Plague is an exceptionally virulent, vector-borne zoonotic disease transmitted from rodents, especially rats, through the bites of infected fleas, most often the rat flea, Xenopsylla cheopsis. Many different species of mammals, including rats, squirrels, mice, prairie dogs and gerbils actually are animal reservoirs for the agent of plague, Yersinia pestis, which persists in the environment as the result of a stable and constant rodent-flea infection cycle, causing a fatal disease in murine and sciuride populations
. The reduction of rodent populations, whether as a consequence of the disease or of rodent control measures, compels fleas to seek new warm-blooded mammalian hosts, incidentally including humans.
The first major epidemic of plague to be historically recorded occurred in China in 224 BC. In Europe, plague was endemic in all of the Roman Empire, with severe outbreaks occurring occasionally, such as the outbreak which occurred in Rome in the third century AD, giving rise to one of the worst persecutions of Christians. Plague later came in long-lasting, dreaded pandemic waves  . The first documented pandemic, the Justinian plague, killed several million people in the Byzantine Empire during the 6th to 8th century. The second pandemic, the "Black Death", started in the middle of the 14th century and persisted over several hundred years, killing about 30% of the European population and culminating with the Great Plague of London in 1665. The third pandemic started in China in the middle of the 19th century and caused 10 million deaths in India alone.
Although the dramatic epidemics of urban plague have disappeared, due to improved sanitation and public health surveillance, plague still is a significant health problem in Africa, Asia and South America, which report around 2 000 cases every year with a global case fatality rate of 5% to 15%. The disease is endemic to Africa, India, and the southwestern states of the USA, and isolated outbreaks continue to this day in many regions of the world    . Africa, mainly The Democratic Republic of Congo and Madagascar, account for 96% of world cases since 1990. The identification of naturally occurring multiple-drug-resistant strains of Y pestis in Madagascar   , as well as the discovery of high frequency conjugative transfer of antibiotic resistance genes to Y pestis in the flea midgut  are matters of serious concern. Moreover, plague has attracted a considerable attention because of its possible use as an agent of biological warfare and terrorism .
Plague assumes three major clinical forms in humans: bubonic, pneumonic, and septicemic. Flea bites usually cause bubonic plague, whose name comes from the bubo, a painful swelling of the bite site-draining lymph nodes which often become hemorrhagic and necrotic (hence the name 'Black Death'). Without prompt antibiotic treatment, approximately 50% of bubonic cases rapidly progress to sepsis and death. About 30% of fleabites directly lead to sepsis, without prior evidence of a bubo  . Sepsis is characterized by circulatory collapse, coagulopathy, hemorrhage, respiratory distress, shock, and organ failure, leading to death in about 40% of cases. The most feared form is pneumonic plague because this form can readily be transmitted from person-to-person via inhalation of contaminated airborne droplets  . Symptoms begin with rigor, severe headache and malaise then quickly advance to fever, difficulty breathing, and cough that yields infectious, bright red sputum teeming with bacteria. The case fatality rate is close to 100% if no antibiotic treatment is given within the first 48 hours following symptoms onset  . The study of experimental Y pestis aerosols in animal models showed that 1µm particle aerosols resulted in both primary pneumonia and infection of the upper respiratory tract whereas 12 µm particles infection resulted in the attack of the nasal mucosa and nasal-associated lymphoid tissues (NALT) prior to bacteremic dissemination and secondary pneumonia .
The pathology of plague is very similar in rodents, nonhuman primates and humans    .
Yersinia pestis, first identified by Alexandre Yersin in 1894, is a Gram-negative, nonmotile bacterium that belongs to the family Enterobacteriacae. Three species in the genus Yersinia are pathogenic for humans: Y pestis, Y pseudotuberculosis and Y enterolytica, the latter two being the cause of self-limiting enteropathogenic infections characterized by diarrhoea, fever and abdominal pain. The extreme virulence of Y pestis mostly results from its virulence factors that impair the host innate immunity response, including phagocytosis, and allow the bacteria to multiply and spread unchecked in the host
The major mechanism that impairs the host phagocytosis response is a 70-kb plasmid (pCD1)-encoded Type III secretion system which is activated by growth of the bacterium at 37°C and whose function is to directly translocate Yersinia outer proteins (Yops) to neighboring host cells, mostly dendritic cells, macrophages and neutrophils, in which they disrupt signaling pathways, suppress cytokine production, debilitate the antibacterial defense mechanisms and promote apoptosis  . The pCD1 plasmid also carries the lcrV gene, which encodes the 37-kD low calcium response virulence antigen, LcrV-Ag, that serves as a positive regulator of the type III secretion system  . Y pestis lacking LcrV is avirulent in mouse models of plague disease. In addition, LcrV can activate Toll-like receptor 2 and trigger the release of IL-10   , a cytokine that suppresses innate immune functions  . LcrV also prevents the release of proinflammatory cytokines tumor necrosis factor (TNF)-? and y-interferon in murine and human macrophages .
