Live Plague Vaccines Development: Past, Present and Future

1. Introduction

Plague is a poly-host and multi-modes of transmission zoonotic infection caused by the gram-negative bacterium Yersinia pestis. Before the widespread use of vaccines and the era of antibiotic therapy plague was one of the deadliest diseases in human history, which claimed the lives of over 200 million individuals. Effective use of the first generation of plague vaccines [1,2,3,4,5] together with successful introduction of antibiotics in plague therapy created a false impression that the devastating plague epidemics remained only in the history of mankind [6]. However, isolation from the natural plague foci of Y. pestis strains resistant to all antibiotics recommended by WHO experts for treatment of plague [7] and non-compliance of previously developed and used plague vaccines with recent WHO requirements [8] indicate the need for development of modern plague vaccines.

At the turn of the 19^th and 20^th centuries it was shown that the inoculation of pathogenic microbes, either attenuated or killed, caused protection against the corresponding infection. A single administration of live vaccines, as a rule, ensured formation of more intense and long-lasting immunity than a similar inactivated vaccine administration. However, live vaccines based on the strains with reduced virulence, unlike inactivated ones, do not exclude the risk of causing an infectious process in immunocompromised individuals. In addition, reversal of virulence is possible as a result of compensatory mutations in the vaccinal strain. Inactivated vaccines also have their own drawbacks. Therefore, research is ongoing to improve both vaccine forms in order to develop the ideal vaccine, combining safety with high protective potency and prolonged immunity [9].

This article briefly reviews the earliest research as well as current and future approaches to the development of live plague vaccines. Readers are encouraged to consult recent reviews for information on trends in the development of modern plague vaccines and their administration regimens [10,11,12,13,14,15,16]. Here, we provide an overview of how to improve live attenuated plague vaccines based on rational considerations [17].

2. Classical Attenuation of Yersinia pestis

Attenuation is a decrease in the virulence of strains of pathogenic microbes for their native hosts as a result of serial passages through naive animals of a host species insensitive to infection, or immune native hosts, or tissue culture/artificial nutrient media, or exposure to bacteriophages, radiation, or chemicals, or lastly due to the site-directed mutagenesis [9,18,19]. Louis Pasteur was the first to generate attenuated strains of pathogens, etiological agents of chicken cholera, anthrax and rabies, and then used them as the basis for vaccine preparations [20,21]. Currently, live vaccines based on attenuated strains of viruses and bacteria continue to be used and developed to prevent such particularly dangerous and/or socially significant infections such as tularemia, brucellosis, anthrax, tuberculosis, rabies, influenza, yellow fever, measles, poliomyelitis, and plague [22,23,24,25,26,27]. Both types of whole-cell vaccines have their own advantages and drawbacks. Thus, after a single inoculation, live vaccines provided immunity that was similar to a post-infection one, but their use could cause an infectious process in persons with a reduced immune status. The dead microbes were unable to cause an infectious process, but numerous doses of inactivated vaccines are generally needed to provide sufficient durable immunity [9].

The empirical and time-consuming classical attenuation of pathogenic bacteria is based on selection of mutants with reduced virulence from a heterogeneous population of a pathogen during its culture under adverse conditions such as long-term passages in animals resistant to the pathogen, in tissue culture and in nutrient media at altered temperatures or when saponin, bile, antibiotics and other substances inhibiting the vital activity of bacteria are added to the nutrient medium. It is possible to generate attenuated strains much faster by treating microorganisms with mutagens, followed by selection of clones with reduced virulence [28]. The passaged population is usually genetically heterogeneous and contains both attenuated and virulent microbes. An important step in the generation of attenuated strains is cloning, that is, isolation of clones with the most reduced virulence from the resulting population of microorganisms, followed by the preparation of a genetically homogeneous bacterial population based on such clones. This methodology was used to obtain attenuated vaccine strains of rabies virus (serial passages from rabbit to rabbit [29]), bacillus Calmette and Guérin (BCG) (passaging on a glycerin-bile-potato mixture for 13 years [30]) and yellow fever virus (virus passages in chicken embryos and tissue culture [31]).

