OmcB PROTEIN EXPRESSED IN ADENOVIRAL VECTOR INDUCES PROTECTION AGAINST CHLAMYDIA MURIDARUM VAGINAL INFECTION Presenting author: Ekaterina A. Koroleva*, Natalia V. Kobets, Dmitrij N. Shcherbinin, Maksim M. Shmarov, Nelly A. Zigangirova. Gamaleya Research Institute for epidemiology and microbiology, Moscow, Russia Abstract Methods Results Conclusion Genital infection with Chlamydia trachomatis is gradually becoming one of the major threats to the reproductive health worldwide with especially high rates of infection in younger adults. Due to a range of the reasons such as widely asymptomatic nature of infection, lack of timely diagnostics and adverse effects on host immunity currently available antimicrobial treatment is not sufficient to restrict the spread of infection. Vaccine development is a highly desirable for more efficient protection of reproductive health of the world population. In this study we present an approach of vaccination that combines the benefits of mucosal delivery of genetically expressed Chlamydia muridarum protein OmcB. OmcB is highly conserved among Chlamydia species, suggesting that it plays a significant role during intracellular chlamydial infection. OmcB may function as an adhesin for chlamydial invasion into host cells, since heparin can block the infectivity of some C. trachomatis serovars by binding to an N-terminal peptide of OmcB [1]. Plasmid pShuttle-CMV, containing gene omcB (Ad-OmcB) was constructed for this study. Expression of omcB gene was confirmed by western blot. To evaluate the protective potential of the constructed vaccine candidate female BALB/c mice were immunized intranasal with a single dose of either 108 plaque forming units (PFU) of Ad-OmcB, 3 weeks prior to intravaginal infection with C. muridarum. Empty vector immunized and primary infected mice were used as controls. Mice were hormonally synchronized by subcutaneous infection with progesterone (“Covinan”) 5 days prior to a vaginal challenge [2]. 4 weeks later treated mice were infected intravaginally with 106 (inclusion-forming units) IFU/mouse of C. muridarum strain Nigg. Infection was monitored by measuring infection forming units (IFU) from vaginal swabs that were collected from day 3 to 20 post challenge. Sera samples were obtained at 14 day post immunization for assessing specific antibodies by immunofluorescence assay (IFA) and ELISA. We designed adenovirus construction expressing OmcB C. muridarum. Expression was carried out according to the commercial protocol of «AdEasy Adenoviral vector system» kit (Stratagene) [3]. Vaginal swab samples were obtained at days 3, 6, 9, 15 and 20 and the protective effects of Ad- OmcB expressed as median lg IFU/ml. At day 3 post infection we observed significant difference between vaccinated and non-vaccinated mice. In control group 100 % mice infected intravaginally with C. muridarum had high levels of Chlamydia inclusions (0,5х106 IFU) in swabs, peaking at day 6. (Fig.1.) Empty vector immunization gave no protection at all, while in the group of omcB immunized mice the level of infection was decreased starting from day 3 post infection. Higher levels of serum anti-chlamydia antibodies were observed in Ad-OmcB group compared to controls at day 14 post immunization (Fig.2 and 3). OmcB expressed in adenoviral vector delivered in a single dose via mucosal surfaces is capable to induce protection against C. muridarum vaginal infection and increase in specific antibodies in response to immunization. Neutralization assay revealed the presence of neutralizing antibodies in sera of mice immunized with Ad-OmcB. Please export the Keynote document as a PDF (File – Save as – PDF – Image Quality – Best) and upload the PDF into the system. Please use the font in the document or a similar one and do not use a font size smaller than 16. Reference 1.Shuping H.L.L., Zhangsheng Y., Manli Q., Quanzhong L. and Zhong G. Chlamydia trachomatis Outer Membrane Complex Protein B (OmcB) Is Processed by the Protease CPAF. J Bacteriol. Mar 2013; 195(5): 951–957. 2. N. Kobets, E. Koroleva, N. Zigangirova. Impaired early intravaginal cell recruitment in response to Chlamydia muridarum in mice pretreated with progesterone., Zh Mikrobiol Epidemiol Immunobiol. April 2012,Vol. 18, Issue Supplement s3, Pages 841 3. Tutykhina I.L., Logunov D.Y., Shcherbinin D.N., Shmarov M.M., Tukhvatulin A.I., Naroditsky B.S., Gintsburg A.L. Development of adenoviral vector-based mucosal vaccine against influenza. J Mol Med. 2011. №89 (4). PP. 331-341. Ad-null IFU/ml C. muridarum Ad-mOmcB Days post infection Figure 1. Vaginal chlamydial shedding was measured post-challenge. Figure 2. Suppression of chlamydia infection in cell culture by serum neutralizing antibodies. Figure 3. Antibody production triggered by vaccination Ad-mOmcB-Fc.