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. 2012 Dec 5;434(1):18-26.
doi: 10.1016/j.virol.2012.07.020. Epub 2012 Aug 11.

A replication-deficient rabies virus vaccine expressing Ebola virus glycoprotein is highly attenuated for neurovirulence

Amy B Papaneri  1 , Christoph Wirblich, Jennifer A Cann, Kurt Cooper, Peter B Jahrling, Matthias J Schnell, Joseph E Blaney
Affiliations

A replication-deficient rabies virus vaccine expressing Ebola virus glycoprotein is highly attenuated for neurovirulence

Amy B Papaneri et al. Virology. .

Abstract

We are developing inactivated and live-attenuated rabies virus (RABV) vaccines expressing Ebola virus (EBOV) glycoprotein for use in humans and endangered wildlife, respectively. Here, we further characterize the pathogenesis of the live-attenuated RABV/EBOV vaccine candidates in mice in an effort to define their growth properties and potential for safety. RABV vaccines expressing GP (RV-GP) or a replication-deficient derivative with a deletion of the RABV G gene (RVΔG-GP) are both avirulent after intracerebral inoculation of adult mice. Furthermore, RVΔG-GP is completely avirulent upon intracerebral inoculation of suckling mice unlike parental RABV vaccine or RV-GP. Analysis of RVΔG-GP in the brain by quantitative PCR, determination of virus titer, and immunohistochemistry indicated greatly restricted virus replication. In summary, our findings indicate that RV-GP retains the attenuation phenotype of the live-attenuated RABV vaccine, and RVΔG-GP would appear to be an even safer alternative for use in wildlife or consideration for human use.

