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ГЕНЕТИЧЕСКИЕ МАРКЕРы ИММУННОГО ОТВЕТА НА АНТИГЕНы YERSINIA PESTIS F1 И V МИКРОКАПСУЛИРОВАННОй ЧУМНОй ВАКЦИНы
© Елена Львовна Назарова1, Иван Алексеевич Дятлов 2, Николай Маркович Поздеев 1, Валентина Тимофеевна Демьянова 1, Игорь Владимирович Парамонов 1, Андрей Валентинович Рылов 1, Михаил Владимирович Храмов 2, Александр Иосифович Борзилов2, Алексей Николаевич Сомов 2, Виталий Григорьевич Абламуниц 3, Андрей Павлович Анисимов 2
1 Кировский научно-исследовательский институт гематологии и переливания крови, 610027, г. Киров, Красноармейская ул., д. 72
2 Государственный научный центр прикладной микробиологии и биотехнологии, 142279, Московская обл., Серпуховский р-н, пос. Оболенск
3 Санкт-Петербургский государственный педиатрический медицинский университет, 194100, Санкт-Петербург, Литовская ул., д. 2
Контактная информация: Андрей Павлович Анисимов — д. м. н., профессор, заместитель директора по научной работе Государственного научного центра прикладной микробиологии и биотехнологии. E-mail: [email protected]
РЕЗЮМЕ: Эффективность иммунного ответа на вакцину зависит от активации антиген-презентирующих клеток и антиген-специфических Т- и В-лимфоцитов, индукции клеток памяти, регуляторных клеток и секрети-рующих антитела плазматических клеток, а также от длительности пребывания антигена в организме. Сила иммунного ответа главным образом зависит от генов, задействованных в этом процессе. Настоящая работа была посвящена изучению полиморфизма генов, который у части людей может быть причиной неудовлетворительного иммунного ответа на молекулярную микрокапсулированную чумную вакцину (ММЧВ). После двух последовательных вакцинаций среди 9 добровольцев у 67 % зафиксированы титры специфических антител не меньше порогового уровня. Однако у 33 %% добровольцев не найдено специфических антител ни к V, ни к F1 (Са^) антигену по истечении 90 суток после последней вакцинации. Мы исследовали 20 одиночных нуклео-тидных полиморфизмов (ОНП) для 14 генов, ответственных за иммунный ответ, с целью найти связь между конкретным аллелем и способностью формировать антитела к антигенам ММЧВ. Гомозиготность гена 1-1р по варианту дикого типа С-3953, либо гомозиготность по мутантному аллелю Т-3953 делали возможной продукцию анти^1 в то время как гетерозиготы оставались серонегативными. Гетерозиготы по гену TLR9 в положении 2848 (A2848G) также реагировали на F1. Среди реагировавших на антиген V (-сМ), продукция ^ наблюдалась лишь у носителей мутантного аллеля 1-4 С-589Т, у носителей мутантного аллеля C-174G гена 1-6, как гомо- так и гетерозиготных, и не наблюдалась у гомозигот по дикому типу аллеля. Отсутствие мутантного аллеля С-819Т гена 1-10 также было связано с нереагирующим фенотипом. Наши результаты позволяют предположить, что потенциально нереагирующие на ММЧВ могут быть опознаны по ОНП в генах 1-1р (С-3953 Т), 1-4 (С-589Т), 1-6 (С-^), 1-10 (С-819Т) и Т^9 (А2848^. Индивидов с нереагирующим генотипом следует включить в программу клинических испытаний новых универсально эффективных чумных вакцин.
КЛЮЧЕВЫЕ СЛОВА: чумная вакцина; иммунный ответ; полиморфизм генов; цитокины; Толл-подобные рецепторы.
GENETIC MARKERS OF IMMuNE RESPONSE TO YERSINIA PESTIS F1 AND V ANTIGENS-LOADED MICROSPHERES VACCINE AGAINST PLAGuE
© Elena L. Nazarova 1, Ivan A. Dyatlov 2, Nikolai M. Pozdeev 1, Valentina T. Demyanova 1, Igor V. Paramonov 1, Andrey V. Rylov1, Mikhail V. Khramov 2, Aleksandr I. Borzilov 2, Aleksey N. Somov 2, Vitaly G. Ablamunits 3, Andrey P. Anisimov 2
1 Kirov Research Institute of Hematology and Blood Transfusion, 72 Krasnoarmejskaja st., Kirov, 610027, Russia
2 State Scientific Center for Applied Microbiology and Biotechnology, Obolensk, Serpukhov district, Moscow Oblast, 142279, Russia
3 Saint Petersburg State Pediatric Medical University, 2 Litovskaya st., Saint Petersburg, 194100, Russia
^ РОССИЙСКИЕ БИОМВДИЦИНСКИЕ ИССЛЕДОВАНИЯ ТОМ 2 № 2 2017
Contact information: Andrey P. Anisimov — MD, professor, deputy director for scientific research of the State Scientific Center for Applied Microbiology and Biotechnology. E-mail: [email protected]
ABSTRACT: 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. This study was focused on the gene polymorphism that could be responsible for insufficient immune response to a plague vaccine (molecular microencapsulated plague vaccine, MMPV) in a proportion of humans. After two subsequent vaccinations of the nine 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 (Caf1) antigen during 90 days of observation post vaccination. We have investigated 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 MMPV antigens. Homozygosity for the I Lip 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 (LcrV) 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. Our data suggest that potential non-responders to the MMPV may be identified by SNP in the ILip (C-3953 T), IL4 (C-589T), IL6 (C-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.
