Casein was removed, antigens were added with a minimum 24 l/well, and plates were incubated 60 min at room temperature with shaking

Casein was removed, antigens were added with a minimum 24 l/well, and plates were incubated 60 min at room temperature with shaking. G. Vaccine efficacy was also assessed using pre-exposure vaccination of mice. EPZ020411 hydrochloride Purified native RABV G induced a RABV neutralizing antibody (rVNA) response with a geometric mean titer of 4.2 IU/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 <0.05 IU/ml and protected <50% of immunized mice. These preliminary results support the hypothesis that immunogenicity may be predicted from the 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: Rabies virus, Vaccine, Glycoprotein, Electrochemiluminescent assay, Antigenicity, Immunogenicity Introduction Rabies virus (RABV) continues to present challenges for vaccine development related to its neurotropism, immune evasion, and antigenic diversity, as well as the access to affordable modern biologics [1-3]. Given the necessity for highly effective pre- and post-exposure prophylaxis (PEP), potency issues are paramount to ensure released lots of RABV vaccine can elicit an adequate level of RABV- neutralizing antibodies (rVNA) in humans and other animals. Rabies vaccines that are applied in human PEP regimes should have a potency of at least 2.5 international units (IU) per dose, as measured by the NIH test [4, 5]. The NIH potency test has been used for over 50 years as a measure of RABV vaccine potency. In this test, groups of 4-week old mice are inoculated intraperitoneally over a two week period. Then RABV is administered intracerebrally in parallel for vaccinated and control mice, to calculate an effective dose (ED50). The ED50 of a standard NIH reference vaccine is divided by the ED50 of the test vaccine to yield its relative potency to permit comparison between different reference vaccine preparations [6]. Shortcomings of the NIH test are numerous and include unnatural routes of vaccination and challenge, the requirement of hundreds of mice per test, the wide confidence interval of results, and the associated time and costs of testing [7]. Refinement and replacement of this historical measure continues to be an issue facing vaccine manufacturers [8]. Alternative methods will require highly robust, reproducible and flexible characteristics to accommodate future development in the field including: greater consideration of rabies as a vaccine-preventable disease for non-occupational, pre-exposure immunization of those at greatest risk; further dose-sparing schedules; development of additional non-inactivated recombinant biologics for both veterinary (including wildlife) and public health applications; and inclusion of other viral antigens towards the production of broader, less expensive pan-lyssavirus vaccines [9]. Various methods have been suggested to replace the NIH test. Single radial immunodiffusion was originally EPZ020411 hydrochloride proposed as a measure of RABV glycoprotein (G) content in vaccine preparations [10, 11]. In addition, ELISA techniques were developed Rabbit Polyclonal to MT-ND5 [12-15]. Of these, an immune-capture (i.e. antigen-capture or sandwich) ELISA using a RABV neutralizing monoclonal antibody (MAb) emerged as a preferred method [16, 17]. Using a MAb that only recognizes the native, trimeric and immunogenic form of RABV G prevents detection of non-immunogenic, soluble, G in vaccines [18-21]. Recently, this method has been further refined using a single-chain variable fragment antibody [22] or a diabody, to replace MAbs for antigen capture [23]. In the current study, an antigen-capture assay was selected to assess the antigen content of various RABV vaccine preparations. The Meso Scale Discovery (MSD) platform was used to quantify the electrochemiluminescence (ECL). The ECL assay is based on the proximity of a sulfo-tag-label to an electrical current on the plate surface, resulting in the release of light which can be measured. An arbitrary unit of counts is assigned to the intensity of the light by the MSD imager. The ECL counts were expressed in terms of the total protein concentration in the test vaccine. This value was compared to the immunogenicity of EPZ020411 hydrochloride the vaccine.