Construction, immunogenicity and protective efficacy in mice of a prototype chimeric Chlamydia trachomatis MOMP vaccine candidate antigen

Background

Urogenital chlamydial infection, caused by Chlamydia trachomatis, is the main sexually transmitted infection in Sweden. Despite active programmes for detection and case finding, nearly 37 000 cases were reported in 2010. Serovar E strains are considered to cause approximately 40-50% of these cases. A vaccine would be highly valuable in order to control the epidemic.
In this project, a chimeric gene construct of C. trachomatis serovar E major outer membrane protein (MOMP) was designed, and expressed as a vaccine candidate antigen.
The construct was based on known T and B cell epitopes located in the variable segment (VS) 2 and 4 loops of MOMP.

Construction and expression of the chimeric MOMP VS2/4 antigen
The PCR primers used to amplify the VS2 and VS4 variable regions of C. trachomatis MOMP serovar E for assembling the chimera were designed from ompA sequence data. The sequence encoding a common flexible linker, [(Gly4Ser)2Gly4] , was introduced and the amplified VS2 and VS4 fragments were then assembled as follows: 5’-VS2 – linker – VS4-3’. The produced genetic construct showed the expected size of 351 bp (Fig. 1).

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The product was then verified by sequencing and cloned into the pET101 vector (containing sequences encoding C-terminal V5- and His-tags). The over-expressed protein was detected using both anti-His antibodies (data not shown) and anti-MOMP antibodies. A band of the expected size (17 kD) was detected with mouse monoclonal antibodies to C. trachomatis MOMP (Acris Antibodies) (Fig. 2).

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For purification of the MOMP chimera using IMAC technology, we scaled-up expression to 2000 ml bacterial culture. The chimeric protein was purified under both native and denaturing conditions. The elution fractions of the chimeric protein, purified under native conditions, analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue, are shown in Fig. 3.

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Pure fractions were pooled and were later used in immunization experiments for verification of immunogenic features of the designed MOMP chimera and for production of anti-MOMP polyclonal serum, as well as for immunization of mice.

Immunization experiments
C57BL/6 mice were immunized intranasally (i.n.) with the chimeric MOMP VS2/4 antigen and Cholera toxin (CT) adjuvant, three immunizations with 10 days intervals. A final boost with the identical antigen/adjuvant preparation was given intravaginally (i.vag.). Challenge with live C. trachomatis was performed 10 days after boost. Antibodies in serum and vaginal washes were determined with the identical chimeric MOMP construct as antigen in ELISAs.
 
All mice in vaccine groups (N=10/group and experiment) developed a strong antigen-specific IgG response in serum, and eight (80%) also had detectable antigen-specific IgG in vaginal washes (Fig 4 a-b). An IgA response, albeit weaker, was detected in some of the mice both in serum (not shown) and in vaginal washes (three mice, 30%) (Fig 4c).

 
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After challenge with C. trachomatis, 80 and 100% of the mice became infected in two experiments, respectively. The vaccinated groups cleared the infection significantly faster than the control groups (Fig 5 a-b).

 
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Conclusions

A new chimeric MOMP VS2/4 antigen was successfully constructed. This construct also gave rise to a significant immune response in mice.
Finally, in challenge and clearance studies the chimeric MOMP VS2/4 antigen gave substantial protection to C. trachomatis infection.
Further studies are in progress.