KEYWORDS: plague vaccine; immune response; gene polymorphism; cytokines; toll-like receptors.
introduction
The causative agent of plague, Yersinia pestis, was the cause of three pandemics, which claimed millions of human lives. Plague is an infection affecting annually more than 3000 people [3]. The live plague vaccine used in the former Soviet Union stimulates intense immunity and protects from lethality caused by infection with Y. pestis [14], but it is associated with local and systemic adverse reactions of various severity in 5-29 % of persons with a normal immune status [44, 45, 53]. Therefore, a search for a better live plague vaccine superior to the existing one in immunogenicity, yet having less side effects, is very important [20-23, 41, 43, 55]. Another approach is to design a subunit (molecular) vaccine based on a few known protective antigens: such vaccines are aimed to minimize immune responses to non-specific components.
An example of such an antigen is a capsular protein F1 (Caf1) — a multifunctional factor of pathogenicity of Y. pestis. Serologic diagnosis of plague is based on detection of anti-F1 antibodies, and this antigen is the most important component of plague vaccines. Its important role in induction of a robust immune response in humans has been demonstrated [2]. The V antigen (LcrV) is another multifunctional factor of pathogenicity and an important protective antigen of Y. pestis [49]. It inhibits chemotaxis of neutrophils and production of pro-inflammatory cy-tokines [35, 52].
A vaccine is supposed to activate many different mechanisms of immune response [7, 19]. However, although vaccines induce an immune response in the majority of individuals, a proportion
of them may respond weakly or remain non-responsive [4, 5, 10, 33]. For instance, induction of a protective antibody response to HIV vaccine depends on particular alleles of HLA class II. The immune response is controlled by various other genes, among which — genes of pattern recognition receptors (PRRs) and genes of cytokines. PRRs are represented by several families which recognize macromolecules characteristic for bacteria and viruses, as well as endogenous molecules that become visible to the immune system as a result of tissue damage. Toll-like receptors (TLR) are most well studied PRRs. Binding of TLRs by their ligands results in activation of nuclear factor-KB (NF-kB) with subsequent transcription of genes of pro-inflammatory cytokines [16].
Genes for PPRs and cytokines have a notable polymorphism in their regulatory loci that influences on expression of the encoded protein and may affect its function. The most common type of mutation of these genes is a single nucleotide polymorphism (SNP) which often leads to amino acid substitution that may influence the immune response [8, 9].
The aim of this pilot study was to investigate a possible role of SNP in heterogeneity of specific humoral immune response to the molecular microencapsulated plague vaccine (MMPV) in normal human volunteers.
MATERIALS AND METHODS Human subjects
All volunteers signed an informed content to participate in this study. The protocol of the study PKI-00-01/2014 was approved
by the local Ethical Committee and was in agreement with Helsinki Declaration (2013). The Protocol was in compliance with the requirements of Good Clinical Practice (GCP) and with the regulations of the State Committee on Sanitary and Epidemiology Surveillance of the Russian Federation of 31.10.1996 No. 33) on the basis of permission No. 190 of the Ministry of Health of the Russian Federation regarding clinical trials of MMPV (16.04.2015). A total of 9 individuals at the age of 38-54 years (median — 44 years), six males and three females participated in the vaccination protocol. Control placebo-treated group consisted of 20 age- and sex-matched healthy volunteers. Immunologic reactivity to the antigens was estimated at the time of screening (three days before the first vaccination) and up to 90 days (or 69 days after the second administration of MMPV).
Vaccine
This study was performed as a part of clinical trial for medical application of MMPV, a lyophilized preparation consisting of two recombinant antigens: F1 (17.7 kDa), and V (37 kDa) both at the dose 25-30 |jg per injection dose. The vaccine was given subcu-taneously twice at an interval of 21 days. The vaccine was manufactured according to the requirements for Good manufacturing Practice (GMP).
Laboratory tests
Genetic studies were based on the DNA obtained from peripheral blood leukocytes as described [24]. The single nucleo-tide polymorphism of immune response genes was carried out using allele — specific primers (NPF LyTech, Moscow) and elec-trophoretic detection of PCR products in agarose gel. Twenty polymorphic sites of 14 genes were amplified: interleukin (IL) -1p ((T-31C), (T-511C), (G-1473C), (C-3953T); IL2 (T-330G); IL4 (C-589T); IL6 (C-174G); IL10 (C-819T, G-1082A); IL17 (G-197A); TNF (G-308A); receptors CD14 (C-159T), FCGR2A (G-166A), TLR2 (G-753A), TLR3 (G-421A), TLR4 (G-299A), TLR4 (C-399T), TLR6 (C-249 E), TLR9 (T-1237C, A2848G). The respective SNP are recorded in the NCBI dbSNP (http://www. ncbi.nlm.nih.gov/projects/SNP/index.html) and have the frequency of minor alleles from 1.0 to 47 %.
