or the fluoroquinolones, usually leads to rapid recovery.

Septicemic Plague. Primary septicemic plague is a clinical diagnosis in an acutely toxic patient with large numbers of Y. pestis in the blood stream, and no identifiable anatomical site of infection. 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 with bleeding, adult respiratory distress syndrome, shock, and organ failure. Owing to the absence of localizing signs, the diagnosis of primary plague sepsis may be delayed with the overall case fatality rate with sepsis approaching 20–40%.

Pneumonic Plague. Some colonization of pulmonary tissues occurs in virtually all untreated fatal cases of plague, but most of these patients do not develop a transmissible plague pneumonia. However, when colonization of the alveolar spaces occurs, a suppurative pneumonia develops, and during the terminal stages of disease there is coughing with the production of a highly infectious, watery, and bloody sputum. Pneumonic plague is the most widely feared form of the disease because it can spread rapidly in respiratory droplets. A number of experiments with animals (guinea pigs, lemurs, and nonhuman primates) have demonstrated the cross-infection of control animals from infected animals with pneumonic disease. 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. In this outbreak, four cases of pneumonic plague were attributed to contact with one patient who had developed secondary pneumonic plague. These 4 infected individuals then transmitted the disease to 11 others, one of whom transmitted the disease to 2 further individuals. In total, 18 individuals contracted pneumonic plague resulting from this single individual and 8 died.

The available evidence indicates that the inhalation of Y. pestis in airborne droplets by susceptible individuals can lead to the development of pneumonic plague within 1–3 days. The rapidity with which the infection spreads between individuals, along with the relatively short incubation period, makes control of the disease difficult and antibiotic therapy may be ineffective once the pulmonary symptoms have developed. Primary pneumonic plague is rare in the United States, with a few cases reported in recent years in veterinarians and owners of domestic cats, exposed to infected wild animals, or by direct exposure to infected wild animals. Nevertheless, the potential for pneumonic plague to spread quickly in human populations is evident for example from small outbreaks in confined communities, for example, in a Northern Congo diamond mine in 2005 or large outbreaks in Tanzania (1980), India (1994), and Madagascar (2017).

Bacteriology

Y. pestis is a Gram-negative bacterium and a member of the family Enterobacteriaceae. The bacterium grows at temperatures between 4°C 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. The genus Yersinia also includes Y. enterocolitica and Y. pseudotuberculosis species, both of which are enteric pathogens of humans, but rarely cause disease with a fatal outcome. ^, Recent studies have shown that Y. pestis evolved from Y. pseudotuberculosis (most probably serotype 1b) between 1500 and 20,000 years ago. A recent report has suggested that the plague-causing bacterium existed long before previous estimates. Bacteria morphologically similar to Y. pestis have been identified on the proboscis and compacted in the rectum of a flea which had been preserved in amber, dating back 20 million years. If these bacteria were indeed Y. pestis they may be ancient strains which evolved as rodent parasites and are now extinct, since genomic data indicate that the animal–flea–human transmission cycle for Y. pestis evolved some 20,000 years ago. Wagner et al. have retrieved teeth from two victims of the Justinian plague and compared the DNA sequence obtained against a database of more than 130 Y. pestis strains from the second and third pandemics. This study identified differences in genome sequences in these ancient strains, from those which caused the Black Death some 800 years later.

However, recent attempts to reconstruct the genome sequence of the medieval Y. pestis from DNA isolated from the teeth of victims of the Black Death, using Y. pestis CO92 as a reference, have not indicated major genetic differences between the medieval and contemporary strains, although this approach would not find any genomic regions now lost in the contemporary strain. The results from independent studies reporting the analysis of DNA from the eras of the Justinian and Black Death plagues classify these samples as most similar to the Orientalis biovar.

An intermediate bacterial species termed Pestoides has been identified. Genome sequencing reveals that Pestoides strains do not contain the full complement of plasmids associated with Y. pestis and that they retain chromosomal loci found in Y. pseudotuberculosis but lost in Y. pestis. ^, Pestoides isolates are virulent, particularly by aerosol routes but can show attenuation by systemic routes and are enzootic strains, maintained in rodents of the superfamily Muroidea comprising rats, mice, hamsters, and gerbils.

