Bernhard Dietzschold Thomas Jefferson University

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

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. • 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

3. 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.

5. Virus Stock Mortality* 0 Passage 10th Mouse Passage SPBNGA 0/10 (10%) 1/10 (10%) SPBNGA-GA 0/10 (10%) 0/10 (10%) SPBNGA-Cyto-C 0/10 (10%) 4/10 (40%) SPBN** 9/10 (90%) ND*** *Mice were infected i.c. with 104 FFU ** SPBN, leathal for adult mice, serves as a control ***ND = not done Reversion to the pathogenic phenotype in GA variants after Virus Passage in suckling mice

6. Nucleotide Sequence Analysis of RV GA after the 5th & 10thMouse Passage single-base change at nucleotide 639 of G: Asn194 Lys194 AATAAG or AAA

7. Asn194 [N]  Lys194 [K] AATAAG Site-directed Mutagenesis of the GA Gene

8. Nonpathogenic G: GAN • aa194 = Asn; aa333 = Glu • 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

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 brainand induction of RV VNA titers inthe 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 • 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 • 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 Thomas Jefferson University, Department of Microbiology & Immunology, Philadelphia, PA • Marie-Luise Faber Molecular Targeting Technologies, Inc., West Chester, PA • Matthias J. Schnell Thomas Jefferson University, Department of Microbiology and Immunology, Philadelphia, PA This work is supported by NIH Grants: R01 AI060686-02 R01 AI045097-08