For serological analysis, blood was taken from the cubital vein in the morning and serum separated. Levels of specific IgG antibodies to the F1 and the V antigens of Y. pestis were determined by ELISA. For that, microtiter plates (Greiner Bio-One, Austria) were coated with 100 ng/well of Caf1 or V protein in 0.1 M sodium bicarbonate buffer (pH 9.6) overnight at 4 °C. Non-specific binding was blocked with 3 % gelatin from cold water fish skin (Sigma) in 0.01 M PBS, pH 7.2. Test sera were added using 2.5-fold serial dilutions in 0.01 M PBS buffer containing 0.05 % Tween-20 (PBST) and incubated for 2 h at 37 °C. After four washes with 0.01M PBST, 100 |jl of sheep anti-mouse IgG conjugated to horseradish peroxidase (GE Healthcare) at the dilution of 1:4000 was added for 1.5 h at 37 °C. The plates were washed with PBST, and 100 jl of 0.01 % o-phenylendiamine-H2O2 was added to each well. The reaction was stopped by the addition of
100 jl of 1 M H2SO4 per well, and OD was read at 450 nm using EVOLIS Twin Plus System (BIO-RAD, USA). The titer of antibodies was estimated as the maximum dilution of serum giving an OD reading that exceeded the background by 0.1. Titer 1:100 was considered as the diagnostic threshold, that is, all individuals who demonstrated positive signal in serum diluted up to 1:100 were considered negative.
Statistical analysis
Titers of specific antibodies were compared between genotype groups for each SNP studied and the differences were analyzed by non-parametric analysis of variance (ANOVA) using GraphPad Prism V software. P<0.05 was considered significant. Correlation between the antibody titer and the number of alleles associated with a responder phenotype was performed using linear regression analysis.
results
Six of nine vaccinated individuals (67 %) developed IgG antibodies to F1 and/or to V antigen (IgG-F1 and IgG-V respectively), and three (33 %) remained seronegative. Five volunteers (55 %) responded to F1 with diagnostic titers > 1:100 (group 1), while 4 others (44 %) remained non-responders. Analysis of SNP showed that serological responders were among individuals with heterozygosity in TLR9 (AG), IL6 (CT), and IL10 (CG) genes. On the other hand, the responders were homozygous for the wild type ILIfi allele (CC), and for the wild type IL4 allele (CC). Carriers of the mutant allele of TLR9 gene (GG) and heterozygotes for the ILIfi gene (CT) appear to be potential non-responders (Figure 1 A-E). Linear regression analysis revealed a positive correlation between the number of responder alleles in an individual and the IgG titer to F1 (Fig. 1F).
Four of nine vaccinated individuals (44 %) developed antibodies to the V antigen, whereas five remained seronegative (55 %). Analysis of SNP revealed that heterozygosity in all the genes studied does not make individuals non-responsive to the antigen. Homozygosity for mutant alleles in IL1fi and IL4 genes (TT), and IL6 gene (GG) was found in responders. On the other hand, homozygosity for a wild type IL4 allele (CC) was associated with non-responsiveness to the V antigen (Fig. 2 A-E). Again, linear regression analysis revealed a positive correlation between the number of responder alleles in an individual and the IgG titer to F1 (Fig. 2F).
The analysis of genotypes of ILIfi (T-31C, G-1473C, T-511C), IL2 (T-330G), IL10 (G-1082A), IL17 (G-197A), TNF (G-308A), CD14 (C-159T), FCGR2A (His166Arg), TLR2 (G-753A), TLR3 (G-421A), TLR4 (G-299A), TLR4 (C-399T), TLR6 (C-249 E), TLR9 (T-1237C) did not reveal an association of polymorphism with antibody responses to MMPV (data not shown).
discussion
Efficiency of novel plague vaccines is a matter of an outmost importance, since the disease is associated with high mortality rates. However, a proportion of vaccinated individuals does
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not respond to one or both F1 and V antigens, therefore, identification of such non-responders on the basis of genetic or other markers could help to avoid unnecessary vaccination, or could promote creation of an alternative vaccine. In this small pilot study we investigated SNP of a number of genes that are known for their role in immune responses. We found that five genes are involved in the strength of specific humoral immune response to MMPV, namely IL1p, IL4, IL6, IL10, and TLR9, and this involvement was associated with SNP at specific positions.