Evolution of Y. pestis has been by the inactivation of a range of genes required for an enteric lifestyle and by the acquisition of new virulence factors, as depicted in Fig. 46.2 . The acquired virulence genes are 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. ^, Y. pestis possesses two further unique plasmids, a 9.5-kb plasmid, pPCP1, and the 100–110 kb pFra plasmid. The pla gene on pPCP1 encodes a surface-bound protease (plasminogen activator), which has potent fibrinolytic activity ( Fig. 46.2 ). It has been observed for some time that inactivation of the O-antigens on Y. pestis lipopolysaccharide was associated with full functioning of pla and recent evidence suggests that by exposing the LPS core, Y. pestis can interact with C-type lectin receptors on host macrophages, thus accelerating its dissemination in the host.

During its evolution from enteric to flea-vectored pathogen, Y. pestis has lost intestinal adhesin and invasin genes, but retained the haem locus and possesses a number of genome-encoded factors such as the ph6 fimbrial and ail adhesins, all of which contribute to its virulence in the human host. The possession of the pPCP1 plasmid has recently been used to date the evolution of Y. pestis from Y. pseudotuberculosis . While the oldest Pestoides strains (E and F) do not possess the pPCP1 plasmid, Angola, and Pestoides A strains which arose later, do contain the pPCP1 plasmid. Furthermore, Angola and Pestoides A strains do not possess a later sequence change in the Pla protein which optimizes its function in disseminating bacteria in vivo; this indicates that the pPCP1 gene was acquired by Y. pestis relatively early in its evolution from Y. pseudotuberculosis , and before it was fully adapted to its flea-vectored lifestyle. The pFra plasmid shows extensive sequence homology with plasmid pHCM2, possessed by some strains of Salmonella enterica serovar Typhi. 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 Fraction 1 (F1) protein capsule expressed by Y. pestis at 37°C.

Pathogenesis

Plague has a complex lifestyle, cycling between arthropod and mammalian hosts, with the primary mammalian hosts being rodents and humans being accidental hosts. As a result of this lifestyle, the organism has had to develop sophisticated strategies to ensure transmission from infected rat to the flea, then from the flea to a new host, or as recently proposed from infected human to body louse and hence to another human. ^, The role of the rat flea in transmission has been the subject of much study. The rat flea ( Xenopsylla cheopis ) 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.

Mutants of Y. pestis lacking the hemin storage locus establish long-term infection of the flea’s midgut and fail to colonize the proventriculus, preventing the efficient transmission of bacteria. The blockage prevents digestion of the blood meal, and further ingestion of blood leads to regurgitation of a bolus of bacteria from the flea to man. In achieving its transmission, the pathogen results in the death of both the animal host and the flea vector. High levels of septicemia in the infected animal are required to allow the infection of a subsequent flea vector, and efficient transmission from the flea is required to infect man. However, in situations in which humans and rodents are in close proximity or when the rodent population is reduced, either as a result of the disease or rodent control measures, humans and other warm-blooded mammals can serve as alternative hosts. Y. pestis is thus an obligate parasite, separating it from other human pathogenic Yersinia species. Y. pestis 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 dipodids such as the Jerboas. Other reservoirs of infection include prairie dogs, rabbits and members of the cat family, including the domestic cat.

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. However the “blocked” flea model alone cannot account for the rates of spread of plague during plague endemics and it is thought that unblocked fleas are also able to transmit infection, by a so-called “early phase transmission” process. This would allow the flea to transmit bacteria to its host in a much shorter time interval after its latest blood meal and would promote sylvatic epizootics and possibly also maintain endemic disease in man at times of low flea burdens or where human flea populations (Pulex irritans) predominate.

An alternative theory has been proposed that Y. pestis can also be transmitted by the human body louse ( Pediculus humanus corporis ), facilitating human-to-human transmission. Such transmission has been demonstrated experimentally in rabbits. Interestingly, only biovar Orientalis, but not Antiqua or Medievalis, could be transmitted in this way. Although the role of the flea in transmission of Y. pestis from rodents to humans is undisputed, there is increasing support of a potential role of body lice as an additional vector for human-to-human transmission during the Black Death. However, vector–pathogen interaction studies have focused mainly on the flea to date, and studies on the interaction of Y. pestis with the body louse are more preliminary.

To date, only three factors have been positively confirmed as essential for transmission from flea to mammal: a murine toxin, an extracellular polysaccharide and a lipopolysaccharide core modification locus. The murine toxin has phospholipase D activity that is essential for survival in the flea midgut, whereas the extracellular polysaccharide and LPS core modification are required for biofilm formation and blockage of the flea. ^, However transcriptional analysis of Y. pestis in the flea gut identified a wide range of genes, such as the insecticidal-like toxin genes, that were differentially regulated, expression of which meant that the bacteria that were regurgitated into a new host had increased resistance to innate immune effectors.

