Rational design of antigens to improve the serodiagnosis of tick-borne borreliosis in central regions of Russia

Abstract

Tick-borne borreliosis (Lyme disease-LD) is caused by pathogenic Borrelia spirochetes that is transmitted through bite of Ixodes ticks to humans and animals. In the Russian Federation, borreliosis registered with an index of 6–7 per 100,000 people annually. In reality, LD morbidity in Russia is much higher because Russian strains develop less erythematous rashes compared to North American strains, thus missed by physicians in most of the early cases, and current serology tests have insufficient sensitivity as well. The aim of this work was to improve the sensitivity and specificity of serology tests for LD in Russia using rationale-designed Borrelia antigens. It was anticipated that sensitivity of LD serodiagnosis will be higher if antigen for test-systems are derived from a strain that is circulated in a geographical region of test application. A large portion of the Russian population lives in the Central region. Thus, effort has been made to create a serological test using antigens from Moscow region, Tula and Ul’janovsk areas. In this study we included wild strains (ultrasonic-treated spirochetes B. garinii H19, B. afzelii P1, B. afzelii P1H13, B. burgdorferi s.s. 39/40, B. burgdorferi s.s. B31), recombinant (expressed in E.coli DbpA, Bgp, Bbk B. garinii, and B. afzelii) antigens and some of their combinations were produced and tested against LD patients and donors serum collected in hospitals of Central regions of Russia by ELISA and Western blotting. Considering sensitivity and specificity, DbpA B. afzelii and DbpA B. garinii recombinant antigens were selected among all probed antigens for regional serology test. As long as DbpA B. afzelii and DbpA B. garinii antigens interacted with LD patient’s serum in a complementary mode, it is possible to combine epitopes DbpA B. afzelii and B. garinii in a single antigen for improving sensitivity. We created recombinant fusion protein DbpA B. afzelii/B using dbpA genes from Russian isolates of B. afzelii and B. garinii in E. coli. Fusion DbpA A + G protein was then used for formulation of fast immunochromatographic serodiagnosis test (LF) in a ‘‘deep-stick’’ format. The trials of LF-test were conducted separately at Institute of Rheumatology Russian Academy of Medical Science (using 325 sera) and at the Borreliosis Reference Center of Ministry of Health RF (using 120 reference sera). The average sensitivity and specificity of LF-test was 80.5 and 100 %, respectively.