Bernhard DietzscholdBernhard Dietzschold

Thomas Jefferson UniversityThomas Jefferson University

Paris, Paris, FranceFrance

May 2007May 2007

Dominance of a non-pathogenic over a pathogenic glycoprotein

gene in rabies virus

The RV G protein plays a The RV G protein plays a major role in the major role in the pathogenesis of RVpathogenesis of RV The RV G is not only the major antigen The RV G is not only the major antigen

responsible for the induction of protective responsible for the induction of protective immunity, but is also a major contributor immunity, but is also a major contributor to the pathogenicity of the virus. to the pathogenicity of the virus.

To abolish the pathogenicity, the To abolish the pathogenicity, the recombinant RVs have been constructed recombinant RVs have been constructed to carry the G gene of SADB19 in which to carry the G gene of SADB19 in which Arg333 is replaced by Glu333. Arg333 is replaced by Glu333.

The Glu333 G protein, referred to as GAN, The Glu333 G protein, referred to as GAN, renders the virus non-pathogenic for renders the virus non-pathogenic for adult mice after i.c. infection adult mice after i.c. infection

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

The non-pathogenic The non-pathogenic phenotype associated with phenotype associated with GAN is not stable GAN is not stable After several passages of After several passages of

SPBNGAN in mice, an Asn → Lys SPBNGAN in mice, an Asn → Lys mutation arose at position 194 of mutation arose at position 194 of GAN resulting in GAK, which was GAN resulting in GAK, which was associated with a reversion to the associated with a reversion to the pathogenic phenotype. pathogenic phenotype.

Reversion to the pathogenic Reversion to the pathogenic phenotype in GA variants after phenotype in GA variants after Virus Passage Virus Passage in suckling micein suckling mice

ND***ND***9/10 (90%)9/10 (90%)SPBN**SPBN**4/10 (40%)4/10 (40%)0/10 (10%)0/10 (10%)SPBNGA-Cyto-CSPBNGA-Cyto-C0/10 (10%)0/10 (10%)0/10 (10%)0/10 (10%)SPBNGA-GASPBNGA-GA1/10 (10%)1/10 (10%)0/10 (10%)0/10 (10%)SPBNGASPBNGA

1010thth Mouse Mouse PassagePassage0 Passage0 Passage

Mortality*Mortality*Virus StockVirus Stock

*Mice were infected i.c. with 104 FFU

** SPBN, leathal for adult mice, serves as a control

***ND = not done

Nucleotide Nucleotide Sequence Sequence Analysis Analysis of RV GA after of RV GA after the 5the 5thth & 10 & 10thth

Mouse PassageMouse Passage

single-base change at single-base change at nucleotide 639 of G:nucleotide 639 of G:

AsnAsn194194 Lys Lys194194

AAAATT AAAAGG

oror

AAAAAA

Site-directed Site-directed Mutagenesis of the GA Mutagenesis of the GA

GeneGene

AsnAsn194194 [N] [N] Lys Lys194194 [K] [K]

AAAATT AAAAGG

Nonpathogenic G: GANNonpathogenic G: GAN– aa194 = Asn; aa333 = Gluaa194 = Asn; aa333 = Glu

Pathogenic G: GAK Pathogenic G: GAK – aa194 = Lys; aa 333= Gluaa194 = Lys; aa 333= Glu

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

Effect of Asn → Lys mutation on Effect of Asn → Lys mutation on the pathogenicity of double G the pathogenicity of double G variantsvariants

Because a RV vaccine candidate Because a RV vaccine candidate containing two GAN genes containing two GAN genes (SPBNGAN-GAN) exhibits (SPBNGAN-GAN) exhibits increased immunogenicity in vivo increased immunogenicity in vivo as compared to the single GAN as compared to the single GAN construct, we tested whether the construct, we tested whether the presence of two GAN genes might presence of two GAN genes might also enhance the probability of also enhance the probability of reversion to pathogenicity reversion to pathogenicity

Construction schematic of recombinant RVs containing one or two modified G genes

encoding either an Asn (GAN) or a Lys (GAK) at position 194

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

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

ConclusionsConclusions

The pathogenicity of an RV The pathogenicity of an RV containing a GAN and a GAK gene containing a GAN and a GAK gene was strongly reduced as was strongly reduced as compared to that of an RV compared to that of an RV containing two GAK genescontaining two GAK genes– This indicates that GAN is dominant This indicates that GAN is dominant

in determining the pathogenicity in determining the pathogenicity phenotype of the RV. phenotype of the RV.

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

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

ConclusionsConclusions

The pathogenicity of an RV correlates The pathogenicity of an RV correlates inversely with its replication rate in inversely with its replication rate in tissue culturetissue culture

Virus production and viral RNA Virus production and viral RNA synthesis were markedly higher in synthesis were markedly higher in SPBNGAN-, SPBNGAK-GAN- and SPBNGAN-, SPBNGAK-GAN- and SPBNGAN-GAK-infected neuroblastoma SPBNGAN-GAK-infected neuroblastoma cells than in the SPBNGAK- and cells than in the SPBNGAK- and SPBNGAK-GAK-infected counterparts, SPBNGAK-GAK-infected counterparts, – This suggests control of GAN dominance at This suggests control of GAN dominance at

the level of viral RNA synthesisthe level of viral RNA synthesis

AcknowledgementsAcknowledgements

Milosz Faber and Jianwei LiMilosz Faber and Jianwei Li Thomas Jefferson University, Department of Microbiology & Immunology, Thomas Jefferson University, Department of Microbiology & Immunology,

Philadelphia, PAPhiladelphia, PA

Marie-Luise FaberMarie-Luise FaberMolecular Targeting Technologies, Inc., West Chester, PA Molecular Targeting Technologies, Inc., West Chester, PA

Matthias J. SchnellMatthias J. SchnellThomas Jefferson University, Department of Microbiology and Thomas Jefferson University, Department of Microbiology and

Immunology, Philadelphia, PAImmunology, Philadelphia, PA

This work is supported by NIH Grants: This work is supported by NIH Grants:

R01 AI060686-02 R01 AI060686-02 R01 AI045097-08R01 AI045097-08