Upon infection of a new host, the plague bacilli are vulnerable to phagocytosis by polymorphonuclear leukocytes or monocytes. However, bacteria from fleas have significantly lower levels of phagocytosis than bacteria grown in vitro, indicating the importance of the complex influence of growth within the flea on pathogenesis. The insecticidal-like toxins are key components in resisting phagocytosis in the early stages of infection, before the antiphagocytic Type III secretion system can be induced.

There is controversial evidence that preferential phagocytosis by monocytes/macrophages may be facilitated by the chemokine receptor Ccr5, prevalent on monocytes/macrophages and dendritic cells, but not on polymorphonuclear leukocytes. 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. Ccr5 is a coreceptor with CD4 facilitating entry of another infection, human immunodeficiency virus (HIV), into host cells.

The plague bacteria are killed within polymorphonuclear leukocytes, but persist within monocytes and express various virulence determinants, thereby allowing growth and eventual release from the monocytes. One such virulence determinant is the pH6 antigen, a fibrillar adhesin induced by the low phagosomal pH (4.5) that enhances resistance to phagocytosis. The F1 capsule plays a key role in avoiding phagocytosis. However, mutants of Y. pestis that are unable to produce F1 antigen are still able to cause disease in the mouse, although they have reduced virulence. It appears that possession of the locus encoding the F1 antigen increases transmissibility from the flea, although this is not observed following needle challenge in a laboratory setting.

The dominant antihost effects are due to induction of the Type III secretion system (TTSS) carried on the large virulence plasmid pYV. Type III system effectors, historically called Yersinia outer membrane proteins (Yops), have cytotoxic and phagocyte regulatory effects and are secreted through an injectisome, elaborated by the bacteria on contact with the surface of a host cell, into target cells. The function of many of the Yops has been determined in this well-characterized secretion system, which serves as a paradigm for bacterial TTSS. For example, the YopE protein is a cytotoxin that is transferred into host cells after bacterial contact ; the YopH protein is a tyrosine phosphatase that has antiphagocytic cell activity. The V antigen plays a pivotal role in TTSS, orchestrating intracellular Yops and low calcium response protein G (lcrG) to elaborate the injectisome and then itself being delivered through this needle-like structure and assembled as a pentamer at the tip. Additionally, V antigen secreted from Y. pestis exerts a local immunomodulatory effect in the host, by down-regulating the production of interferon-γ and tumor necrosis factor-α. ^,

Plasminogen activator (Pla) is another major virulence factor in Y. pestis ( Fig. 46.2 ). Pla is an outer membrane-located protease, and breaks down the physical barriers of connective tissue in the host, thus promoting the systemic dissemination of the bolus of Y. pestis injected by the flea. The requirement for Pla has driven the selection in Y. pestis of a “rough” phenotype of LPS which lacks an O antigen. ^, This phenomenon is rare among Gram-negative bacteria, but it is necessary for Pla to be functional. When O antigen expression is forced in Y. pestis , it blocks the activity of Pla, and is thus attenuating by both dermal and respiratory routes of infection ^, but not following intravenous inoculation. Pla interacts with the DEC-205 receptor on host antigen-presenting cells to promote uptake, and it appears that dissemination only occurs after phagocytosis.

The bacteria disseminate from the site of primary infection into regional lymph nodes that drain these tissues. Within 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 due to the TTSS, which is highly expressed in the lymph node. An ability to proliferate in the bubo requires efficient iron acquisition systems. Y. pestis possesses 10 iron acquisition systems that are actively utilized in the bubo.

Eventually, the bacteria are disseminated by the lymphatic system, gain access to the blood stream, 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, which triggers septic shock in the host. However, the precise mechanisms that lead to death of the host have not been identified. Systemic induction of nitric oxide synthase may be the terminal event, as seen with other Gram-negative septicemias.

While pigmentation-negative strains of Y. pestis are usually avirulent and attenuated, the risk of reversion to virulence was recently highlighted by the fatal case of a laboratory worker who was unknowingly suffering from hemochromatosis and was exposed to the attenuated pgm- Y. pestis laboratory strain KIM. This individual developed and died from plague, presumably due to the complementation of the haem deficiency by the individual’s medical condition.

