Potential human immunotherapeutics for plague

Abstract

Two monoclonal antibodies directed to the V antigen of Yersinia pestis have been tested for protective efficacy in a murine model of bubonic plague. Mice were infected with a current clinical isolate from Madagascar, designated Y. pestis 10-21/S. Mab7.3, delivered to mice intra-periteoneally at either 24 h prior to, or 24 h post-infection, was fully protective, building on many studies which have demonstrated the protective efficacy of this Mab against a number of different clinical isolates of Y. pestis. Mab 29.3, delivered intra-peritoneally at either -24 h or +24 h, protected 4/5 mice in either condition; this has demonstrated the protective efficacy of this Mab in vivo for the first time. These results add to the cumulative data about Mab7.3, which is currently being humanized and highlight its potential as a human immunotherapeutic for plague, which is an enduring endemic disease in Madagascar and other regions of Africa, Asia, and South America.

Keywords: Plague; clinical isolate; immunotherapeutic; passive therapy; protection.

Graphical abstract

Figure 1.

SDS-Page for Mab 7.3. For the nonreduced sample, Mab7.3 was diluted to 5 µg in a final volume of 15 µl using NuPage LPS 4× sample buffer (Novex, NP007) in accordance with the manufacturer’s instructions. The reduced sample was prepared similarly, but with the addition instead of NuPage 10× sample reducing agent (Novex, NP004) and heating of the sample (95°C for 15 min). SDS-PAGE was performed after loading 10 µl of antibody samples and 5 µl of SeeBlue Plus2 marker (Invitrogen, LC5925) into a NuPAGE 4–12% gradient Bis-Tris gel. Electrophoresis conditions were 200 V for 20 min. The gel was stained with SimplyBlue Safe Stain (Invitrogen, LC6065) for 1 h and destained in distilled water overnight.

Figure 2.

Limit of detection of V antigen by Mab 7.3. V antigen was coated to a microtitre plate in the dilution range 5 µg/ml to 85 pg/ml in triplicate (0.1 ml/well) and then probed with a fixed concentration (800 ng/ml) of biotinylated Mab 7.3. After washing in PBS/Tween 20 and blocking with 2% milk powder in PBS (Blotto), binding was detected with streptavidin peroxidase (Pierce High Sensitivity Streptavidin-HRP, 21132) at 1:10,000 in Blotto and developed with 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS; SeraCare, 5120-0041) and the signal was read at OD₄₁₄. The LOD was 20 ng/ml.

Figure 3.

Survival of mice dosed with Mab 7.3. or Mab 29.3 or macaque immune serum pre- or post-infection with 95 cfu Y. pestis 10/21-S.