Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development

Many candidate subunit vaccine antigens are membrane proteins. Historically, these have been difficult to produce and purify in sufficient quantities. With this technique, we're able to create antigens of interest embedded within a lipid membrane to help ensure a native-like confirmation.

This process also facilitates formulation with adjuvants of interest. Cell-free expression enables rapid production of proteins of interest in their native tagless structure. This technique is also scalable and we're already working with commercial companies for developing vaccine formulations using this process.

We demonstrate the process here for a chlamydia antigen, but the process could be easily adapted to produce antigens of interest regardless of whether or not they're membrane proteins. This process may be applicable to both pathogen and cancer vaccine development. To begin, prepare MOMP-tNLPs by employing a cell-free method.

Defrost the reconstitution buffer from the cell-free protein expression kit two hours before setting the reaction and add EDTA-free protease inhibitor to the buffer. Use a kit designed to run five one-milliliter reactions. For each one-milliliter reaction, add 525 microliters of reconstitution buffer to the E.coli lysate bottle and roll it to dissolve.

Add 250 microliters of the reconstitution buffer to the reaction bottle containing the additive and dissolve by rolling. Next, add 8.1 milliliters of reconstitution buffer to the reaction feed bottle and roll it to dissolve. To a bottle of amino acid mixture, add three milliliters of reconstitution buffer to dissolve it.

To another bottle of methionine, add 1.8 milliliters of reconstitution buffer, dissolve it, and store it on ice. Add 225 microliters of the reconstituted mixture, 270 microliters of the reconstituted amino acid mixture without methionine, and 30 microliters of reconstituted methionine to the E.coli lysate bottle. Further, add 400 microliters of the DMPC/telodendrimer mixture 15 micrograms of MOMP plasmid, and 0.6 micrograms of delta-49ApoA1 to the mixture and mix it.

Aliquot 20 microliters of the total solution in a 1.5-milliliter tube For a GFP-expressing control reaction. Prepare a feed solution by adding 2.65 milliliters of the reconstituted amino acid mixture without methionine and 300 microliters of reconstituted methionine. Transfer one milliliter of the reaction solution to the inner reaction chamber in the cell-free reaction kit and seal it.

Fill the outer chamber of the reaction vessel with 10 milliliters of the feed solution and close it. Add 0.5 microliters of the GFP control plasmid to the 20-microliter aliquots of the reaction mixture. Put the reaction in a shaker at 300 rotations per minute for 18 hours at 30 degrees Celsius.

Monitor the reaction under a UV light after 15 minutes for fluorescence due to GFP synthesis. Purify the MOMP-tNLP nanoparticle complex from the cell-free reaction mixture by immobilized nickel affinity chromatography using the HIS-tag on the delta-49ApoA1 protein. Transfer one milliliter of the 50%slurry of the HIS-tag purification resin to a 10-milliliter disposable chromatography column, and add three milliliters of binding buffer to equilibrate.

Afterward, drain the buffer and add 250 microliters of binding buffer to the resin. Extract 20 microliters of the cell-free reaction mixture for SDS-PAGE analysis. Add the remaining cell-free reaction mixture with the equilibrated resin and incubate on a laboratory rocker for one hour at four degrees Celsius.

Remove the column cap and wash it with 500 microliters of binding buffer. Add the washing liquid to the column and collect the flow-through for SDS-PAGE analysis. Wash the column with one milliliter of wash buffer with 20-millimolar imidazole six times and collect the fractions.

While washing for the second time, use a one-milliliter pipette to agitate the resin. Elute the MOMP-tNLPs in six 300-microliter fractions of elution buffer I containing 250-millimolar imidazole and a final elution with 300 microliters of elution buffer II containing 500-millimolar imidazole. On the second elution, vigorously agitate the resin by pipetting up and down using a one-milliliter pipette.

Use a gel imager to capture the images at 600 nanometers. Then, quantify the amount of different proteins in the nanoparticles through SDS-PAGE analysis using a protein standard. Draw a standard curve using the densities of the MOMP bands.

Then, determine the MOMP component of the nanoparticles with the standard. Resolve the samples by SDS-PAGE and transfer the gel stacks using a commercial dry blotting system for Western blot analysis. Remove the blots from the stack and incubate them overnight at four degrees Celsius in a blocking buffer containing 0.2%TWEEN 20 and 0.5 micrograms per milliliter MAb40, or 0.2 micrograms per milliliter MAbHIS antiHIS-tag antibody directed against the HIS-tag from delta-49ApoA1 protein.

Wash each blot with PBST three times for five minutes per wash. Incubate the blots for one hour in a blocking buffer containing secondary antibody conjugated to a fluorophore in a dilution of 1:10, 000. Repeat the washes with PBST and capture the image using a fluorescence imager after the final wash.

Using a dot blot apparatus, blot three micrograms of MOMP-tNLP and empty tNLP. Develop and block the blots using the same method for Western blotting. Freeze the mixed solution on dry ice and lyophilize it overnight using a lyophilizer.

Store the dried formulations at minus 20 degrees Celsius. When needed, reconstitute the lyophilized tNLPs with endotoxin-free water and roll it to dissolve and rehydrate. Dialyze the solution with PBS and remove the trehalose with a 3.5-kilodalton-cutoff dialysis membrane.

Centrifuge the nanoparticle solution in a vacuum concentrator before adding the adjuvant. Monitor the sample volume after 20 to 30 minutes to prevent complete drying. In a biosafety cabinet, add the adjuvant under sterile conditions.

Using analytical size exclusion chromatography, analyze the formulation for successful incorporation. Store the adjuvanted MOMP-tNLPs and empty tNLP at four degrees Celsius for up to 14 days. Analyze the stability of the tNLP formulation with size exclusion chromatography.

The SDS-PAGE analysis of MOMP-tNLP was carried out through nickel affinity chromatography, which demonstrated that the cell-free reaction mixture has high levels of expression for both the MOMP and the delta-49ApoA1 proteins. By employing gel densitometry, the MOMP concentration was quantified using a purified recombinant MOMP with a known concentration as the standard. To determine the oligomer formation, SDS-PAGE MOMP and MOMP-tNLP were treated with heat and DTT, which showed distinct bands for MOMP and delta-49ApoA1.

Western blot analysis using an antibody MAb40 was employed against MOMP protein, which revealed a banding pattern confirming the oligomer formation by MOMP protein in its non-denatured state. A dot blot assay was carried out in the presence of MAb40 and MAbHIS antibodies, which indicated the formation of MOMP-tNLP. However, empty tNLP showed a positive signal for MAbHIS.

Sera from the immunized mice injected with adjuvanted MOMP-tNLP demonstrated strong MOMP binding and indicated that MOMP-tNLP could elicit an immune response. Since this protocol generates protein from DNA, it is essential to avoid contaminating the reaction with with DNase, RNase, as well as the extraneous DNA and RNA. Any materials or reagents used in the reactions should be free of these type of molecules.

After expression, purification, and addition of adjuvants, these vaccine candidates can be evaluated in appropriate animal models for the pathogens of interest to assess immune activation markers or evaluate protection during a challenge. For chlamydia, the major outer membrane protein, or MOMP, has been considered a leading candidate antigen for subunit vaccines for many years, but it's been difficult to produce at scales necessary for vaccine application. Cell-free co-expression of this membrane protein is a viable way to start moving this protein toward evaluation in animal models such as mice, and eventually going into human testing.