1. Bernhard Dietzschold Thomas Jefferson University Paris, France May 2007 Dominance of a non-pathogenic over a pathogenic glycoprotein gene in rabies virus

2. The RV G protein plays a major role in the pathogenesis of RV The RV G is not only the major antigen responsible for the induction of protective immunity, but is also a major contributor to the pathogenicity of the virus. The RV G is not only the major antigen responsible for the induction of protective immunity, but is also a major contributor to the pathogenicity of the virus. To abolish the pathogenicity, the recombinant RVs have been constructed to carry the G gene of SADB19 in which Arg333 is replaced by Glu333. To abolish the pathogenicity, the recombinant RVs have been constructed to carry the G gene of SADB19 in which Arg333 is replaced by Glu333. The Glu333 G protein, referred to as GAN, renders the virus non-pathogenic for adult mice after i.c. infection The Glu333 G protein, referred to as GAN, renders the virus non-pathogenic for adult mice after i.c. infection

3. An ARG ILE mutation in the RV G results in a loss of pathogenicity for adult mice An ARG ILE mutation in the RV G results in a loss of pathogenicity for adult mice

4. The non-pathogenic phenotype associated with GAN is not stable After several passages of SPBNGAN in mice, an Asn Lys mutation arose at position 194 of GAN resulting in GAK, which was associated with a reversion to the pathogenic phenotype. After several passages of SPBNGAN in mice, an Asn Lys mutation arose at position 194 of GAN resulting in GAK, which was associated with a reversion to the pathogenic phenotype.

5. Reversion to the pathogenic phenotype in GA variants after Virus Passage in suckling mice ND*** 9/10 (90%) SPBN** 4/10 (40%) 0/10 (10%) SPBNGA-Cyto-C SPBNGA-GA 1/10 (10%) 0/10 (10%) SPBNGA 10 th Mouse Passage 0 Passage Mortality* Virus Stock *Mice were infected i.c. with 10 4 FFU ** SPBN, leathal for adult mice, serves as a control ***ND = not done

6. Nucleotide Sequence Analysis of RV GA after the 5 th & 10 th Mouse Passage single-base change at nucleotide 639 of G: Asn 194 Lys 194 Asn 194 Lys 194 AAT AAG AAT AAG or or AAA AAA

7. Site-directed Mutagenesis of the GA Gene Asn 194 [N] Lys 194 [K] AAT AAG AAT AAG

8. Nonpathogenic G: GAN Nonpathogenic G: GAN –aa194 = Asn; aa333 = Glu Pathogenic G: GAK Pathogenic G: GAK –aa194 = Lys; aa 333= Glu

9. Mortality (A), clinical score (B), and body weight (C) of Swiss-Webster mice infected i.c. with different ratios of SPBNGAN and SPBNGAK

10. Effect of Asn Lys mutation on the pathogenicity of double G variants Because a RV vaccine candidate containing two GAN genes (SPBNGAN- GAN) exhibits increased immunogenicity in vivo as compared to the single GAN construct, we tested whether the presence of two GAN genes might also enhance the probability of reversion to pathogenicity Because a RV vaccine candidate containing two GAN genes (SPBNGAN- GAN) exhibits increased immunogenicity in vivo as compared to the single GAN construct, we tested whether the presence of two GAN genes might also enhance the probability of reversion to pathogenicity

11. Construction schematic of recombinant RVs containing one or two modified G genes encoding either an Asn (GAN) or a Lys (GAK) at position 194

12. Mortality (A), clinical score (B), and body weight (C) of Swiss-Webster mice infected i.c. with SPBNGAN-GAN, SPBNGAK-GAK, SPBNGAK-GAN, or SPBNGAN-GAK

13. Synthesis of genomic RV RNA (A) in the brain Synthesis of genomic RV RNA (A) in the brain and induction of RV VNA titers in the serum (B) of mice infected with double-G RV variants

14. Conclusions The pathogenicity of an RV containing a GAN and a GAK gene was strongly reduced as compared to that of an RV containing two GAK genes The pathogenicity of an RV containing a GAN and a GAK gene was strongly reduced as compared to that of an RV containing two GAK genes –This indicates that GAN is dominant in determining the pathogenicity phenotype of the RV.

15. Single-step virus growth curves (A) and mitochindrial respiration (B) in NA cells infected with double G RV variants

16. Transcription of viral mRNA (A) and viral genomic RNA (B) in NA cells infected with the single or double G recombinant RVs

17. Conclusions The pathogenicity of an RV correlates inversely with its replication rate in tissue culture The pathogenicity of an RV correlates inversely with its replication rate in tissue culture Virus production and viral RNA synthesis were markedly higher in SPBNGAN-, SPBNGAK-GAN- and SPBNGAN-GAK-infected neuroblastoma cells than in the SPBNGAK- and SPBNGAK-GAK-infected counterparts, Virus production and viral RNA synthesis were markedly higher in SPBNGAN-, SPBNGAK-GAN- and SPBNGAN-GAK-infected neuroblastoma cells than in the SPBNGAK- and SPBNGAK-GAK-infected counterparts, –This suggests control of GAN dominance at the level of viral RNA synthesis

18. Acknowledgements Milosz Faber and Jianwei Li Milosz Faber and Jianwei Li Thomas Jefferson University, Department of Microbiology & Immunology, Philadelphia, PA Marie-Luise Faber Marie-Luise Faber Molecular Targeting Technologies, Inc., West Chester, PA Matthias J. Schnell Matthias J. Schnell Thomas Jefferson University, Department of Microbiology and Immunology, Philadelphia, PA This work is supported by NIH Grants: This work is supported by NIH Grants: R01 AI060686-02 R01 AI060686-02 R01 AI045097-08 R01 AI045097-08