The first attempts to attenuate the plague pathogen were made by its discoverer A. Yersin. To do this, he used culturing at elevated temperatures and/or addition of ethanol at 0.5-5.0% to the nutrient medium. The combination of these approaches made it possible to obtain almost avirulent cultures in two months of passages [4]. In addition, he demonstrated that live attenuated plague microbes protected small laboratory rodents significantly better than killed ones. The protective superiority of live plague vaccines was also shown by other researchers who generated a number of attenuated strains of the plague pathogen [5,32], some of which are presented in Table 1. Later, it was also shown that a greater degree of residual virulence of attenuated cultures was accompanied by a greater degree of protection, while complete attenuation led to a significant decrease or total loss of immunogenicity. Thus, W. Kolle and R. Otto [33] showed that a single injection of attenuated in varying degrees of spontaneous Y. pestis mutants to mice, rats, guinea pigs or monkeys caused the death of some of them. However, the surviving animals were protected from death after subsequent infection with a highly virulent strain to a greater extent and for a longer time than those repeatedly vaccinated with killed bacteria. It was also noted that a greater degree of attenuation was usually accompanied by lower immunogenicity [34]. Attenuated strains are also found among closely related microorganisms circulating in nature. In our case, these are representatives of the microti subspecies of Y. pestis, which are as a rule avirulent for humans and guinea pig but being highly pathogenic for laboratory mouse and their main host, vole [35,36].

At the beginning of the 20^th century, it was persuasively shown that vaccine preparations containing exclusively inactivated Y. pestis and its waste products couldn't provide a high level of protection to vaccinated people as well as to protect inoculated guinea pigs. There was only a slight increase in resistance to experimental infection of the latter, while the live vaccine, in very small doses, completely protected cavies from Y. pestis infection. Mass vaccination campaigns against plague carried out in Java, Madagascar and South Africa have shown the high effectiveness of live plague vaccines without any accidents among several million human vaccinations [18,41]. It should be noted that the vaccine strains used were derived from different parental strains, attenuated using different procedures based on different theoretical backgrounds (Table 1).

Five conclusions can be drawn summarizing the results of this stage of research on the development of a plague vaccine:

• Live plague vaccines are superior to killed ones in terms of intensity and duration of induced immunity.
• The simultaneous use of two independently attenuated Y. pestis strains potentiates the protective potency of each of them.
• Immunizing properties are dependent on more than one antigen. Different antigens fluctuate in their ability to induce immunity in diverse mammalian species. The immunogenicity of plague vaccines should be evaluated in mice, guinea pigs and monkeys.
• Vaccines based on live attenuated Y. pestis strains may cause the death of some immunized mammal individuals with weakened immunity or metabolic disorders.
• A mixed vaccine including the strains with special efficacy in rats and in guinea pigs protects both animal species better than the monocomponent ones.

3. Discovery of Y. pestis Virulence Determinants and Subcellular Fractions Responsible for Pathogenicity and/or Immunogenicity

The disadvantages of killed and live whole-cell plague vaccines have initiated the search for bacterial subcellular components that induce the development of a protective immune response. H. Schütze [42,43,44] described two antigens that are components of Y. pestis. The antiphagocytic envelope antigen (envelope substance, capsular antigen, fraction I, F1, antigen A, antigen 3 [38], Caf1 [45]) was contained in the gelatinous capsule formed at 37°C, while the water-insoluble somatic antigen (basic somatic antigen (BaSoAn), “residue antigen” [32,46]) was a part of the somatic portion of bacterium. The envelope antigen possessed significant prophylactic power for rats, mice and monkeys [32], while the water-insoluble “residue” antigen remaining after saline extraction of acetone-treated bacteria [38,47,48] and consisting of 70-75% of the whole bacterium [49] was protective for guinea pigs and monkeys [32,50].

In 1956, T.W. Burrows described the virulence antigen Vi [51], and later renamed as V [38]. Like F1, it was also antiphagocytic, but when the culture temperature changed from 28°C to 37°C, its activity appeared several hours earlier than the comparable activity of F1 [38]. Immunization of rabbits induced higher titers of antibodies to the V antigen compared with similar indicators in guinea pigs and mice. Furthermore, immune rabbit serum provided passive protection of mice from infection with the wild-type strain of Y. pestis. Guinea pig immune serum was not protective.