Other Y pestis virulence factors include the F1 pilus antigen, a 17 kD polypeptide encoded by the caf gene that is carried on a large 100-kb plasmid (pMT1, or pFra). F1 is the major protein component of the outer capsule encompassing Y pestis bacilli and is believed to help avoid phagocytosis  . YopH, a protein tyrosine phosphatase that is part of the type III secretion system  , Pla, a plasminogen activator protease encoded by plasmid pPCP1, as well as murein (or Braun) lipoprotein (Lpp), which links the outer bacterial membrane to the peptidoglycan layer in Enterobacteriacae  are also virulence factors, as judged by the fact that their mutation or deletion attenuates the virulence of Y pestis in rodents, which is currently used as a basis for the development of live attenuated vaccine strains.
The first widely used plague vaccine was developed by Haffkine in 1897 using a heat-killed culture of Y pestis. The vaccine conferred significant protection against bubonic plague but induced severe adverse reactions including high fever in the majority of vaccinees. Moreover, later studies in rodents and nonhuman primates showed that the vaccine was unable to elicit protection against pneumonic plague. A formalin-killed whole-cell vaccine was developed in the mid-20th century in the USA and used to protect US military personnel against bubonic plague during the Vietnam War
, but it also caused severe adverse reactions and was unable to elicit protection against pneumonic plague. Its use was discontinued in 1999
The development of live attenuated plague vaccines began at the beginning of the 20th century using partially attenuated, pigmentation negative (pgm negative) Y pestis strains such as the Girard and Robic EV strain   and later derivatives  . Between 1934 and 1940, mass vaccination campaigns in Madagascar dramatically reduced the annual plague incidence (from 3500 to 200 cases). In spite of frequently reported side effects and residual virulence in nonhuman primates  , the live attenuated EV 76 and EV 88 strains are still in use in Russia and Central Asian republics today  , both for protecting humans and camels. Other live attenuated plague vaccines that are in early development include a DeltaYopH strain, which protected mice against high-dose parenteral or aerosol challenge after a single intranasal administration , a Deltalpp mutant  and a YadC mutant 123] , as well as an IpxM mutant of the already partially attenuated EV strain of Y pestis  . In addition, the use of Yersinia pseudotuberculosis as a Jennerian vaccine against plague has been entertained because it shares high genetic identity with Y pestis, is less virulent and can be administered by the oral route .
The development of subunit plague vaccines started in the 1950s, focusing on the use of the capsular F1 (Caf1) pilus antigen. Vaccination with F1 protected rats, mice and nonhuman primates against subcutaneous and aerosol challenge with virulent Y pestis    . However, Y pestis variants lacking caf1 were found which not only were fully virulent in animal models of bubonic and pneumonic plague, but also broke through the immune responses generated with F1 subunit vaccines   .
Unlike F1, the Lcr V antigen was found to be critical for Y pestis virulence    and, when used as a subunit vaccine, generated high titers of antibodies that conferred protective immunity against bubonic and pneumonic plague in mice, guinea pigs and nonhuman primates, whether the strain used for challenge was F1 positive or not     . Passive immunization with an anti-Lcr V monoclonal antibody was shown to protect mice against aerolized Y pestis, even when administered 48 h postinfection  . Finally, anti-Lcr V antibodies were demonstrated to neutralize Y pestis-mediated macrophage cytotoxicity in a dose-dependent manner, which could be used as an in vitro assay as a correlate of protective immunity  . In view of the immune modulatory properties of Lcr V, concerns were raised regarding its safety as a vaccine in humans. A truncated version of the Lcr V antigen, V10, which lacks amino acids 271 to 300, was developed that showed reduced immune modulatory properties while offering full protection of mice against bubonic and pneumonic plague    and could be used advantageously in place of the full molecule.