Models of Disease and Protection

Y. pestis causes disease in a wide variety of laboratory animals, including mice, Brown Norway rats, guinea pigs, and nonhuman primates. Additionally, several species such as the black-footed ferret and the prairie dog have been actively immunized against plague. Most experimental work has been carried out in the murine model; however, the model may not faithfully mimic human disease because of the susceptibility of mice and rats to the murine exotoxin. Although this limitation might be resolved by using the guinea pig model of disease, the protracted nature of the disease in the guinea pig suggests that the mouse model is a better indicator of the infection that occurs in animal reservoirs. In a comparative study with mice and guinea pigs, it was concluded that mice were more suitable for the evaluation of plague vaccines and this species was approved by the U.S. Public Health Service for the testing of plague vaccines. Disease arising from the delivery of Y. pestis by the subcutaneous (s.c.) route (median lethal dose [MLD], 1–2 cfu ) is considered to mimic bubonic plague, whereas the exposure of mice to the bacteria via inhaled aerosols results in the pneumonic form of the disease (MLD, 2 × 10 ⁴ 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).

Diagnosis of Plague

The clinical diagnosis of plague is supported by laboratory tests. Bacteriological diagnosis of plague is usually made by analysis of aspirates taken from a bubo, blood, or sputum samples. This involves Gram- or Wyson-staining of air-dried smears on microscope slides. ^, After staining, Y. pestis cells appear as small Gram-negative rods with a characteristic bipolar staining (safety pin appearance). Culture on Congo Red agar, ideally at 26–28°C, results in so-called “bull’s-eye” colonies, which have a red pigmented central region and paler margins. However, culture methods are generally considered to be too slow (typically taking 48 hours) in the context of the rapid progression of the disease (especially pneumonic plague) and a presumptive diagnosis should be made before such tests are completed.

A variety of confirmatory tests for Y. pestis have been proposed, including the use of fluorescent antibody to the capsular F1 antigen, passive hemagglutination tests, the use of the polymerase chain reaction (PCR), and enzyme-linked immunoassay (ELISA). ^{, ,} These tests can be used on bubo aspirates for the rapid diagnosis of disease. An immunogold-chromatography dipstick has been used for the direct analysis of bubo, serum, or urine samples and fielded to endemic countries to evaluate for the rapid detection of plague.

Treatment and Prevention With Antimicrobials

The successful treatment of plague is dependent on the prompt commencement of therapy during the early stages of the disease. 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 FDA for the treatment of plague. ^, For the treatment of plague meningitis, intravenously administered chloramphenicol is the antibiotic of choice because of its ability to cross the blood–brain barrier. ^, However, chloramphenicol is difficult to procure and alternatives include streptomycin (or gentamicin) with or without doxycycline or a fluoroquinolone such as ciprofloxacin or levofloxacin. ^{, ,} The fluoroquinolones, ciprofloxacin and levofloxacin have been licensed by the FDA for the indication of inhalational plague and subsequent data in African green monkeys have shown that administration of the antibiotics needs to be started 10 hours or less after fever onset, to be fully effective. For all antibiotics, a 10-day course is recommended with commencement as early as possible, based on data from murine models. In mice infected with Y. pestis by the airborne route, successful treatment of disease with ciprofloxacin was dependent on the commencement of antibiotic dosing within 24 hours of exposure to the pathogen.

Improvements in the condition of patients suffering from bubonic plague are seen within 2–3 days, but buboes may remain for several weeks, although culture-negative. Clinical isolates of plasmid-mediated, antibiotic-resistant Y. pestis have previously been reported from Madagascar and Peru. ^, The failure of a patient to respond to treatment with one or more of the antibiotics tetracycline, streptomycin, chloramphenicol, and sulfonamides should alert the clinician to the possibility of an antibiotic-resistant strain of Y. pestis . All sensitivity testing and culture of Y. pestis should be done carefully by a person experienced in manipulating dangerous pathogens safely, with biocontainment of the organism and protection of the operator.

Family members and other close contacts with individuals 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 antibiotics prophylactically for 7 days in these individuals, especially if the primary case is pneumonic plague. The prophylactic use of antibiotics is recommended even in individuals who have been previously vaccinated with the KWC vaccine and who might have been exposed to airborne Y. pestis because of the limited ability of this vaccine to provide protection against pneumonic disease.