Identification of antibodies specific for at least two different lyssaviruses

#This disclosure concerns methods of identifying lyssavirus-specific antibodies and the use of such antibodies in the treatment and prophylaxis of rabies.

#Rabies is an inevitably fatal but preventable disease. In developing countries rabies remains a significant endemic disease burden. World-wide approximately 55,000 people die of rabies each year (WHO Expert Consultation on Rabies, 2004). Rabies is preventable with proper early post-exposure treatment. Currently, post-exposure prophylaxis includes thorough wound-washing with soap and water followed by administration of vaccine and anti-rabies virus immunoglobulin (RIG) of human or equine origin. RIG administered shortly after exposure at the wound site provides passive immunity which neutralizes rabies virus and prevents its spread until the patient's immune response following vaccination is elicited. Deaths due to post-exposure prophylaxis failure are most commonly attributed to deviations from the recommended regimen such as late initiation of post-exposure prophylaxis or no administration of RIG (Wilde, Vaccine, 25:7605-7609, 2007). In developing countries, availability of RIG is extremely low with only 1-2% of post-exposure prophylaxis being performed using RIG (Sudarshan et al., Int J Infect Dis, 11:29-35, 2007; WHO Consultation on a Monoclonal Antibody Cocktail for Rabies Post Exposure Treatment, 2002). In the United States, only human derived RIG is administered due to the risk of anaphylactic shock from exposure to equine immunoglobulins, but the concern of blood-born pathogen transmission in human RIG remains.

#Human monoclonal antibodies (mAbs) that neutralize rabies virus have long been recognized as an alternative to overcome the limitations of RIG (Dietzschold et al., J Virol, 64:3087-3090, 1990). Adequate supplies of cell-cultured human mAbs could be produced in a cost-effective manner (Prosniak et al., J Infect Dis, 188:53-56, 2003). In addition, the use of human mAbs reduces the likelihood of an adverse immune response (Weiner, J Immunother, 29:1-9, 2006) and has been shown to be as effective as RIG in preventing rabies in animals (de Kruif et al., Annu Rev Med, 58:359-368, 2007). When a cocktail of two rabies-neutralizing, human mAbs was given with a rabies vaccine to animals experimentally infected with rabies, dose-dependent survival was observed, and all animals receiving the highest dose survived (Goudsmit et al., J Infect Dis, 193:796-801, 2006). The same cocktail was recently shown to be safe to administer to healthy humans in two phase-one clinical trials (Bakker et al., Vaccine, 26:5922-5927, 2008). However, selection of human monoclonal antibodies to include in such a cocktail has some limitations. The diversity of mAbs produced depends significantly on the diversity of viral antigens used to immunize human donors.

#FIG. 1: Construction of ERA-3G. The G333 mutation is introduced into the ERA backbone and two transcriptional (trans) units are added. The transcriptional units are introduced between the P and M genes and between the G and L genes. The MOKV and WCBV G genes are cloned into the transcriptional units to form a recombinant ERA rabies virus with three glycoprotein genes (ERA-3G).

#FIG. 2: Results of whole virus ELISA. Relative binding of selected human variable heavy (VH) domain phage display clones to recombinant ERA RABV expressing glycoprotein (G) from RABV, MOKV and WCBV. The left bar for each clone shown indicates clones selected using the whole recombinant virus ERA RABV expressing glycoprotein (G) from RABV, MOKV and WCBV. The right bar for each clone shown indicates clones selected using glycoproteins purified from the recombinant ERA RABV expressing glycoprotein (G) from RABV, MOKV and WCBV. Using a standard ELISA method, raw absorbance (420 nm-650 nm) was fixed relative to a known clone binding to its cognate antigen (positive control).

#FIG. 3: Results of glycoprotein ELISA. Relative binding of selected human variable heavy (VH) domain phage display clones to glycoproteins purified from recombinant ERA RABV expressing glycoprotein (G) from RABV, MOKV and WCBV. Left bars for each clone indicate clones selected using the whole recombinant virus ERA RABV expressing glycoprotein (G) from RABV, MOKV and WCBV. Right bars for each clone indicate clones selected using glycoproteins purified from the recombinant ERA RABV expressing glycoprotein (G) from RABV, MOKV and WCBV. Using a standard ELISA method, raw absorbance (420 nm-650 nm) was fixed relative to a known clone binding to its cognate antigen (positive control).

#The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The Sequence Listing is submitted as an ASCII text file, created on Jul. 24, 2015, 102 KB, which is incorporated by reference herein. In the accompanying sequence listing:

#SEQ ID NOs: 1-43 are the nucleotide sequences of human variable heavy (VH) domain phage display clones selected using a recombinant ERA RABV expressing glycoprotein (G) from RABV, MOKV and WCBV.

#SEQ ID NOs: 44-110 are the nucleotide sequences of human variable heavy (VH) domain phage display clones identified by sequential selection on cells expressing LBV glycoprotein, cells expressing MOKV glycoprotein, cells expressing WCBV glycoprotein and cells expressing DUVV glycoprotein.