Development of a Mouse Monoclonal Antibody Cocktail for Post-exposure Rabies Prophylaxis in Humans

As the demand for rabies post-exposure prophylaxis (PEP) treatments has increased exponentially in recent years, the limited supply of human and equine rabies immunoglobulin (HRIG and ERIG) has failed to provide the required passive immune component in PEP in countries where canine rabies is endemic. Replacement of HRIG and ERIG with a potentially cheaper and efficacious alternative biological for treatment of rabies in humans, therefore, remains a high priority. In this study, we set out to assess a mouse monoclonal antibody (MoMAb) cocktail with the ultimate goal to develop a product at the lowest possible cost that can be used in developing countries as a replacement for RIG in PEP. Five MoMAbs, E559.9.14, 1112-1, 62-71-3, M727-5-1, and M777-16-3, were selected from available panels based on stringent criteria, such as biological activity, neutralizing potency, binding specificity, spectrum of neutralization of lyssaviruses, and history of each hybridoma. Four of these MoMAbs recognize epitopes in antigenic site II and one recognizes an epitope in antigenic site III on the rabies virus (RABV) glycoprotein, as determined by nucleotide sequence analysis of the glycoprotein gene of unique MoMAb neutralization-escape mutants. The MoMAbs were produced under Good Laboratory Practice (GLP) conditions. Unique combinations (cocktails) were prepared, using different concentrations of the MoMAbs that were capable of targeting non-overlapping epitopes of antigenic sites II and III. Blind in vitro efficacy studies showed the MoMab cocktails neutralized a broad spectrum of lyssaviruses except for lyssaviruses belonging to phylogroups II and III. In vivo, MoMAb cocktails resulted in protection as a component of PEP that was comparable to HRIG. In conclusion, all three novel combinations of MoMAbs were shown to have equal efficacy to HRIG and therefore could be considered a potentially less expensive alternative biological agent for use in PEP and prevention of rabies in humans

Neutralization pattern of candidate MoMAbs (E559.9.14, 62-7-13, 727-5, 777-16) or other MoMAbs (1112-1, D1) with RABV (E559.9.14 antigenic site II escape mutant, 10⁴ FFU/ml) under varying VNA titres (IU/ml).

<p>Absence (−) and presence (+) of viable virus is indicated.</p

Protection of hamsters challenged with RABV following treatment with MoMAb combination cocktails during three independent <i>in vivo</i> studies.

<p>Survivorship of hamsters after challenge with a Mexican (2004), Thai (2006), or Indian (2008) canine RABV variant and subsequent treatment with 1∶1 cocktail formulations of MoMAbs to simulate passive immunization in PEP.</p

Coomassie blue staining of purified MoMabs (5 µg/well) demonstrating appropriate size of light chain and heavy chain.

<p>Ma–molecular weight marker in kD; lane1, E559 (batch # 603-02); lane 2, 62-7-13 (batch # 604-26); lane 3, 1112-1 (batch # 604-26); lane 4, M777-16-3 (batch # 605-03); lane 5, M777-16-3 (batch # 605-12); lane 6, M727-5-1 (batch # 605-03); lane 7, M727-5-1 (batch # 605-19).</p

<i>In vitro</i> neutralization pattern of individual candidate MAbs.

<p>Tests were conducted in comparison to standard rabies immunoglobulin (SRIG) against lyssa- (gt 1–7) and putative lyssavirus gts. MoMAbs M777-16 and M727-5 were used purified and the remaining as cell culture supernatants. For MAb 62-7-13, three different harvests were tested. Figures in boxes show the minimum MoMAb concentration in IU/ml at which complete neutralization was observed. Boxes with cross (+) represent presence of viable virus.</p

Available technical information for candidate MoMAbs.

<p>Legend: aa–amino acid, CVS 11–Challenge virus standard 11, DMEM–Dulbeccos' minimum essential medium, ELISA–enzyme linked immunosorbent assay, ERA–Evelyn Rokitniki Abelseth SAD derived RABV strain, FCA–Flouricon-CA Assay, HB–hybridization medium, SAD–Street Alabama Dufferin strain of RABV. Media specification: Iscove's DMEM 1 = Iscove's modified DMEM + HAM F12 (1∶1) + 10% FCS; Iscove's DMEM 2 = Iscove's modified DMEM + ITS + antibiotics/antimycotics + L-glutamine + 5% FCS.</p

<i>In vitro</i> neutralization pattern of equal amounts of MoMAbs in combination cocktails.

<p><i>In vitro</i> neutralization pattern of equal mixes of MoMAbs in combination cocktails adjusted to 2000 IU/10 ml in comparison to SRIG against lyssaviruses of gt 1–7 and putative lyssavirus gts. Figures in boxes show the minimum MoMAb concentration in IU/ml at which complete neutralization was observed. Boxes with cross (+) represent the presence of viable virus.</p

Neutralization results obtained after batch production under GLP conditions.

<p>Geometric mean (GEO) VNA titres and standard deviation (SD) of 1 mg/ml of purified MoMAbs as determined by RFFIT in three independent tests and subsequent estimation of the yield of supernatant of the five hybridomas in comparison to a negative control and the WHO standard rabies immunoglobulin (SRIG).</p