Cytokines are involved in intercellular cooperation and regulation of the immune response. They are produced mostly by activated leukocytes [1, 6, 42]. Some of them play a key role in maintaining of balance between cellular (Th1) and humoral (Th2) immune responses, where Th1 cells provide defense against in-tracellular pathogens, while Th2 cells — protection against extracellular agents [28]. Genes encoding cytokines and their receptors are characterized by allelic polymorphism, which may have an impact on the function of their respective products [12]. There are more than seven million SNP with a frequency of the minor allele within 5-10 %. Some of these SNP are synonymous, but others affect expression or function of the gene product. Therefore, functionally different alleles can define the risk of a poor immune response [42].
IL-1P is a pro-inflammatory cytokine synthesized mostly by the activated macrophages, keratinocytes, fibroblasts, and stimulated B-lymphocytes. The IL1fi gene is a part of the cluster of genes located on the long arm of chromosome 2. It consists of 22 exons and nine introns. SNP of the ILIfi gene at positions -511, —31 and +3953 are well documented [11]. IL1fi gene wild type allele has C at the position +3953, and a mutant allele has T [30]. This mutation is associated with an excess in IL-1P production [12]. According to our data, the presence of both normal and mutant alleles in homozygosity correlates with humoral immune response to both F1 and V antigens (Fig. 1C and 2C). On the contrary, the heterozygous state was associated with a low or no response to both vaccine components. It has been shown that the structure of highly polymorphic regions of ILIfi gene affects specific immune response to malaria [26]. A disadvantage of heterozygotes observed in our study is difficult to explain and requires further elucidation.
TLR9 is a receptor capable of prokaryotic DNA recognition due to the presence of non-methylated CpG motifs [13]. TLR9 expressing germinal center plasma cells participate in differentiation of T lymphocytes towards Th1 or Th2 subpopulations [40, 50]. Polymorphism of TLR9 gene may influence immune response [34, 46]. Some haplotypes of TLR9 gene are associated with a decreased level of the gene expression [48], which may result in a reduced production of immunoglobulins [29]. In our work, the heterozygotes for TLR9 gene at the position-2848 were respond-ers to the vaccine, whereas homozygotes for either wild type, or mutant allele were non-responders.
The IL4 gene is located in a cluster of genes (together with IL3, IL5, IL13 and CSF2) on the long arm of chromosome 5 and contains several polymorphic sites, some of which
participate in regulation of IL-4 production. This locus controls Th2 responses [27]. There is evidence that certain haplotypes of IL4 gene in the position — 590C/T up-regulate its transcription and result in hyper-production of IL-4 [17, 47]. It has been reported that SNP at this gene position influences production of antibodies to malaria [47]. Thus, IL4 haplotype may be important for subsequent generation of Th2 cells producing various amounts of IL-4 [36]. Bartova et al. [15] have shown that IL4 polymorphism may be important for IL-6 production.
The IL6 gene is located on the short arm of chromosome 7 and consists of five exons and four introns. The polymorphic locus of C-174G the regulatory area of the gene is the most studied one [11]. Our data demonstrate that the presence of mutant allele (C-174G) is associated with a responder phenotype, whereas the wild type allele is not. Effect of IL6 polymorphism on antibody response has been demonstrated. Patel et al. demonstrated that SNP at position 174 modulates IL-6 production in response to RCV so that in carriers of the wild type allele IL-6 secretion is inhibited. Authors concluded that RSV, perhaps, has the ability to suppress IL6 gene transcription and the mutant allele provides an escape mechanism from this inhibition, but this phenomenon requires further studies [38]. Poland et al. showed that IL6 polymorphism were associated with a more efficient synthesis of antibodies to hemagglutinin in response to inactivated vaccine against seasonal influenza [39].
IL-6 inhibits production of TNF and IL-1 and stimulates secretion of IL-10 [17]. IL-10 is an anti-inflammatory cytokine which inhibits synthesis of Th1 of cytokines, expression of HLA class II antigens on macrophages, increases the number of IL-4 producing T-cells and enhances survival and proliferation of B cells, and hence — antibody production. The IL10 gene is located on the long arm of chromosome 1 and contains five exons. The most studied SNPs of this gene are G-1082A, C-819T and C-592A [29]. We found that the absence of the mutant allele at gene position 819 was associated with the responder phenotype to both antigens. Polymorphism of IL10 gene acts at the level of gene transcription and subsequent protein biosynthesis [11]. It has been reported that polymorphism at the position 1082 of IL10 gene was associated with variability of humoral immune response to EBV [25].
The strategy of creating anti-plague vaccines was focused mostly on induction of protective antibodies specific to Y. pes-tis targets [3, 44, 45, 49]. The important role of humoral immune response has been demonstrated by passive transfer of immunity with sera from recovered or vaccinated animals, and also by monoclonal antibodies, specific to F1 and V [51]. Unfortunately, the majority of these and relevant studies have an experimental character and their conclusions are based on mouse models [32, 37, 51, 54].
Anti-plague immunity in humans also depends on both humoral and cellular immune responses to other antigens (for example, Yop) which are not a part of these subunit vaccines [31, 54]. Participation of specific CD4+ and CD8 + T-lymphocytes [25, 38], and subpopulations of Th17 and Th1 cells that may attenuate
or enhance vaccines [32] should be taken into consideration for creation of better vaccine in the future.
