Single-domain adapted antibodies against Chlamydia trachomatis, preserving the development of chlamidic infection in vitro

Abstract

1 Институт биологии гена, Москва; эл. почта: tillib@genebiology.ru 2 Национальный исследовательский центр эпидемиологии и микробиологии им. акад. Н.Ф. Гамалеи, Москва Технология генерирования однодоменных рекомбинантных моноклональных антител (наноантител), базирующаяся на иммунизации верблюда, клонировании индуцированных последовательностей, кодирующих однодоменные антиген-узнающие фрагменты неканонических верблюжьих антител, а также на функциональной селекции клонов наноантител методом фагового дисплея, была использована с целью получить новые эффективные инструменты для более эффективной диагностики хламидийной инфекции и для разработки новых подходов для эффективной терапии. Были получены два перспективных наноантитела, которые показали эффективное связывание с внеклеточными и внутриклеточными формами Chlamydia trachomatis, а также обладали активностью, подавляющей развитие хламидийной инфекции в условиях in vitro.Ключевые слова: рекомбинантное однодоменное антитело, хламидия, урогенитальная инфекция

Anti-Idiotypic Nanobodies Mimicking an Epitope of the Needle Protein of the Chlamydial Type III Secretion System for Targeted Immune Stimulation

“…Both whole bacterial cells of C. trachomatis (strain Bu-434), inactivated by UV irradiation, and a protein complex without LPS, prepared from the outer membrane of the chlamydia cell wall, were used as antigens for the immunization. As a result of this work, we were able to obtain one particularly promising nanobody (aChlNP, Figure 1) that recognized the TC_0037 protein (the "Chlamydia injectisome needle" protein) while exhibiting very specific binding to chlamydiae, including intracellular chlamydiae, forming inclusions (similar staining for the two originally derived nanobodies, as well as all methodological procedures, was described earlier [33]). The initially selected coding sequence of the aChlNP nanobody was further formatted according to the traditional protocol of our laboratory, as described previously [34].…”

“…Earlier, we described the preparation of a number of promising nanobodies that effectively bind to extracellular and intracellular forms of Chlamydia trachomatis, as well as have activity that inhibits the development of chlamydial infection under in vitro conditions [33]. We were unable to identify with which particular Chlamydia antigen the obtained nanobodies bind.…”

“…The nanobody aChlNP at a concentration of 5 µg/mL specifically bound antigens of closely related Chlamydia species, demonstrated using a micro-immunofluorescence assay on eukaryotic McCoy cells infected with C. trachomatis or C. muridarum (Figure 1d). The inhibitory effect of the aChlNP nanobody at concentrations of 10 µg/mL preincubated with C. trachomatis elementary bodies on their intracellular development in vitro was demonstrated [33]. We found this aChlNP nanobody in the trimerizing format to be the most promising for use as an idiotype for the creation of anti-idiotypic nanobodies (ai-Nbs) mimicking an epitope of the Chlamydial Type III Secretion System Needle Protein.…”

“…The preparation of a VHH-cDNA-library and the selection of aChlNP-specific binders using phage display was performed as previously described [33,34]. Nanobodies with different (non-Chlamydial) recognition specificity containing the same conserved framework and amino acid sequences added to their C-terminus as in the case of formatted aChlNPs (e.g., trimerized Nbs [34], available in our laboratory), were used in selection procedures for initial subtraction of nonspecifically bound sticky VHH-phage particles, and then as competitors or blockers of interactions other than anti-idiotype (aiChlNP) with idiotype (aChlNP).…”