The summarized results of 60 years of research from several laboratories on Y. pestis strains differing in virulence allowed T.W. Burrows to propose the concept of virulence determinants. The absence of one or more of these biomolecules or properties in a pathogen is accompanied by its attenuation [38,48,52]. T.W. Burrows perfectly understood the limitations of the concept he put forward, based on the study of non-isogenic strains and experimentally unsupported assumptions. Direct proof of the importance of any determinant in the pathogenesis of plague would require demonstrating that a "wild-type" strain mutated in the gene encoding that virulence determinant would decrease its virulence, and complementation of the non-functioning gene would restore virulence to its original high level (Table 2). In an attempt to prove this direct evidence, he observed several challenges including:

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the possibility of losing not only the determinant, but also some unidentified genes, and;

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the absence at that time of tools for genetic exchange using models that would allow complementation. As for the evidence that a particular factor plays a minor role in virulence, it suffices to show that its loss does not reduce the virulence of the pathogen [48]. Another problem he mentioned is that the overwhelming majority of researchers did not use standard operating procedures when assessing immunity to plague, which prevents a reliable comparative assessment of the results obtained in different laboratories using different vaccine strains and different test-infecting strains administered at different doses, etc. Unfortunately, the latter situation has survived to this day [53].

Further studies of plague pathogenesis and immunogenesis in molecular biology, genomic and post genomic eras [60,61,62,63,64,65,66] allow scientists to identify a significant number of additional pathogenicity factors necessary for full virulence (Pla, PsaA, Ymt, Ail, LPS, etc.), but among them there were no antigens comparable in protectiveness to the F1 and V antigens [67]. This was the reason for the vast majority of researchers to switch their focus to developing various versions of low-component subunit vaccines based on F1 and V antigens. In principle, vaccines based on nucleic acids [68,69], viral or bacterial vectors [70,71], plant-derived plague vaccine [72], nanoparticles [73] and microencapsulated preparations [74,75] can also be included into the group of subunit vaccines, since they all induce an immune response to only one to three target immunodominant antigens.

The main advantage of subunit vaccines is their non-infectious nature. Other benefits include low reactogenicity and relative ease of authentication control. However, they have no or limited innate defense triggers and do not guarantee formation of the good immune memory. Their immunogenicity can be increased through the use of adjuvants (for example, adsorption on aluminum hydroxide) [76].

Different mammalian species, subspecies and even populations including diverse racial/ethnic groups of humans vary in their susceptibility to plague [5,46,55,77,78,79,80,81,82,83] and other severe bacterial infections [84]. They also differ in their ability to respond to different antigens of bacterial pathogens causing the humoral and/or cellular species-specific immune response [83,85]. The reason for this is the differences between mouse and human immunology [86,87,88].

In addition, age and sex differences in the immunopathogenesis of infectious diseases have also been recorded. Susceptibility to and mortality from many infections is higher among men, and the immune response to various types of vaccinations is usually stronger in women, which is likely due to stronger humoral responses. The major sex steroid hormones have opposing effects on cells of both the adaptive and innate immune systems: estradiol generally enhances, while testosterone suppresses the response [89]. Recently, sex differences in plague-vaccine efficacy, and the immunological factors that differ between male and female BALB/c mice were shown. The protectivity of various experimental plague vaccines was higher in female mice. Males had a higher bacterial load and differed in cytokine expression patterns and serum antibody titers. The synergy between vaccination and antibiotic treatment repeatedly observed in female mice was absent in male mice [90]. Taking into account the above and, above all, the fact that the vast majority of experiments assessing the protectiveness of candidate plague vaccines are carried out using a mouse model, their results must be extrapolated to humans with great caution.

In addition to differences in the interspecies-specific and intraspecies-specific host response to Y. pestis antigens, it is also necessary to take into account Y. pestis antigenic variability. To evade host immune defenses, pathogens evolve by producing isoforms of immunodominant antigens, bacterial factors absolutely necessary for the virulence of the pathogen [91,92], or reversibly [93,94] or even completely [95] losing the ability to produce these antigens if their loss does not reduce virulence.

In closing this section, the following should be emphasized:

Two-component subunit plague vaccines based on the F1 and V antigens provide good protection in mice [96,97], but their efficacy varies significantly among non-human primate species, and antibody titers do not correlate with protection [10,14,98].

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These antigens induce primarily antibody-mediated immune responses in humans [99,100,101] with widely varying antibody titers and induce weak cellular responses [102], which play a decisive role in human immune protection against plague.