Our study pores light on the possibility that genetic pre-screen-ing may allow for selection of human individuals that would likely respond to the F1/V subunit MMPV vaccination by mounting a specific humoral protective immunity. Humans that fall into the group of non-responders would benefit from an alternative method of protection, including an alternative vaccine, to be designed yet. If our findings hold true in a study of a larger cohort, we could recommend genotyping candidate individuals for SNP in genes of IL1p (C-3953T), IL4 (C-589T), IL6 (C-174G), IL10 (C-819T), and TLR9 (A2848G). In addition, we would suggest that individuals with non-responder genotype should be included in the future clinical trials assessing novel, universally effective plague vaccines.
This work was performed as a part of Governmental Contract # 334/0K/2013/DRGZ of 06.05.2013 (State Registration # 01201366243).
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13. Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat. Immunol. 2001; 2 (8): 675-80.
14. Anisimov A. P., Lindler L. E., Pier G. B. Intraspecific diversity of Yersinia pestis. Clin. Microbiol. Rev. 2004; 17 (2): 434-64.
15. Bartova J., Borilova Linhartova P., Podzimek S. et al. The effect of IL-4 gene polymorphisms on cytokine production in patients with chronic periodontitis and in healthy controls. Mediators Inflamm. 2014; Article ID 185757: 11 pages. Available at: http://dx.doi.org/10.1155/2014/185757 (Accessed 09.02.2016). doi: 10.1155/2014/185757.
16. Bauer S., Muller T., Hamm S. Pattern recognition by Toll-like receptors. Adv. Exp. Med. Biol. 2009; 653: 15-34.
17. Cantagrel A., Navaux F., Loubet-Lescoulie P. et al. Interleu-kin-1beta, interleukin-1 receptor antagonist, interleukin-4, and in-terleukin-10 gene polymorphisms: relationship to occurrence and severity of rheumatoid arthritis. Arthritis Rheum. 1999; 42 (6): 1093-1100.
18. Corey L., Gilbert P. B., Tomaras G. D. et al. Immune correlates of vaccine protection against HIV-1 acquisition. Sci. Transl. Med. 2015; 7 (310): 310-317. doi: 10.1126/scitranslmed.aac7732.
19. Dentovskaya S. V., Ivanov S. A., Kopylov P. Kh. et al. Selective protective potency of Yersinia pestis AnlpD mutants. Acta Naturae. 2015; 7 (1): 102-108.
20. Derbise A., Cerdà Marin A., Ave P. et al. An encapsulated Yersinia pseudotuberculosis is a highly efficient vaccine against pneumonic plague. PLoS Negl Trop Dis. 2012; 6 (2): e1528. 11 pages. doi: 10.1371/journal.pntd.0001528.
21. Feodorova V. A., Pan'kina L. N., Savostina E. P. et al. A Yersinia pestis lpxM-mutant live vaccine induces enhanced immunity against
bubonic plague in mice and guinea pigs. Vaccine. 2006; 25 (44): 7620-28.
22. Flashner Y., Mamroud E., Tidhar A. et al. Generation of Yersinia pestis attenuated strains by signature-tagged mutagenesis in search of novel vaccine candidates. Infect. Immun. 2004; 72 (2): 908-15.
23. Garbom S., Forsberg A., Wolf-Watz H., Kihlberg B.-M. Identification of novel virulence-associated genes via genome analysis of hypothetical genes. Infect. Immun. 2004; 72 (3): 1333-40.
24. Graham D. E. The isolation of high molecular weight DNA from whole organisms or large tissue masses. Anal. Biochem. 1978; 85
(2): 609-613.
25. Helminen M., Lahdenpohja N., Hurme M. Polymorphism of the interleukin-10 gene is associated with susceptibility to Ep-stein-Barr virus infection. J. Infect. Dis. 1999; 180 (2): 496-99.
26. Israelsson E. Host genetic factors and antibody responses with potential involvement in the susceptibility to malaria. Stockholm; 2008.
27. Kaslow R. A., McNicholl J., Hill A. V. S., eds. Genetic Susceptibility to Infectious Diseases. Oxford: Oxford University Press; 2008.
28. Keen L. J. The extent and analysis of cytokine and cytokine receptor gene polymorphism. Transpl. Immunol. 2002; 10 (2-3): 143-146.
29. Kikuchi K., Lian Z. X., Kimura Y. et al. Genetic polymorphisms of toll-like receptor 9 influence the immune response to CpG and contribute to hyper-IgM in primary biliary cirrhosis. J. Autoimmun. 2005; 24 (4): 347-52.
30. Kornman K. S., di Giovine F. S. Genetic variations in cytokine expression: a risk factor for severity of adult periodontitis. Ann. Periodontal. 1998; 3 (1): 327-38.
31. Li B., Du C., Zhou L. et al. Humoral and cellular immune responses to Yersinia pestis infection in long-term recovered plague patients. Clin. Vaccine Immunol. 2012; 19 (2): 228-34. doi: 10.1128/CVI.05559-11.
