An electrochemiluminescence assay for analysis of rabies virus glycoprotein content in rabies vaccines

Abstract

Vaccine potency testing is necessary to evaluate the immunogenicity of inactivated rabies virus (RABV) vaccine preparations before human or veterinary application. Currently, the NIH test is recommended by the WHO expert committee to evaluate RABV vaccine potency. However, numerous disadvantages are inherent concerning cost, number of animals and biosafety requirements. As such, several in vitro methods have been proposed for the evaluation of vaccines based on RABV glycoprotein (G) quality and quantity, which is expected to correlate with vaccine potency. In this study an antigen-capture electrochemiluminescent (ECL) assay was developed utilizing anti-RABV G monoclonal antibodies (MAb) to quantify RABV G. One MAb 2-21-14 was specific for a conformational epitope so that only immunogenic, natively folded G was captured in the assay. MAb 2-21-14 or a second MAb (62-80-6) that binds a linear epitope was used for detection of RABV G. Vaccine efficacy was also assessed in vivo using pre-exposure vaccination of mice. Purified native RABV G induced a RABV neutralizing antibody (rVNA) response with a geometric mean titer of 4.2IU/ml and protected 100% of immunized mice against RABV challenge, while an experimental vaccine with a lower quality and quantity of G induced a rVNA titer<0.05IU/ml and protected <50% of immunized mice. These preliminary results support the hypothesis that in vivo immunogenicity may be predicted from the in vitro measurement of RABV G using an ECL assay. Based upon these results, the ECL assay may have utility in replacement of the NIH test.

Keywords: Antigenicity; ECL; ED(50); Electrochemiluminescent assay; G; Glycoprotein; IM; IU; Immunogenicity; MAb; MSD; Meso Scale Discovery; RABV; RFFIT; Rabies virus; Vaccine; effective dose; electrochemiluminescent; glycoprotein; international units; intramuscular; monoclonal antibody; p.i.; post challenge or post infection; rVNA; rabies virus; rabies virus neutralizing antibody; rapid fluorescent focus inhibition test.

Figure 1. ECL counts over total protein concentration

Antigen was captured and detected using the 2-21-14 αRABV G MAb, and ECL was measured for eight 5-fold serial dilutions of different rabies vaccine preparations. ECL counts were plotted against the total protein concentration on logarithmic scales. Means of at least four statistical replicates from at least two biological replicates are shown. Shown in decreasing order are purified RABV G ERA G lot 2005 (■), Medicago^® (✲), ERA G lot 2012 (◆), RabAvert^® (■), RabAvert^® depleted (•), Imovax^® (−), CVS G lot 2011 (×), ERA G lot 2010 (▲), Fraunhofer^® (+), Imovax^® depleted (◆), and Fraunhofer^® Adjuvant (•).

Figure 2. Linear regressions of ECL counts over total protein concentration

Antigen was captured and detected using the 2-21-14 αRABV G MAb, and ECL was measured for eight 5-fold serial dilutions of different rabies vaccine preparations. The linear regression of the ECL counts was plotted against the total protein concentration on a linear scale. ECL counts μg⁻¹ ml⁻¹ were calculated based on the linear regression at 25 μg/ml. As an example, linear regressions are shown for RabAvert^®, 4000 counts μg⁻¹ ml⁻¹ (dotted line); RabAvert^® diluted, 3000 counts μg⁻¹ ml⁻¹ (dashed line); and RabAvert^® depleted, 1000 counts μg⁻¹ ml⁻¹ (solid line).

Figure 3. Comparison of rVNA and antigenicity measured by the ECL assay

The geometric mean rVNA titer, measured 30 days after immunizing mice with selected rabies vaccines, was plotted against the log transformed ECL counts μg⁻¹ ml⁻¹. Vaccines were considered immunogenic (+) if the rVNA titer was >0.5 IU/ml, and non-immunogenic (—) if the rVNA titer was <0.5 IU/ml. The statistical cut-off for antigenicity was 440 counts μg⁻¹ ml⁻¹ (2.64 log₁₀). The ECL assay was able to distinguish between vaccine lots that were close to the cut-off values for rVNA and antigenicity.