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Such low-component vaccines will be of little effectiveness against infection by strains producing serologically distinct isoforms of V [91] and F1 [92] antigens or by virulent mutants that fail to form an envelope from the capsular consisting of F1 antigen [103].

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Taking into account the above considerations, it is hoped that a whole cell vaccine, containing a complete or almost complete set of pathogen antigens, can provide protection against both “classical” and antigen-altered variants.

4. Recent Progress in the Development and Use of Live Plague Vaccines

4.1. Current Plague Vaccination

Recent progress in high-throughput “omics” methodologies and the public accessibility of complete genome sequences of hundreds of strains of many species of pathogenic bacteria have fundamentally changed the scope for picking out new vaccine candidates. In the course of development of plague vaccines, a number of promising vaccine strains were generated, some of which were successfully used as a live vaccine for large-scale immunization involving several million people (more detailed information about these studies can be found in the reviews [10,11,12,13,14,15,67,104,105,106,107,108]), but only one of them, based on the NIIEG line of the EV76 strain, is currently approved for vaccination of people in a limited number of countries.

In the Soviet Union, more than 10,000,000 people were vaccinated with the live vaccine based on the NIIEG line of the EV strain [109]. An analysis of the results of vaccination with the live vaccine based on EV line Saigon of the population of six provinces of South Vietnam involving 2,089,388 people indicates that vaccination did not fundamentally affect the reduction of morbidity among those vaccinated, but the course of the disease was milder and complications in the form of secondary pneumonia were less frequent [110,111]. According to N.I. Nikolaev, in 1945, during the period of exacerbation of the epidemiological situation in Inner Mongolia, the use of the live plague vaccine made it possible to reduce the incidence in the vaccinated group (0.25 per 1000 people) compared to the unvaccinated (28.8 per 1000 people) by 100 times [109]. High efficiency has also been demonstrated with the use of the Y. pestis EV vaccine strain in the Belgian Congo and South Africa [112,113].

Currently live plague vaccine based on the NIIEG line of the EV76 strain is used annually in quantities of several tens of thousands of doses in Russia and Kazakhstan to immunize the anti-plague laboratories’ staff and the population living in enzootic areas. Over the last five years, 90,822 people have been vaccinated against plague in the Altai Republic [109,114].

It is obvious that the live vaccine based on the NIIEG line of the EV strain will continue to be used for specific plague prevention until a new drug is developed that is superior to the existing one [115] in at least some of the parameters noted in the WHO target product profile for plague vaccines [8].

4.2. Criteria for Selecting Candidate Vaccine Strains

The development of any vaccine candidate follows general rules, but each development has its own specifics depending on the type of vaccine (live/killed/subunit/DNA/peptide), pathogen-host interaction at the population level, target product profile, target population and availability of an existing vaccine. To include a vaccine candidate in the drug development cycle, it must meet a number of criteria. However, international standard operating procedures for assessing plague immunity are still lacking, which does not allow for a reliable comparative assessment of the results obtained in different laboratories using different candidate vaccine strains and different test-infecting strains administered at different doses [19,116,117].

In Russia, all trials of attenuated plague strains that are promising as vaccine strains are conducted in comparison with the reference Y. pestis vaccine strain EV line NIIEG [118]. In abbreviated form and in English, these requirements are discussed in the article [97]. According to this publication, candidate vaccine strains of Y: pestis must match or exceed the reference vaccine strain EV in terms of immunogenicity. In addition, it should match the control strain in terms of harmlessness and reactogenicity or be safer, and may differ from the EV strain in some insignificant characteristics but still maintain it as a representative of the species of Y. pestis.

The candidate vaccine strain being studied must:

• be lysed by the plague diagnostic bacteriophage L-413C [119];
• be typical in its cultural and morphological properties [120];
• the F1 titer of the studied strain must not be less than the similar indicator obtained with the culture of the control strain Y. pestis EV grown under similar conditions;
• the proportion of calcium-independent mutants in the population of Y. pestis cultures [121] passaged through the body of laboratory animals and not subjected to long-term storage or any physical impacts, must not exceed 0.3%;
• must not be inferior to the control strain in fibrinolysin-coagulase activity [122];
• the studied and control strains must not have the ability to pigment sorption (pigmentation) [123];
• the studied vaccine strains, similar to the reference strain EV, should have 3 bands of plasmid DNA on the electropherogram, corresponding to pFra (60 MDa), pCad (47 MDa) and pPst (6 MDa) [120].