32. Lin J.-S., Kummer L. W., Szaba F. M., Smiley S. T. IL-17 contributes to cell-mediated defense against pulmonary Yersinia pestis infection. J. Immunol. 2011; 186 (3): 1675-84. doi: 10.4049/jimmu-nol.1003303.
33. McMichael A. Prospects for an AIDS vaccine. Clin. Med. 2003; 3
(3): 269-72.
34. Misch E. A., Hawn T. R. Toll-like receptor polymorphisms and susceptibility to human disease. Clinical Science. 2008; 114 (5): 34760. doi: 10.1042/CS20070214.
35. Nakajima R., Brubaker R. R. Association between virulence of Yersinia pestis and suppression of gamma interferon and tumor necrosis factor alpha. Infect. Immun. 1993; 61 (1): 23-31.
36. Nakashima H., Miyake K., Inoue Y. et al. Association between IL-4 genotype and IL-4 production in the Japanese population. Genes Immun. 2002; 3 (2): 107-9.
37. Parent M. A., Berggren K. N., Kummer L. W. et al. Cell-mediated protection against pulmonary Yersinia pestis infection. Infect. Immun. 2005; 11: 7304-10.
38. Patel J.A., Nair S., Ochoa E. E. et al. Interleukin-6-174 and tumor necrosis factor a-308 polymorphisms enhance cytokine
production by human macrophages exposed to respiratory viruses. J. Interferon Cytokine Res. 2010; 30 (12): 917-21. doi: 10.1089/jir.2010.0033.
39. Poland G. A., Ovsyannikova I. G., Jacobson R. M. Immunogenetics of seasonal influenza vaccine response. Vaccine. 2008; 26 (Suppl. 4): D35-D40.
40. Rissoan M. C., Soumelis V., Kadowaki N. et al. Reciprocal control of T helper cell and dendritic cell differentiation. Science. 1999; 283 (5405): 1183-86.
41. Rosenzweig J. A., Chopra A. K. The future of plague vaccines: hopes raised by a surrogate, live-attenuated recombinant vaccine candidate. Expert. Rev. Vaccines. 2012; 11 (6): 659-61. doi: 10.1586/erv.12.34.
42. Senda T. Structure and function of cytokines and their receptors. Protein Nucl. Acid and Enzyme. 1999; 44 (4): 318-24.
43. Sha J., Agar S. L., Baze W. B. et al. Braun lipoprotein (Lpp) contributes to virulence of yersiniae: potential role of Lpp in inducing bubonic and pneumonic plague. Infect. Immun. 2008; 76 (4): 13901409. doi: 10.1128/IAI.01529-07.
44. Smiley S. T. Immune defense against pneumonic plague. Immunol. Rev. 2008; 225 (1): 256-71. doi: 10.1111/j.1600-065X.2008.00674.x.
45. Sun W., Roland K. L., Curtiss R. 3rd. Developing live vaccines against plague. J. Infect. Dev. Countries. 2011; 5 (9): 614-27.
46. Suzuki H., Suzuki Y., Narita I. et al. Toll-like receptor 9 affects severity of IgA nephropathy. Am. Soc. Nephrol. 2008; 19 (12): 238495. doi: 10.1681/ASN.2007121311.
47. Tangteerawatana P., Perlmann H., Hayano M. et al. IL4 gene polymorphism and previous malaria experiences manipulate anti-Plasmodium falciparum antibody isotype profiles in complicated and uncomplicated malaria. Malar. J. 2009; 8 (286): 10 pages. doi: 10.1186/1475-2875-8-286.
48. Tao K., Fujii M., Tsukumo S. et al. Genetic variations of Tolllike receptor 9 predispose to systemic lupus erythematosus in Japanese population. Ann. Rheum. Dis. 2007; 66 (7): 905-09.
49. Une T., Brubaker R. R. Roles of V antigen in promoting virulence and immunity in yersiniae. J. Immunol. 1984; 133 (4): 2226-30.
50. Wagner H. The immunobiology of the TLR9 subfamily. Trends Immunol. 2004; 25 (7): 381-86.
51. Wang S., Goguen J. D., Li F., Lu S. Involvement of CD8+ T cell-mediated immune responses in LcrV DNA vaccine induced protection against lethal Yersinia pestis challenge. Vaccine. 2011. 29 (39): 6802-09. doi: 10.1016/j.vaccine.2010.12.062.
52. Welkos S., Friedlander A., McDowell D. et al. V antigen of Yersinia pestis inhibits neutrophil chemotaxis. Microb. Pathol. 1998; 24 (3): 185-96.
53. Welkos S., Pitt M. L. M., Martinez M. et al. Determination of the virulence of the pigmentation-deficient and pigmentation-/plasminogen activator-deficient strains of Yersinia pestis in non-human primate and mouse models of pneumonic plague. Vaccine. 2002; 20 (1718): 2206-14.