Candidate vaccines containing either a combination of F1 and Lcr V antigens or a recombinant F1-Lcr V fusion protein in alum or alhydrogel formulations have been developed that efficiently protect mice against pulmonary Y pestis challenge       and elicit long-lasting protective antibodies able to neutralize Y pestis-mediated cytotoxicity of macrophages in cynomolgus macaques  . The F1-V vaccine also protected black-footed ferrets against oral challenge with Y pestis  . Both vaccines appeared to be safe and immunogenic in human trials   . A spray-freeze-dried F1-V fusion protein powder vaccine was recently developed that could be administered either by the IM or the ID route with similar protective efficacy in mice. The vaccine could also be administered by the intranasal route but an extra dose was required to achieve the same level of protection  . The DynPort Vaccine Company (DVC) is managing the advanced development of a rF1-V vaccine for the US Department of Defense (DOD) .
The possibility of developing a dual vaccine against anthrax and plague was investigated in a murine model by combining equal amounts of the anthrax rPA antigen and the Y pestis F1-V antigen. The vaccine was able to elicit a robust IgG and IgG1 response in mice against both antigens when administered by the SC route and a robust IgG2 response when administered by the intranasal route with appropriate adjuvants. Circulating antibody levels were still detectable at 6 months post primary immunization .
The US Army Medical Research Institute of Infectious Diseases (USAMRIID) demonstrated however that while the F1/V vaccines efficiently protected cynomolgus macaques against aerosolized Y pestis challenge, they failed to do so in African green monkeys, which raises the question of their eventual efficacy in humans  . A number of approaches are underway to increase the efficacy of the subunit F1/V vaccines  , such as the introduction of point mutations in the V antigen  or the use of other adjuvant formulations than alhydrogel. Thus, the use of flagellin as an adjuvant was tested by generating a flagellin-F1-V triple fusion protein that elicited robust antigen-specific humoral immunity in mice and two species of nonhuman primates and fully protected mice against intranasal Y pestis challenge  . The flagellin-F1-V antigen showed remarkable stability at temperatures between 4° and 25°C. In another approach, a promising intranasal vaccine against pneumonic plague was developed using lipid A mimetics as adjuvants, which showed high protective efficacy in both mice and rats  . In still another approach, rF1 and V antigens were separately microencapsulated in polymeric microspheres, mixed together and used to immunize mice by either the IM or intranasal route, resulting in high levels of serum IgGs, secretion of cytokines by the spleen and draining lymph nodes and protection against high level Y pestis challenge after a single immunization .
Protective efficacy of DNA vaccines was studied using plasmids that encoded the F1 and V antigens together with interleukin 12 (IL-12) as an adjuvant. DNA vaccines were administered either by the intranasal or the IM route, but protection was reached only after three weekly doses followed by protein boosts  .
Improving the efficacy of F1- and/or Lcr V-based vaccines by delivering the antigens via live attenuated recombinant vectors was also attempted using attenuated Salmonella enteritica serovar Typhimurium (Salomonella typhimurium) as a vector      . A single oral dose of a Samonella-F1-antigen recombinant protected mice against bubonic plague challenge, but not against pneumonic plague challenge. Protection against pneumonic plague challenge required the dual expression of both the F1 and the V antigen by the Salmonella vector  . The use of a vesicular stomatitis virus (VSV) vector expressing the V antigen and administered in a prime-boost regimen also protected mice against intranasal challenge with Y pestis  . The same protective efficacy was obtained with a single-dose IM immunization with an adenovirus recombinant expressing the V antigen .
Mice immunized by SC immunization with F1-V antigen purified from Nicotiana tabacum leaves expressing the F1-V fusion antigen in chloroplasts were boosted by oral delivery of the transgenic plants, which induced effective protection against aerosolized Y pestis challenge .
In spite of all these efforts, and the wealth of investigational approaches, we still currently lack a safe, effective and licensed vaccine for pneumonic plague, which is nearly always fatal and can be intentionally transmitted by weaponized strains of Y pestis. None of the live attenuated or live recombinant plague vaccine candidates is ready yet for an application for a license, which will have to be in accordance with the FDA 'Animal Rule' that requires safety and immunogenicity data in humans along with robust efficacy data in more than one animal model.