When reading these requirements, it seems that they were developed to obtain strains that are closest in properties to the EV vaccine strain. In fact, attenuation can be carried out not only by eliminating the pgm locus from the genome [123], but also by knocking out other genes encoding pathogenicity factors [67]. A change in the structure of lipopolysaccharide during attenuation due to shortening of the core oligosaccharide [124] should prevent the interaction of the diagnostic phage with the surface of the bacterial cell [125]. Cultural and morphological properties can most likely change when editing the genes encoding the surface structures of the bacterium [126]. The production of plasminogen activator does not contribute to the development of intense immunity and it is advisable to remove the gene encoding it from the genome [55,127].

There is no doubt that despite the variety of candidate vaccine strains, all of them must meet at least the following criteria:

• absolute safety;
• high vaccine protectivity.

The methods used to test these criteria should be standardized and their number reasonably minimized.

The most accessible way to assess specific immunity in vaccinated individuals is to determine the level of specific antibodies, but the presence of specific antibodies even to immunodominant antigens F1 and/or V does not completely and not in all mammals correlate with the host’s protection from infection [128], since the leading role in the formation of anti-plague immunity belongs to cellular factors of the immune system. systems [129].

4.3. Yersinia pestis Natural Strains Selectively Virulent or Non-Pathogenic (Conditionally Pathogenic) for Human

Y. pestis species includes a group of strains (pestoides group, vole’s strains, bv. Microtus, subsp. microti) circulating in populations of various species of voles or Mongolian pikas. These strains are highly virulent in their natural hosts and laboratory mice, but they are generally avirulent in larger mammals, including humans [35,46]. In fact, these strains are already candidates for vaccine strains.

Evaluation of the protective efficacy of the subsp. microti strain 201 showed that this strain is highly attenuated by subcutaneous route, elicits an F1-specific antibody titer similar to the EV and provides a protective efficacy similar to the EV against bubonic plague in Chinese-origin rhesus macaques [130]. It would seem that the problem of a live plague vaccine in terms of obtaining candidate vaccine strains has been solved, since natural plague foci (including those in which conditionally pathogenic for human vole’s strains circulate) are a virtually inexhaustible source of Y. pestis isolates. However, there are several "buts":

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vole’s strains are characterized by polymorphism of amino acid sequences of a number of proteins (including F1 [92] and V antigens [91]); several laboratory-confirmed cases of isolation of microti strains from humans have been described [46];

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the possibility of increasing subcutaneous virulence for guinea pigs to levels similar to that of strains of the main subspecies was shown through testicular passages [131].

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So, in order to be confident in the safety of vole’s strains in the event of their use as vaccines, it is necessary to introduce into their genome one or two additional alternative attenuating mutations.

4.4. Selective Protective Potency of Yersinia pestis

The majority of plague experts believe that differences in the clinical form and severity of plague infection are not determined by age, gender or ethnicity, but are due only to differences in exposure to infection [132,133]. Nevertheless, a number of data indicate that the different severity of the infection and the intensity of immunity is determined by the genotype (ethnicity) of the host [134,135,136] in interaction with the specific phylogenetic group (subspecies) of the pathogen [35].