54. Williamson E. D. The role of immune correlates and surrogate markers in the development of vaccines and immunotherapies for plague. Adv. Prev. Med. 2012; Article ID 365980: 7 pages (Accessed 17.02.2016). doi: 10.1155/2012/365980.
55. Zhang X., Qi Z., Du Z. et al. A live attenuated strain of Yersinia pestis AyscB provides protection against bubonic and pneumonic plagues in mouse model. Vaccine. 2013; 31 (22): 2539-42. doi: 10.1016/j.vaccine.2013.03.054.
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13. Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat. Immunol. 2001; 2 (8): 675-80.
14. Anisimov A. P., Lindler L. E., Pier G. B. Intraspecific diversity of Yersinia pestis. Clin. Microbiol. Rev. 2004; 17 (2): 434-64.
15. Bartova J., Borilova Linhartova P., Podzimek S. et al. The effect of IL-4 gene polymorphisms on cytokine production in pa-
tients with chronic periodontitis and in healthy controls. Mediators Inflamm. 2014; Article ID 185757: 11 pages. Available at: http://dx.doi.org/10.1155/2014/185757 (Accessed 09.02.2016). doi: 10.1155/2014/185757.
16. Bauer S., Muller T., Hamm S. Pattern recognition by Toll-like receptors. Adv. Exp. Med. Biol. 2009; 653: 15-34.
17. Cantagrel A., Navaux F., Loubet-Lescoulie P. et al. Interleu-kin-1beta, interleukin-1 receptor antagonist, interleukin-4, and in-terleukin-10 gene polymorphisms: relationship to occurrence and severity of rheumatoid arthritis. Arthritis Rheum. 1999; 42 (6): 1093-1100.
18. Corey L., Gilbert P. B., Tomaras G. D. et al. Immune correlates of vaccine protection against HIV-1 acquisition. Sci. Transl. Med. 2015; 7 (310): 310-317. doi: 10.1126/scitranslmed.aac7732.
19. Dentovskaya S. V., Ivanov S. A., Kopylov P. Kh. et al. Selective protective potency of Yersinia pestis AnlpD mutants. Acta Naturae. 2015; 7 (1): 102-108.
20. Derbise A., Cerdà Marin A., Ave P. et al. An encapsulated Yersinia pseudotuberculosis is a highly efficient vaccine against pneumonic plague. PLoS Negl Trop Dis. 2012; 6 (2): e1528. 11 pages. doi: 10.1371/journal.pntd.0001528.
21. Feodorova V. A., Pan'kina L. N., Savostina E. P. et al. A Yersinia pestis lpxM-mutant live vaccine induces enhanced immunity against bubonic plague in mice and guinea pigs. Vaccine. 2006; 25 (44): 7620-28.
22. Flashner Y., Mamroud E., Tidhar A. et al. Generation of Yersin-ia pestis attenuated strains by signature-tagged mutagenesis in search of novel vaccine candidates. Infect. Immun. 2004; 72 (2): 908-15.
23. Garbom S., Forsberg A., Wolf-Watz H., Kihlberg B.-M. Identification of novel virulence-associated genes via genome analysis of hypothetical genes. Infect. Immun. 2004; 72 (3): 1333-40.
24. Graham D. E. The isolation of high molecular weight DNA from whole organisms or large tissue masses. Anal. Biochem. 1978; 85 (2): 609-613.
25. Helminen M., Lahdenpohja N., Hurme M. Polymorphism of the in-terleukin-10 gene is associated with susceptibility to Epstein-Barr virus infection. J. Infect. Dis. 1999; 180 (2): 496-99.
26. Israelsson E. Host genetic factors and antibody responses with potential involvement in the susceptibility to malaria. Stockholm; 2008.
27. Kaslow R. A., McNicholl J., Hill A. V. S., eds. Genetic Susceptibility to Infectious Diseases. Oxford: Oxford University Press; 2008.
28. Keen L. J. The extent and analysis of cytokine and cytokine receptor gene polymorphism. Transpl. Immunol. 2002; 10 (2-3): 143-146.
29. Kikuchi K., Lian Z. X., Kimura Y. et al. Genetic polymorphisms of toll-like receptor 9 influence the immune response to CpG and contribute to hyper-IgM in primary biliary cirrhosis. J. Autoimmun. 2005; 24 (4): 347-52.
30. Kornman K. S., di Giovine F. S. Genetic variations in cytokine expression: a risk factor for severity of adult periodontitis. Ann. Periodontal. 1998; 3 (1): 327-38.
31. Li B., Du C., Zhou L. et al. Humoral and cellular immune responses to Yersinia pestis infection in long-term recovered plague
patients. Clin. Vaccine Immunol. 2012; 19 (2): 228-34. doi: 10.1128/CVI.05559-11.
32. Lin J.-S., Kummer L. W., Szaba F. M., Smiley S. T. IL-17 contributes to cell-mediated defense against pulmonary Yersinia pestis infection. J. Immunol. 2011; 186 (3): 1675-84. doi: 10.4049/jimmu-nol.1003303.