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Figures

Figure 1
Figure 1
Recombinant viruses. (A) Negative sense RNA genomes are illustrated for the parental rabies vaccine, RVA(p), which was previously termed BNSP. (B) The further attenuated derivative, RVA, contains an attenuating mutation at amino acid 333 of RABV G resulting in an Arg→Glu change, which restricts neurovirulence of RABV vaccines in adult mice. (C) RV-GP encodes EBOV Mayinga strain GP between RABV N and P from the RVA vector. (D) RVΔG-GP contains a deletion in the RABV G gene and is propagated on a complementing cell line, BSR-G cells, which express G.
Figure 2
Figure 2
Virus yields in cell culture. (A) Vero cells were infected with indicated viruses at an MOI of 5. BSR-G cells (a BHK cell derivative that expresses RABV G) were infected at an MOI of 0.01. Infected monolayers were incubated at 37°C for indicated time, and samples were removed for determination of virus yield. Virus concentrations were determined by viral focus assay. Dashed line indicates limit of detection.
Figure 3
Figure 3
Addition of GP to RVA does not increase neuroinvasiveness in immunodeficient mice. Groups of 8 4–6 week-old SCID mice were injected i.m. in the hind leg with 1x106 FFU of the indicated virus and monitored daily for survival for 84 days.
Figure 4
Figure 4
RV-GP and RVΔG-GP are avirulent after intracerebral (i.c.) inoculation of adult mice. Groups of eight four week-old mice were injected i.c. with 1x105 FFU of the indicated virus and monitored daily for survival for 28 days.
Figure 5
Figure 5
Replication of vaccine candidates in suckling mouse brain assayed by quantitative PCR. Five-day-old Swiss Webster mice were inoculated i.c. with 1x105 FFU of the indicated virus. On days 1, 3, 5, 7, 9, 14, and 21, three mice per group were sacrificed and brain homogenates were generated. The level of viral genomic RNA was determined by a RABV NPspecific quantitative RT-PCR assay.
Figure 6
Figure 6
Replication of RVΔG-GP in suckling mouse brain determined by virus focus assay. Five-day-old Swiss Webster mice were inoculated i.c. with 1x105 FFU of the indicated virus. On days 1, 3, 5, 7, 9, 14, 21, 28, 35, and 42, three mice per group were sacrificed and brain homogenates were generated. The level of infectious virus was determined by focus-forming assay on (A) Vero cells or (B) BSR-G cells).
Figure 7
Figure 7
Histopathological and immunohistochemical analysis of RVΔG-GP in suckling mouse brain. Five-day-old Swiss Webster mice were inoculated i.c. with 1x105 FFU of the indicated virus. On days 1, 3, 5, 7, 9, 14, and 21, three mice per group were sacrificed and brains were processed for (A, B) H&E or (C, D) anti-RABV NP IHC analysis as described in the Materials and Methods. Inflammation and immunohistochemical staining were semi-quantitatively scored in the (A, C) cerebellum and (B, D) hippocampus in each of three brains per group using a 0–3 scale (0 = none, 1 = mild, 2 = moderate, and 3 = severe). The mean severity score is indicated. X indicates that all mice from the RVA group had succumbed to infection and were unavailable for analysis.
Figure 8
Figure 8
Histopathology (H&E) and anti-RABV NP immunohistochemistry (IHC) of the cerebellum. Representative H&E (A, B) and IHC (C, D) of cerebellum from suckling mice inoculated i.c. with RVA (A, C) or RVΔG-GP (B, D). Size indicated is the objective magnification. A: Marked infiltration and disruption of the Purkinje cell layer (yellow dotted line) and granule cell layer (area between the yellow and green dotted lines), and to a lesser extent the molecular layer (pink area to the right of the yellow dotted line), by large numbers of neutrophils and lymphocytes. Also note dilatation and congestion of the capillaries (asterisks). B: Essentially normal cerebellum with well-delineated molecular, Purkinje, and granule cell layers, and normal capillaries (see A for explanation of annotations). C: Anti-RABV NP IHC showing numerous immunopositive (brown) neurons and inflammatory cells. D: Anti-RABV NP IHC showing minimal immunopositivity (brown).
Figure 9
Figure 9
Histopathology (H&E) and anti-RABV NP immunohistochemistry (IHC) of the hippocampus. Representative H&E (A, B) and IHC (C, D) of hippocampus from suckling mice inoculated i.c. with RVA (A, C) or RVΔG-GP (B, D). Size indicated is the objective magnification. A: Marked infiltration and disruption of the CA3-CA4 regions (boxed area) by neutrophils and lymphocytes. Also note rarefaction (paleness) of the surrounding neuropil and prominent capillaries lined by reactive endothelium. Inset shows higher magnification of the CA3-CA4 region containing large numbers of neutrophils, lymphocytes, and prominent capillaries. B: Essentially normal hippocampus. C: Anti-RABV NP IHC of the CA3-CA4 region showing large numbers of immunopositive (brown) inflammatory cells. D: Anti-RABV NP IHC of the CA3-CA4 region showing no significant immunopositivity (brown).
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References

    1. Bermejo M, Rodriguez-Teijeiro JD, Illera G, Barroso A, Vila C, Walsh PD. Ebola outbreak killed 5000 gorillas. Science. 2006;314:1564. - PubMed
    1. Blaney JE, Wirblich C, Papaneri AB, Johnson RF, Myers CJ, Juelich TL, Holbrook MR, Freiberg AN, Bernbaum JG, Jahrling PB, Paragas J, Schnell MJ. Inactivated or live-attenuated bivalent vaccines that confer protection against rabies and Ebola viruses. Journal of virology. 2011;85:10605–10616. - PMC - PubMed
    1. Bradfute SB, Dye JM, Jr, Bavari S. Filovirus vaccines. Hum Vaccin. 2011;7:701–711. - PMC - PubMed
    1. Bukreyev AA, Dinapoli JM, Yang L, Murphy BR, Collins PL. Mucosal parainfluenza virus-vectored vaccine against Ebola virus replicates in the respiratory tract of vector-immune monkeys and is immunogenic. Virology. 2010;399:290–298. - PMC - PubMed
    1. Cleaveland S, Fevre EM, Kaare M, Coleman PG. Estimating human rabies mortality in the United Republic of Tanzania from dog bite injuries. Bull World Health Organ. 2002;80:304–310. - PMC - PubMed

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