An efficient immune response to a vaccine depends on activation of antigen-presenting cells and antigen-specific T and B lymphocytes, induction of memory cells, regulatory cells and antibody-secreting plasma cells, as well as on the persistence of the antigen in the body. The strength of immune response largely depends on the genes participating in this process. It was shown that gene polymorphism could be responsible for insufficient immune response to a plague vaccine in a proportion of humans. After two subsequent vaccinations of human volunteers, 67 % of them developed specific antibody titers equal to or exceeding the threshold level. However, 33 % of the subjects did not develop specific antibodies to either V, or F1 antigen during 90 days of observation post vaccination. Investigation of 20 single nucleotide polymorphisms (SNP) in 14 of immune response genes in an attempt to find an association between a particular allele and the ability to develop antibodies to Y. pestis antigens was performed. Homozygosity for the IL1β gene wild type allele variant C-3953, or homozygosity for a mutated allele T-3953 allowed to produce anti-F1 IgG, whereas heterozygotes remained seronegative. Heterozygotes for the TLR9 gene at position 2848 (A2848G) were also responders to F1. Among responders to the V antigen, IgG production was observed only in carriers of mutant IL4 allele C-589T and in carriers of mutant allele of IL6 gene C-174G, either homozygotes or heterozygotes, and was not observed in homozygotes of the wild type allele. The lack of a mutant allele of IL10 gene C-819T was also associated with a non-responder phenotype. The results of experiment suggest that potential non-responders to the plague vaccine may be identified by SNP in the IL1β (С-3953 Т), IL4 (C-589T), IL6 (С-174G), IL10 (C-819T), and TLR9 (A2848G) genes. Individuals with non-responder genotype should be included in the clinical trials assessing novel, universally effective plague vaccines [136].

5. Strategies Aimed at Increasing Genetic Stability

Currently, in countries where there are no licensed plague vaccines, the main efforts of researchers are aimed at developing new remedies that meet WHO requirements [8]. In Russia and Kazakhstan, countries of the former Soviet Union, where the live plague vaccine based on the EV vaccine strain continues to be constantly used to immunize tens of thousands of people annually, most research studies have been aimed at preserving the properties of the initial vaccine strain in terms of safety and protective activity during prolonged storage of stock culture. To a large extent, this problem was solved by using freeze drying and storing dry vaccine in low-temperature refrigerators [137].

Genetic variations of live attenuated plague vaccine strains (Y. pestis EV76 lineage) during laboratory passages in different countries [138] caused accumulation of mutations that reduced the protectiveness of the vaccine strain [137].

This problem can be solved in several ways as below:

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Comparative tests were carried out on all lines of the EV vaccine strain supported in the USSR. The freeze-dried NIIEG lineage retained maximum protective activity and was grown and packaged as stock cultures for the subsequent production of live plague vaccine [137]. Currently, in countries where there are no licensed plague vaccines, the main efforts of researchers are aimed at developing new remedies that meet WHO requirements [8].

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Animalization of the vaccine strain is carried out with the aim of purifying its population from mutants that have reduced viability.

"Animalization" of the Y. pestis EV vaccine strain was carried out by its intravenous injection to a rabbit and subsequent (after 3-4 hours) isolation of the vaccine strain from its internal organs. A comparative assessment of the original and animalized bacterial cultures showed a significantly greater number of viable microbes in the preparations of the latter, combined with a significantly greater degree of protection when infected with a virulent strain [139].

Another option for animalizing the vaccine strain involves three testicular passages in guinea pigs [140].

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Stabilization of the genome of the vaccine strain by genetic engineering methods is also possible. Recombinase RecA is responsible for most acts of homologous genetic recombination in bacteria [141,142]. To overcome unwanted homologous recombination that destabilizes the genome of vaccine strains of various bacterial species, researchers create recA deletion mutants, since RecA is mainly involved in recombination in bacteria [143,144].

Deletion of the BCG recA gene lead to a complete loss of recombination between homologous sequences located on the chromosomes, as well as between sequences located on the plasmid and on the chromosomes. Mutant BCG ΔrecA was as effective as wild-type in protecting mice from intravenous challenge with virulent Mycobacterium tuberculosis, indicating that the loss of the SOS-mediated DNA repair mechanism does not compromise the immunological properties of BCG. The availability of genetically stable, fully immunogenic BCG is important for the future development of BCG as a live vaccine [145].

Construction of a recA knockout mutant was successfully used to stabilize the genome of Francisella tularensis subsp. holarctica vaccine strain 15 lineage NIIEG. Compared to the parent strain, the constructed 15/23-1ΔrecA mutant multiplied in macrophage-like J774A.1 cells 8-10 times slower. BALB/c mice responded to immunization with strain 15/23-1ΔrecA with a smaller weight loss (~ 2%) than to strain 15 (~ 14%). F. tularensis strain 15/23-1ΔrecA, which has reduced reactogenicity, has been proposed as the basis for the creation of a stable and safe live tularemia vaccine [146]. It makes sense to try to stabilize the genome of candidate Y. pestis vaccine strains in a similar way.