33. McMichael A. Prospects for an AIDS vaccine. Clin. Med. 2003; 3 (3): 269-72.
34. Misch E. A., Hawn T. R. Toll-like receptor polymorphisms and susceptibility to human disease. Clinical Science. 2008; 114 (5): 34760. doi: 10.1042/CS20070214.
35. Nakajima R., Brubaker R. R. Association between virulence of Yersinia pestis and suppression of gamma interferon and tumor necrosis factor alpha. Infect. Immun. 1993; 61 (1): 23-31.
36. Nakashima H., Miyake K., Inoue Y. et al. Association between IL-4 genotype and IL-4 production in the Japanese population. Genes Immun. 2002; 3 (2): 107-9.
37. Parent M. A., Berggren K. N., Kummer L. W. et al. Cell-mediated protection against pulmonary Yersinia pestis infection. Infect. Immun. 2005; 11: 7304-10.
38. Patel J. A., Nair S., Ochoa E. E. et al. Interleukin-6-174 and tumor necrosis factor a-308 polymorphisms enhance cytokine production by human macrophages exposed to respiratory viruses. J. Interferon Cytokine Res. 2010; 30 (12): 917-21. doi: 10.1089/jir.2010.0033.
39. Poland G. A., Ovsyannikova I. G., Jacobson R. M. Immunogenetics of seasonal influenza vaccine response. Vaccine. 2008; 26 (Suppl. 4): D35-D40.
40. Rissoan M. C., Soumelis V., Kadowaki N. et al. Reciprocal control of T helper cell and dendritic cell differentiation. Science. 1999; 283 (5405): 1183-86.
41. Rosenzweig J. A., Chopra A. K. The future of plague vaccines: hopes raised by a surrogate, live-attenuated recombinant vaccine candidate. Expert. Rev. Vaccines. 2012; 11 (6): 659-61. doi: 10.1586/erv.12.34.
42. Senda T. Structure and function of cytokines and their receptors. Protein Nucl. Acid and Enzyme. 1999; 44 (4): 318-24.
43. Sha J., Agar S. L., Baze W. B. et al. Braun lipoprotein (Lpp) contributes to virulence of yersiniae: potential role of Lpp in inducing bubonic and pneumonic plague. Infect. Immun. 2008; 76 (4): 13901409. doi: 10.1128/IAI.01529-07.
44. Smiley S. T. Immune defense against pneumonic plague. Immunol. Rev. 2008; 225 (1): 256-71. doi: 10.1111/j.1600-065X.2008.00674.x.
45. Sun W., Roland K. L., Curtiss R. 3rd. Developing live vaccines against plague. J. Infect. Dev. Countries. 2011; 5 (9): 614-27.
46. Suzuki H., Suzuki Y., Narita I. et al. Toll-like receptor 9 affects severity of IgA nephropathy. Am. Soc. Nephrol. 2008; 19 (12): 238495. doi: 10.1681/ASN.2007121311.
47. Tangteerawatana P., Perlmann H., Hayano M. et al. IL4 gene polymorphism and previous malaria experiences manipulate anti-Plasmodium falciparum antibody isotype profiles in complicated and uncomplicated malaria. Malar. J. 2009; 8 (286): 10 pages. doi: 10.1186/1475-2875-8-286.
48. Tao K., Fujii M., Tsukumo S. et al. Genetic variations of Toll-like receptor 9 predispose to systemic lupus erythematosus in Japanese population. Ann. Rheum. Dis. 2007; 66 (7): 905-09.
49. Une T., Brubaker R. R. Roles of V antigen in promoting virulence and immunity in yersiniae. J. Immunol. 1984; 133 (4): 2226-30.
50. Wagner H. The immunobiology of the TLR9 subfamily. Trends Immunol. 2004; 25 (7): 381-86.
51. Wang S., Goguen J. D., Li F., Lu S. Involvement of CD8+ T cell-mediated immune responses in LcrV DNA vaccine induced protection against lethal Yersinia pestis challenge. Vaccine. 2011; 29 (39): 6802-09. doi: 10.1016/j.vaccine.2010.12.062.
52. Welkos S., Friedlander A., McDowell D. et al. V antigen of Yersinia pestis inhibits neutrophil chemotaxis. Microb. Pathol. 1998; 24 (3): 185-96.
53. Welkos S., Pitt M. L. M., Martinez M. et al. Determination of the virulence of the pigmentation-deficient and pigmentation-/plasminogen activator-deficient strains of Yersinia pestis in non-human primate and mouse models of pneumonic plague. Vaccine. 2002; 20 (1718): 2206-14.
54. Williamson E. D. The role of immune correlates and surrogate markers in the development of vaccines and immunotherapies for plague. Adv. Prev. Med. 2012; Article ID 365980: 7 pages (Accessed 17.02.2016). doi: 10.1155/2012/365980.
55. Zhang X., Qi Z., Du Z. et al. A live attenuated strain of Yersinia pestis AyscB provides protection against bubonic and pneumonic plagues in mouse model. Vaccine. 2013; 31 (22): 2539-42. doi: 10.1016/j.vaccine.2013.03.054.