6. Synergy of Action of Multi-Strain Vaccines

As noted above, the synergistic effect of combined immunization with two differently attenuated Y. pestis strains resulted in reliable protection against plague [4,147]. caused by both wild-type and nonencapsulated Y. pestis strains. Such double-strain vaccines include both bacteria covered with an envelope formed from the F1 antigen, and bacterial cells whose outer membrane antigens (LcrV, YopD, YpkA, etc.) are open for interaction with the host immune system.

7. Future of Plague Live Vaccines

The need to improve an existing commercial plague vaccine and/or develop new effective and safe vaccines can be realized in several directions of research. Regardless of the chosen approach, it is desirable that the vaccine being developed presents to the immune system multiple antigens, and induce both humoral and cellular immune responses, since both arms of the immune response appear to be critical in anti-plague vaccine strategies. Unfortunately, vaccines not only protect against infectious diseases but also can cause both general and local post-vaccination adverse reactions [148,149], one of the causes of which may be allergies [150]. These reactions are most often associated with the non-microbial components of the vaccine, but in many cases the specific ingredient causing the reaction cannot be definitively identified [148]. A high level in the blood of IgE, which is responsible for the formation of allergic reactions, was noted in people who were repeatedly vaccinated against plague [151]. A direct dependence of the frequency of allergic diseases on the frequency of vaccination of employees of anti-plague institutions has also been revealed [152]. In silico analysis of 3256 proteins made it possible to identify 170 (5.22%) probable allergenic proteins of the vaccine strain Y. pestis EV line NIIEG. 38 allergenic proteins belonging to the extracellular and outer membrane groups have been identified as the most promising targets for the creation of hypoallergenic vaccines by removing the genes encoding them from the genome of the vaccine strain EV.

Another group of researchers to optimize the EV vaccine strain proposed elimination from its genome the gene encoding the plasminogen activator Pla [127]. The product of this gene is not significantly protective, but is one of the main pathogenicity factors of Y. pestis [122]. It is likely that the removal from Y. pestis genome of other ballast components may become one of the directions for improving both existing and newly constructed live plague vaccines.

Taking into account the poly-host nature, several clinical forms of plague and the multi-modes of its transmission, it can be assumed that in different mammalian species different antigens of the plague pathogen can be protective. Research into creation of combined plague vaccines based on derivatives of one or several Y. pestis strains, attenuated in various ways, which have shown a different breadth of protection against various virulent strains in a model of one or several species of laboratory animals, was initiated in the first half of the last century. Live vaccines #46-S, M # 74, or MP-40 (F1⁻), when administered together with EV in the form of two-component mixture, significantly potentiated each other’s protective activity [4]. The bivalent vaccine composed of Y. pestis strains 1 and 17 competed in protection with the vaccine based on the EV strain, but turned out to be more reactogenic [11,153]. More recently, it was shown that immunization with a precisely attenuated Y. pestis strain accompanied by the administration of a protein subunit vaccine was significantly more protective than administration of individual preparations [147].

In a recent study an adenoviral vector-based (genes encoding YscF, F1, and V) and live-attenuated ΔlppΔmsbBΔail Y. pestis CO92 derivative vaccines were successfully used in mixture, with each vaccine inducing a distinct cellular immune response [154].

Y. pestis ΔnlpD mutant has recently been shown to be able to protect mice against bubonic and pneumonic plague better than the EV vaccine strain [155]. Comparative testing of a set of ΔnlpD strains of different origin confirmed that immunization of mice with ΔnlpD mutants induces immunity 10⁵ times more potent than the one induced by the administration of the EV vaccine strain. Simultaneously, NlpD-deficient bacteria failed to protect guinea pigs in the case of a subcutaneous challenge with Y. pestis, inducing a 10⁶ times less potent protection compared with that conferred by immunization with the EV vaccine strain [126]. It would be advisable to evaluate the protective activity of a mixture of ΔnlpD and Δpgm strains in these two animal species.

One of the recently developed and most attractive ways to attenuate bacterial pathogens giving them ability to persist in the body of a vaccinated individual in quantities and for periods sufficient to form long-term and intense immunity but not enough to cause infection is a set of technologies based on regulated delayed gene expression. Their review is presented in [156], but we would recommend that readers read these publications in the original [157,158,159,160,161,162,163,164,165]. They deserve it [161].